bluuebunny/crossref_metadata_2025 huggingface dataset:
KIR/HLA-I Mismatching Predicts Risk of Relapse in Pediatric Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Scquizzato Elisa , Zambello Renato , Baesso Ilenia , Varotto Stefania , Albergoni Maria Paola , Teramo Antonella , Boscaro Elisa , Cesaro Simone , Pillon Marta , Calore Elisabetta , Gazzola Maria Vittoria , Semenzato Gianpietro , Messina Chiara , Trentin Livio
Published: October 2019

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for a variety of hematologic malignancies. Recent studies have indicated the presence of donor-derived alloreactive NK cells as an additional factor influencing transplantation outcome, NK cells being regulated by signals delivered from multiple activating and inhibitory receptors. In the allogeneic HSCT setting, several studies considered donor-recipient HLA-I mismatch as the basis for NK alloreactivity. The implication of the KIR ligand incompatibility model is that donor’s NK cells exhibit the relevant inhibitory KIR for the self HLA-I ligands. It is known, however, that the KIR genes and the HLA genes segregate independently of each other in normal mendelian fashion. This allows the possibility that individuals lack HLA ligands for their KIR receptors and indicates that NK cells alloreactivity can occur also in HLA-matched or HLA-identical transplants. Recent investigations suggest that relapse and KIR-driven alloreactivity in the transplantation setting might be better predicted if the donor KIR genotype is considered in addition to the HLA genotype of the recipient. The aim of this study was to investigate at genetic level the prognostic impact of recipient HLA-I lacking for donor KIR on allotransplanted patients outcome. We analyzed donors KIR genotype and HLA genotype of 60 pediatric patients who received related (n.15) or unrelated (n.45) transplantation. When patients were grouped on the basis of the KIR gene type involved in the KIR/HLA-I mismatch, we did not observe any relapse in the group of patients characterized by mismatches involving only inhibitory KIR. On the contrary, all relapses were observed in patients showing at least one activating gene involved in the mismatch (p<0.05). Although the biological mechanism accounting for this putative genetic rule is still to be clarified, we suggest that a careful survey of KIR/HLA-I mismatching should be taken into account in the selection of donor in related and unrelated HSCT.
A High Long-Term Survival Were Produced by Early Sequential Intensive Consolidation Chemotherapy Followed by Autologous Stem Cell Transplantation in First Complete Remission in Patients with Adult Acute Lymphoblastic Leukemia.
Authors: Zou Dehui , Qiu Lugui , Li Yenan , Zhao Yaozhong , Han Mingzhe , Feng Sizhou
Published: October 2019

Objective: To investigate the efficiency of our designed protocol with early sequential intensive consolidation chemotherapy followed by autologous stem cell transplantation (ASCT) in first complete remission (CR1)on adult acute lymphoblastic leukemia (ALL) patients who lack of matched sibling donors.

Methods: The ALL patients were given a median 4 intensive/ consolidation therapy courses including high-dose of methotrexate, cytarabine and/or cyclophosphamide in CR1, followed by ASCT with total body irradiation (TBI)-based conditioning regimen. A 1 to 1.5 year maintenance chemotherapy with 6-mercaptopurine, methotrexate and vincristine-prednisone in combination with interleukin-2/IFN-α immunotherapy were administered after adequate hematological recovery.

Results: A total of 83 patients were enrolled in this study. 78 patients reconstituted hematopoiesis except 5 patients died of early toxicity. By a median 47(3– 218) months follow-up after ASCT, the 5-year probabilities of leukemia free survival (LFS) and overall survival (OS) were 53.2%±5.9% and 54.4%±5.8%, respectively. The 5-year cumulative relapse rate (RR) was 35.7%±6.1%, and no patients relapsed more than 3 years after transplantation. According to the adverse features, age ≥35 years, WBC >30×109/L for B-cell ALL or >100×109/L for T-ALL, Pro-B and early T immunotyping, more than 4 weeks to attain remission, and high-risk karyotypes including t(9;22)/BCRABL, t(4;11)/ALL1-AF4 or t(1;19)/E2A-PBX1, the patients were identified in 3 prognostic risk groups of standard (24, 29.0%), intermediate(32, 38.5%), and high(27, 32.5%) with 0, 1, 2 or high-risk karyotypes adverse features, respectively. The 5-year probabilities of DFS were 80.36%±9.0%, 59.39%±9.2% and 24.78%±8.9% in these 3 group s, respectively. While, the 5-year cumulative incidences of relapse was 16.7%,37.5% and 70.4%, respectively.. The standard and intermediate -risk groups had significantly better OS and LFS than the high-risk group.

Conclusion: Theses results indicated our designed protocol with early sequential intensive consolidation chemotherapy followed by autologous stem cell transplantation (ASCT) in first complete remission (CR1) could produced a relatively high log-term survival in standard and intermediate-risk group of adult ALL patients.
Hematopoiesis in Chronic Phase CML emerges from Hematopoietic Stem Cells with a Transcriptional Phenotype Resembling Normal Myeloid Progenitor Cells.
Authors: Bruns Ingmar , Czibere Akos , Cadeddu Patrick , Roels Frederik , Fischer Johannes C , Büst Sebastian , Brünnert Daniela , Steidl Ulrich , Brors Benedict , Gattermann Norbert , Kronenwett Ralf , Haas Rainer
Published: October 2019

Introduction: Composition of cell subsets within the CD34+ cell population is markedly altered in chronic phase (CP) CML. Specifically, megakaryocyte-erythrocyte progenitors (MEP) show a proportional increase and the percentage of granulocyte-macrophage progenitors (GMP) is significantly lower in comparison to healthy bone marrow. To understand the molecular basis for this, we have used microarray technology to analyze transcriptional differences between distinct subsets from normal and CP CML bone marrow.

Methods: We examined highly purified HSCs, CMPs, GMPs and MEPs from patients with chronic phase CML and healthy volunteers by FACS for subset analyses and applied high-speed cell-sorting to obtain the distinct CD34+ subsets. Then, we performed gene expression analyses (HU-133A 2.0) of each separate subset. Further assays included quantitative RT-PCR, FISH, adhesion and migration assays and transient transfection experiments.

Results: Subset analyses showed a significant proportional expansion of CMPs and MEPs and a decrease of HSCs in chronic phase CML. When comparing the concentration of each subset by means of absolute cell counts, HSC counts were similar whereas counts for other subsets were significantly higher in CML ranging between 2.8 and 7.7-fold. When looking at the gene expression data, it was surprising to see that the CML HSC has a transcriptional profile, which is similar to CML progenitors and healthy CMP as determined by Euclidian Distance Analysis. In contrast, for normal individuals the cluster tree clearly resembled the current model for hierachical development with the HSC sitting at the top of the hierarchy, and the more differentiated subsets of MEP and GMP at the bottom. Given that differences between the determined progenitors were minor, we focused on the characterisation of the CML HSC. We found 614 genes differentially expressed including downregulation of genes encoding for adhesion molecules, transcription factors and regulators of stem cell fate as well as upregulation of proliferation-associated genes in CP CML. Impaired adhesive and migratory capacity and a novel role of the nuclear receptors NR4A1 and NR4A3 for the transcriptional regulation of c-Jun and JunB was functionally corroborated in CML HSC.

Conclusion: Based on these data we propose a loss of quiescence of CML HSC upon detachment from the niche leading to expansion of myeloid progenitors as determined by quantitative analysis.
Significance of Factor VIII Inhibitors by ELISA in Patients with Hemophilia A.
Authors: Brewer Jamie R , Harris Sandra , Green David , Zakarija Anaadriana
Published: October 2019

Background: The Bethesda assay traditionally has been used to detect Factor VIII inhibitors in patients with Hemophilia A, but recent evidence suggests that it is not sensitive to all inhibitors, particularly non-inhibitory or low-titer antibodies.

Methods: Patients with Hemophilia A without prior history of inhibitor were recruited. Study questionnaire collected demographic and clinical information, bleeding history and factor usage over the preceding 6 months. Functional status was assessed by the Hemophilia Activities List (HAL). Factor VIII inhibitor was assessed by both the Bethesda assay and Factor VIII inhibitor ELISA (GTI Diagnostics). T-test was performed to assess statistical significance.

Results: Data is available for 26 patients, 19 severe, 2 moderate and 7 mild. All subjects had a negative Bethesda assay, but 10 (39%) had a detectable inhibitor by ELISA. 9/10 inhibitor patients had severe hemophilia, while one had mild hemophilia. In severe hemophiliacs, there were no differences in age, HIV status, CD4 count, Hepatitis C positivity or viral load between those with and those without inhibitors. Inhibitors were more frequent in those using plasma-derived concentrates 4/5 (80%), than in those using recombinant products 6/14 (43%), p=0.15. There was no difference in bleeding frequency or functional status in patients with or without inhibitors, although those with inhibitors had more frequent infusions.(Table). In patients on prophylaxis, those with inhibitors had a higher bleeding frequency compared to those without an inhibitor, (p =0.2). 11 patients were not on prophylaxis and had a higher bleeding frequency (p = 0.02) than patients on prophylaxis irrespective of inhibitor presence. However those with inhibitors required more factor doses per bleed compared to those without an inhibitor (4.4 vs. 1.5, p=0.16) even though the mean factor dose was the same (25.3 units/kg vs 25.2 units/kg).

Conclusions: The Factor VIII ELISA assay detected inhibitors in 39 % of Hemophilia A patients who had undetectable inhibitors by standard Bethesda assay. This data suggests that these inhibitors may be clinically relevant, given that inhibitor patients who are not on prophylaxis require more doses of factor per bleeding event. Further study is necessary to determine mechanism and clinical significance of these Factor VIII inhibitors.

Table. Characteristics of severe hemophilia patients with and without ELISA Factor VIII inhibitor

All severe (n=19) Inhibitor (n=9) No inhibitor (n=10) Age 43.4 40.8 45.7 Plasma-derived factor 5 (26.3%) 4 (44.4%) 1 (10%) Total bleeds/6 months 8.6 8.1 9.0 Muscle bleeds/6 months 1.3 0.8 1.7 Joint bleeds/6 months 7.3 7.3 7.3 Factor infusions/6 months 50.9 57.2 45.2 On prophylaxis 8 (42%) 4 (44.4%) 4 (40%) Total bleeds/6 months 4.3 5.8 2.8 Not on prophylaxis 11 (58%) 5 (55.6%) 6 (60%) Total bleeds/6 months 11.7 10 13.2 Factor doses/bleed 2.9 4.4 1.5
Health-Related Lifestyle among Adult & Pediatric Patients with Idiopathic Thrombocytopenic Purpura in the United Kingdom.
Authors: Sarpatwari Ameet , Watson Shirley , Anderson Howard , Provan Drew , Newland Adrian
Published: October 2019

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune condition characterized by autoantibody-mediated platelet destruction and suboptimal megakaryocytic production. Primarily acute (< 6 months) in duration among children, ITP manifests predominantly chronically among adults, increasing susceptibility to bleeding events.

Despite recent growth, published literature on health-related quality of life in ITP remains limited. The objective of our investigation was to identify lifestyle concerns associated with ITP among adult and pediatric patients in the United Kingdom.

In collaboration with ITP specialists, a 43 question, closed-field lifestyle survey was developed, addressing social engagement, work and school performance, sports and activities, treatment, and travel. Patient members of the United Kingdom ITP Support Association (N = 1,767) were asked to complete and return mailed surveys. Pearson’s chi-square and Fisher’s exact tests were used to evaluate differences in dichotomized variables between groups.

790 (45%) completed surveys were returned. As illustrated in the table below, roughly one-quarter of adults (≥ 16 years) and one-fifth of children reported ‘always’, ‘sometimes’, or ‘often’ missing school or work owing to fatigue and having encountered difficulty obtaining insurance. Nearly one-third of adults further revealed having an elective surgery delayed owing to a low platelet count.

Disparities were noted across gender with regard to bruise concealment (adults: p < 0.01; children: p = 0.03) and suspicions of subjection to violence (adults: p < 0.01; children: p = 0.49). In contrast with adults, pediatric patients reported being more likely to having requests for referral denied (p = 0.03).

This study represents the largest quality of life investigation of ITP to date. Although the survey utilized remains to be validated, its findings nonetheless successfully highlight avenues for future investigation.

Subgroups: ‘Yes’ % Adults Children Question Total ‘Yes’ % N = 790 Male N = 199 Female N = 497 Male N = 51 Female N = 43 [1] Total number of responses recorded for the given question. Have you ever been unable to go to work or school because of tiredness and fatigue? 29%   710[1] 26%  186 31%  435 20%  49 30%  40 Have you had difficulty obtaining or been refused travel and life insurance? 29%  611 30%  540 18%  71 Have you ever had surgery (other than splenectomy for ITP) postponed or delayed because of a low platelet count? 30%  620 34%  158 30%  391 13%  39 19%  32 Do you try to hide your bruises? 29%  756 10%  190 37%  475 20%  50 42%  41 Are people ever suspicious that the bruises are a result of physical violence? 17%  750 5%  193 19%  468 31%  49 38%  40 Have you ever been refused a referral to an ITP specialist or hospital of 5%  574 3%  144 4%  358 10%  41 10%  31
Sensitive Detection of Survival of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Red Blood Cells, Granulocytes and Monocytes by High Resolution Multiparameter Flow Cytometry.
Authors: Movalia Mayur K , Illingworth Andrea
Published: October 2019

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by intravascular hemolysis due to GPI-deficient red blood cells sensitive to complement-mediated lysis. Accurate and sensitive detection of PNH-type cells has become important not only to diagnose PNH but also because studies have shown PNH-type cells may indicate favorable response to therapy and favorable prognosis in patients with aplastic anemia and myelodysplastic syndrome. Previous studies have suggested optimal testing for PNH-type cells by flow cytometry should be limited to within 48 hours after collection of whole blood. Our laboratory has developed a very sensitive and specific high resolution flow cytometric method for detecting PNH-type cells based on testing over 3,000 patients with known PNH, aplastic anemia, myelodysplastic syndromes and other bone marrow failure syndromes. The aim for this study was to determine the longevity of PNH clones in whole blood samples, the day-to-day variability of these clones and the rate of deterioration of the PNH clones compared to normal blood cells. We analyzed 10 whole blood samples from patients known to have PNH-type cells on seven consecutive days utilizing a two-color assay with GPA-CD59 for the red blood cells, a 5-color assay with FLAER-CD24-CD14-CD15-CD45 for the granulocytes and a 5 color assay with FLAER-CD33-CD14-CD64-CD45 for the monocytes. The results are summarized in the table below. The initial PNH clone sizes ranged from 0.02% to 90.8%. The PNH cells showed an overall similar level of deterioration to the normal blood cells with even minor PNH clones of 0.02% able to be detected at day 7. The day-to-day variability of PNH clone sizes was generally less than 10%, with smaller clone sizes showing a higher degree of variation, up to 20%, due to their smaller absolute numbers. Interestingly, Type III PNH red blood cells showed slightly better overall survival than normal red blood cells and were detected in modestly increasing percentages throughout the study. Based on this data, we propose that accurate detection of PNH type cells can be achieved up to seven days after collection of whole blood when utilizing high resolution flow cytometry. PNH Clone Size on Sequential Days as Percentage of Original PNH Clone Size

Original PNH Clone Sizes PNH Clone Sizes as Percentage of Original PNH Clone Size Cell Type Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Type III RBCs .02%–58.6% 102% 106% 107% 104% 108% 103% Granulocytes .29%–90.8% 100% 100% 93% 89% 79% 86% Monocytes .52%–89.9% 96% 96% 92% 94% 97% 85%
GATA-1 Enhances the Transcription of PIGM and Is Responsible for Increased GPI Expression in Erythrocytes in Inherited Glycosylphosphatidylinositol Deficiency: A Model for Tissue-Specific Housekeeping Gene Transcription Regulation
Authors: Costa Joana S , Nolasco Madalena , Almeida Antonio M , Roberts Irene , Layton Mark , Karadimitris Anastasios
Published: October 2019

Inherited glycosylphophatidylinositol (GPI) deficiency (IGD), characterized by thrombosis and epilepsy but minimal hemolysis, is caused by a c>g mutation at position −270 that disrupts binding of the transcription factor (TF) Sp1 to the core promoter of the mannosyltransferase-encoding gene PIGM. In IGD, the level of GPI expression varies considerably between hematopoietic cells of different lineages; for example, while in erythrocytes it is near normal, accounting thus for the absence of significant intravascular hemolysis, GPI synthesis in granulocytes is severely deficient suggesting erythroid-specific transcriptional regulation of PIGM. We tested the hypothesis that the erythroid-megakaryocytic-specific TF GATA-1 is critical regulator of PIGM transcription and it can overcome the transcriptional block imposed by the c>g mutation. Consistent with this hypothesis, we found that in luciferase assays a 2kb PIGM promoter construct had at least 3-fold higher activity when transfected in erythroid (K562) rather than myeloid (HL60), B lymphoid or epithelial (HeLA) cell lines. Co-transfection of the 2kb WT or c>g mutated construct with GATA-1 resulted in proportional increase of the transcriptional activity which was completely abolished when shorter, 0.6kb WT or c>g constructs were used, implying the presence of GATA-1 binding elements (BE) between −0.6 and −2kb. The functional significance of 3 such GATA-1 BE initially identified by bioinformatic analysis was investigated next. In vitro binding to each of these BE was shown in electromobility shift assays using appropriate oligos, nuclear extracts from K562 cells and anti-GATA-1 antibody while in vivo binding was demonstrated by chromatin immune-precipitation assays. The relative transcriptional activity of the 3 GATA-1 BE was tested in luciferase assays by co-transfecting into HeLA cells WT or c>g 2 kb constructs with single, double or triple BE disrupted by site-directed mutagenesis. Overall the results suggested that the proximal of the 3 GATA-1 BE is the most active and that the transcription-enhancing effect of GATA-1 is diminished in the presence of the disease causing c>g mutation. Thus, as well as explaining the near normal synthesis of GPI in erythrocytes in IGD, our results also provide an important paradigm of co-operative function of a generic (Sp1) and tissue-specific (GATA-1) TF in the transcriptional regulation of a house-keeping gene such as PIGM.
Incidence of SCN-Assoicated Gene Mutations in Severe Congenital Neutropenia Patients in North America
Authors: Xia Jun , Bolyard Audrey Anna , Rodger Elin , Stein Steven , Aprikyan Andrew AG , Dale David C. , Link Daniel C.
Published: October 2019

Severe congenital neutropenia is a genetically heterogeneous syndrome associated with mutations in several different genes including ELA2, HAX1, GFI1, WAS, and CSF3R. The goal of this study was to define the mutation frequency of these genes in the North American SCN patient population. We also sequenced SBDS, since mutations of SBDS have been associated with congenital and acquired neutropenia. A total of 159 patients were identified in the North American Severe Chronic Neutropenia International Registry (SCNIR) for whom informed consent and genomic DNA samples adequate for sequencing were available. To accommodate our semi-automated high-throughput sequencing pipeline, 94 samples were chosen for sequencing. Since ELA2 sequencing had already been performed in most cases, preference was given to those samples without known ELA2 mutation. Among the samples, 73 were from patients with SCN, 4 with cyclic neutropenia, 10 with idiopathic neutropenia, 2 with Shwachman-Diamond Syndrome (SDS), and 3 with Barth syndrome. Two samples were excluded because of poor sequence quality. Singleton cases with validated mutations of GFI1 (N382S) and WAS (L270P) were observed. The N382S GFI1 mutation was associated with striking monocytosis. A novel nonsense mutation of GFI1 (R412X) was detected in one additional case. As expected, compound heterozygous mutations of SBDS were present in the two cases of SDS. In addition, heterozygous mutations of SBDS (84Cfs3X and Q94X) were observed in two cases of SCN. Typical truncation mutations of CSF3R were detected in 4 cases, all developing MDS or AML. Surprisingly, no mutations of HAX1 were detected. Considering only patients with a diagnosis of SCN who were from North America (125 of the total 159 cases), the incidence of ELA2 mutations was 68%. Eleven novel ELA2 mutations were identified. In 28.8% of cases, no mutation of any gene were detected. Based on these data, we recommend that ELA2 genotyping be performed in all patients with suspected SCN. In the North American population mutations in HAX1, GFI1, SBDS, and WAS are rare and routine genotyping is not indicated. Finally, the data suggest that there are yet undiscovered genetic causes of SCN.
Gfi-1 Represses CDKN2B Encoding p15INK4B through Interaction with Miz-1
Authors: Liu Qingquan , Basu Suchitra , Qiu Yaling , Dong Fan
Published: October 2019

Zinc finger (ZF) transcriptional repressor Gfi-1 plays an important role in hematopoiesis and inner ear development, and also functions as an oncoprotein that cooperates with c-Myc in lymphomagenesis. Gfi-1 represses transcription by directly binding to the consensus DNA sequence in the promoters of its target genes. We report here an alternative mechanism by which Gfi-1 represses CDKN2B encoding the cyclin-dependent kinase inhibitor p15INK4B. Gfi-1 did not directly bind to CDKN2B, but interacted with Miz-1 and, via Miz-1, was recruited to the core promoter of CDKN2B. The C-terminal zinc finger domains of Gfi-1 and Miz-1 are involved in the interaction. Miz-1 is a POZ-ZF transcription factor that has been shown to mediate transcriptional repression by c-Myc. Like c-Myc, upon recruitment to the CDKN2B promoter, Gfi-1 repressed transcriptional activation of CDKN2B by Miz-1 and in response to TGFb. Notably, Gfi-1 and c-Myc formed a ternary complex with Miz-1 and were both recruited to the CDKN2B core promoter via Miz-1, and acted in collaboration to repress CDKN2B. Consistent with its role in repressing CDKN2B transcription, knockdown of Gfi-1 in human leukemic cells resulted in augmented levels of p15INK4B, which was associated with attenuated cell proliferation. The expression of p15INK4B was also significantly higher in Gfi-1−/− mouse bone marrow (BM) cells than in Gfi-1+/+ BM cells. Our data reveal a novel mechanism of transcriptional repression by Gfi-1 and also identify CDKN2B as a new Gfi-1 target gene. The findings may have important implications for understanding the role of Gfi-1 in normal development and the cooperation between Gfi-1 and c-Myc in lymphomagenesis.
Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by Tyrosine Kinase Inhibitors
Authors: Marit Michael , Chohan Manprit , Matthew Natasha , Huang Kai , Barber Dwayne
Published: October 2019

Mutation and activation of JAK2 is a common event in Myeloproliferative Disease as JAK2 V617F and related deletion mutations are observed in Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. In addition, the TEL-JAK2 chromosomal translocation is a rare event in Acute Myeloid Leukemia and Chronic Myelomonocytic Leukemia. We reasoned that activated alleles of JAK2 would develop resistance to JAK inhibitors in a clinical setting, similar to the development of Imatinib resistance in BCR-ABL-mediated Chronic Myeloid Leukemia. The objective of this study was to develop a random mutagenesis screen to isolate mutations of JAK2 resistant to tyrosine kinase inhibitors. We selected JAK Inhibitor-1 for this study, since the crystal structure of this inhibitor bound to the JAK2 JH1 kinase domain has been reported and would allow for mapping of confirmed mutations. TEL-JAK2(5–12) and JAK2 V617F were subcloned into retroviral expression vectors and random libraries of mutations were generated by transformation into XL-1 Red strain of E. coli, a strain defective in pathways of DNA repair. High titer retroviral supernatants were generated and used to transduce Ba/F3 (for TEL-JAK2) or Ba/F3-EPO-R (for JAK2 V617F). Control experiments were performed with “wild type” versions of each JAK2 allele. Inhibitor-resistant clones were identified and DNA sequencing was performed to identify JAK2 JH1 kinase domain mutations that confer resistance to inhibitor. We have restricted the analysis of JAK inhibitor-resistant mutants to those that map within the kinase domain of JAK2 for purposes of this study. We have confirmed that E864K, V881A, N909K, G935R, R975G confer resistance to JAK inhibitor-1 in growth assays. In addition, M929I (analogous to BCR-ABL T315I) mediates resistance to JAK inhibitor-1, relative to wild-type activated JAK2 alleles. All mutations result in increased phosphorylation of STAT5, Akt and Erk in the presence of inhibitor. We are currently testing the catalytic activity of each mutant to determine whether JAK2 kinase domain mutations have similar enzymatic activity or whether mutation also affects catalysis. We have mapped each mutation within the JAK2 JH1 crystal structure and models for how each mutant affects inhibitor binding will be presented. Importantly, many of these residues are highly conserved in other JAK tyrosine kinases and within BCR-ABL. We are extending these observations by testing clinically relevant inhibitors in our screen. At the conclusion of this study we will be able to identify common residues critical for resistance by JAK2 inhibitors and unique residues that are inhibitor-specific. Random mutagenesis screening offers an excellent strategy to identify JAK2 residues that may be relevant in the clinic and also serve in enhancing our knowledge regarding JAK kinase activation and regulation.
Immune-Related and Inflammatory Conditions Likely Play a Role in the Development of Lymphoplasmacytic Lymphoma/Waldenström’s Macroglobulinemia
Authors: Kristinsson Sigurdur Y , Bjorkholm Magnus , Koshiol Jill , Goldin Lynn R. , McMaster Mary L. , Turesson Ingemar , Landgren Ola
Published: October 2019

Introduction: Certain autoimmune and infectious conditions are associated with increased risks of subtypes of non-Hodgkin lymphomas (NHL). A few prior studies suggest that chronic immune stimulation may particularly elevate risk for the NHL subtype lymphoplasmacytic lymphoma (LPL)/Waldenström’s macroglobulinemia (WM). To improve our understanding on the role of immune-related and inflammatory conditions in LPL/WM pathogenesis, we conducted a large population-based study including close to 2,500 LPL/WM patients diagnosed in Sweden and almost 10,000 matched controls.

Methods: Using both the central Cancer registry and local hospital-based registries, we identified all LPL/WM patients diagnosed in Swedish hospitals 1958–2005. From the Swedish Population Registry we identified four matched controls per LPL/WM patient. Through data linkage with the central Inpatient registry, we gathered information on hospital inpatient discharges that listed autoimmune-, infectious-, and other inflammatory/allergic diseases present at least 1 year prior to LPL/WM. Using Poisson regression, we calculated rate ratios (RR) and 95% confidence intervals (CI) adjusted for categorical year of birth, date of diagnosis, gender, and county.

Results: A total of 2,470 LPL/WM patients (647 LPL and 1,823 WM), and 9,698 population-based matched controls were included in the study. We found an increased risk of developing LPL/WM among individuals with a prior history of systemic sclerosis (RR=4.7; 1.4–15.3), Sjögren’s syndrome (RR=12.1; 3.3–45.0), autoimmune hemolytic anemia (AIHA) (RR=24.2; 5.4–108.2), polymyalgia rheumatica (RR=2.9; 1.6–5.2), and temporalis arteritis (RR=8.3; 2.1–33.1). We also found excess risk of LPL/WM among persons with a history of pneumonia (RR=1.4; 1.1–1.7), septicaemia (RR=2.4; 1.2–4.3), pyelonephritis (RR=1.7; 1.1–2.5), sinusitis (RR=2.7; 1.4–4.9), herpes zoster (RR=3.4; 2.0–5.6), and influenza (RR=2.9; 1.7–5.0). Importantly, when we assessed the associations by latency (time between immune-related or inflammatory conditions and LPL/WM), for most autoimmune- and infectious diseases the excess LPL/WM risk remained significant at >5 years latency. We found no significant increased risk for LPL/WM among individuals with prior chronic inflammatory or allergic conditions.

Conclusions: In the largest investigation of risk factors for LPL/WM to date, we found a personal history of certain autoimmune and infectious diseases to be associated with excess LPL/WM risk. Immune-related conditions might act as potential LPL/WM triggers or they could represent premalignant immune disruptions preceding LPL/WM. Our results provide novel insights into the as yet unclear pathogenesis of LPL/WM.
Are Mast Cells a Biologic Predictor of Outcome in Follicular Lymphoma?
Authors: Howard Matthew T , Pohlman Brad , Jin Tao , Hsi Eric
Published: October 2019

Background: The tumor microenvironment is important in the biology of follicular lymphoma (FL). In addition to macrophages, which have been shown to be associated with outcome in FL, mast cells (MCs) have the ability to influence the microenvironment through secretion of numerous biologically active molecules such as growth factors and cytokines/chemokines. Increased numbers of tumor-infiltrating MCs have been suggested as an unfavorable prognostic marker that also supercedes the predictive value of lymphoma-associated macrophages (LAM) in FL patients treated with immunochemotherapy. This study examines the effects of tumor-infiltrating MCs in FL patients at Cleveland Clinic.

Design: Tissue microarrays were constructed from 94 newly diagnosed cases of FL from 09/01/1985 to 12/13/2002 that had been previously characterized for CD68+ LAMs and demonstrated that extrafollicular (EF) LAMs (>16.8/high power field) were a poor prognostic marker (Kelley T et al 2007). Immunohistochemistry for tryptase was performed on these microarrays to enumerate MCs per high powered (400x) field. In each core, all available lymphomatous tissue was evaluated (mean of 8.13 hpf/case, range 1–9). The cell counts were compared to the available clinical follow-up data, using overall survival as the measurable outcome.

Results: The FL patients (44 women, 50 men) had a mean age at diagnosis of 59 years and a mean follow-up of 95.8 months. The patients were treated heterogeneously; the great majority did not receive immunochemotherapy as initial treatment. MC data was available in 91 patients. The mean MC count was 4.14 MCs/hpf (range 0–17.33). As shown previously, > 16.8 EF LAMs/hpf was a predictor of poor overall survival; hazard ratio of 2.28, 95% CI: 1.22–4.24, P = .009. In the entire cohort, MCs were not associated with overall survival. However, in the subgroup with low LAM (CD68 <16.8/hpf), increased MCs (>3.3/hpf) was associated with better overall survival, HR = 0.40, 95% CI: 0.16–0.99, P = .049.

Conclusion: In our data set, increased LAMs in FL are associated with poor overall survival. Overall, MCs do not appear to predict outcome; however, in the subgroup of patients with low LAMs, increased MCs are associated with a favorable overall survival. This suggests a complex interaction between cellular constituents of the tumor microenvironment that affects patient outcome. The application of immunochemotherapy or other immunomodulatory therapies may alter these interactions and outcomes. Further work is needed to better understand these interactions and to validate biomarkers in therapy specific contexts.
Identification of ERG Specific Target Genes by Genome-Wide Screening in T-Lymphoblastic Leukemia
Authors: Mochmann Liliana H , Neumann Konrad , Bock Juliane , Tanchez Jutta Ortiz , Bohne Arend , Thiel Eckhard , Hofmann Wolf K , Baldus Claudia D
Published: October 2019

The Ets related gene, ERG, encodes a transcription factor with a vital role in hematopoiesis. Recent findings have shown that ERG knockout mice require a minimum of one functional allele to ensure embryonic blood development and adult stem cell maintenance. Moreover, it was earlier reported that enforced expression of ERG induced oncogenic transformation in 3T3 cells. Overexpression of ERG, observed in a subset of acute T-lymphoblastic and acute myeloid leukemia patients, was associated with an inferior outcome. However, the impact of ERG contributing to this unfavourable phenotype has yet to be determined, as downstream targets of ERG in leukemia remain unknown. Herein, we conducted a genome-wide analysis of ERG target genes in T-lymphoblastic leukemia. Chromatin immunoprecipitation-on-chip array (ChIP-on-chip) analyses were performed using two ERG specific antibodies for the enrichment of ERG-bound DNA templates in T-lymphoblastic leukemia cells (Jurkat) with input DNA or IgG precipitated DNA as controls. Enriched DNA templates and control DNA were differentially labelled and co-hybridized to high resolution promoter chip arrays with 50–75mer probes (770,000) representing 29,000 annotated human transcripts (NimbleGen). Based on two independent ChIP-on-chip assays, bioinformatic analysis (ACME) yielded statistically significant enriched peaks (using a sliding window of 1000 bp, and a P-value < 0.0001) identifying promoter regions of 365 potential ERG target genes. From these genes, clustering by functional annotation was performed using the DAVID database and subsequently genes related to leukemia were further selected for quantitative PCR validation. The design of promoter primers included the highly conserved ETS GGAA DNA binding site. Genes with greater than two-fold enrichment (ERG ChIP versus control) included WNT2 (17-fold), OLIG2 (14-fold), WNT11 (7-fold), CCND1 (5-fold), WNT9A (4-fold), CD7 (3-fold), EPO (3-fold), ERBB4 (3-fold), RPBJL (3-fold), TRADD (3-fold), PIWIL1 (2-fold), TNFRSF25 (2-fold), TWIST1 (2-fold), and HDAC4 (2-fold). Interestingly, enriched target genes involved in developmental processes (WNT2, WNT9A, WNT11, TWIST1, PIWIL1, ERBB4, and OLIG2) have shown oncogenic potential when mutated or overexpressed. Thus, we hypothesize that overexpression of ERG may contribute to T-cell leukemogenesis by the deregulation of these oncogenic targets. Further disclosure of ERG directed downstream pathways may contribute to the design of specific treatment strategies (such as WNT inhibitors) with particular effectiveness in ERG deregulated leukemia.
Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia
Authors: Thomas Daniel , Woodcock Joanna , Powell Jason A , Barry Emma F , Lopez Angel F , Guthridge Mark A
Published: October 2019

New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays.

Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts.

Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.
Factor H Binds to Von Willebrand Factor (VWF) and Regulates Its Cleavage by ADAMTS-13
Authors: Cruz Miguel A. , Gushiken Francisca C. , Rumbaut Rolando , Patel Kavita N , Turner Nancy , Nolasco Leticia , Han Hyojeong , Pickering Matthew , Dong Jing-fei , Moake Joel L. , Afshar-Kharghan Vahid
Published: October 2019

Factor H is a plasma protein that regulates activation of the alternative complement system. Mutations in the factor H gene are associated with a rare form of thrombotic microangiopathy, known as atypical hemolytic uremic syndrome. Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are characterized by systemic (TTP) or renal (HUS) microvascular thrombosis. The clinical presentations of TTP and HUS have some common clinical features, including the presence of thrombocytopenia, intravascular hemolysis, and mechanical injury to red cells. We investigated whether factor H has any interaction with proteins involved in the pathogenesis of thrombotic thrombocytopenic purpura, namely von Willebrand Factor (VWF) and ADAMTS-13. We found that factor H binds to the A1 and A2 domains of VWF, and inhibits cleavage of VWF by ADAMTS-13 under static and flowing conditions. Factor H deficient mice had a delayed thrombous formation after vessel wall injury, and showed a lower mortality rate and a higher platelet count after Shiga toxin/lipopolysaccharide challenge compared to their wild-type littermates. We concluded that factor H, in addition to its complement regulatory activity, might regulates cleavage of VWF by ADAMTS-13. Presence of an abnormal factor H that is unable to control complement activity on cell surfaces but still capable of inhibiting cleavage of VWF might contribute to the pathogenesis of thrombotic microangiopathies.
Activity of SYK and PLCγ2 Predict Apoptotic Response of Chronic Lymphocytic Leukemia Cells to SRC Tyrosine Kinase Inhibitor Dasatinib.
Authors: Wang Y. Lynn , Song Zibo , Lu Pin , Furman Richard R , Leonard John P. , Martin Peter , Tyrell Lauren , Lee Francis Y. , Knowles Daniel M. , Coleman Morton
Published: October 2019

Abstract 1249

Poster Board I-271

Purpose

B-cell receptor (BCR) signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking BCR signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in pre-clinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL.

Experimental Design

Fresh CLL B cells were treated with dasatinib and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of BCR signaling molecules as well as with molecular and cytogenetic prognostic factors.

Results and Conclusions

Among 44 CLL cases, dasatinib treatment reduced cell viability by 2-90%, with an average reduction of 48% on day four of culture. The drug induced CLL cell death via the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good as well as poor responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and PLCγ2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with PLCγ2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment.

Disclosures

Lee: Bristol-Myers Squibb Research & Development: Employment. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.
Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.
Authors: Jallu Vincent , De Brevern Alexandre , Panzer Simon , Torchet Marie-Francoise , Kaplan-Gouet Cecile
Published: October 2019

Abstract 1312

Poster Board I-336

Introduction

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3.

Patient and methods

Genomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling.

Results

7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect.

Conclusion

Seven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction.

Disclosures

No relevant conflicts of interest to declare.
Barriers to Reduced-Intensity Conditioning (RIC) Transplant in Patients with Chronic Lymphocytic Leukemia.
Authors: Jaglowski Samantha M. , Lin Thomas S. , Elder Patrick , Scholl Diane , Byrd John C. , Devine Steven M. , Andritsos Leslie
Published: October 2019

Abstract 1374

Poster Board I-396

Introduction:

Allogeneic stem cell transplantation is the only potentially curative therapeutic option for patients with CLL. However, in spite of recent improvements in transplant-related mortality due to the introduction of reduced-intensity conditioning (RIC), only a proportion of patients go on to receive a stem cell transplant. The aim of this study was to quantify how many patients are evaluated for transplant versus how many go on to transplant, and to examine the reasons why transplantation was not performed.

Patients and methods:

A list of all patients with CLL who have been referred to the Ohio State University for a transplant evaluation from January 2003 to the present was obtained. Two electronic databases, E-Results and TransChart, were reviewed to determine if a consultation was performed, if patients were subsequently transplanted, and if not, why.

Results:

The outcomes of the referrals are summarized in Table 1. From January 2003 to the present, 205 patients with CLL were referred to our center for a transplant evaluation. Of those, 34 (16.6%) underwent transplantation. For patients who did not receive a transplant, it was not indicated at the time of consult according to current standard of care in 37 patients (18%), and in 29 (14.1%), transplant was indicated but patients were unable to obtain a remission in order to proceed. The differences between patients who went on to transplant and those who could not obtain remission were analyzed more closely. The median age in the transplant group was 55, and the median age in the group that could not obtain remission was 58. Both groups received a median 2 lines of treatment prior to transplant evaluation. The cytogenetic abnormalities of the groups are summarized in Table 2.

Conclusions:

The majority of patients who did not undergo transplantation did not have an indication for transplant, reflecting previous standards of care prior to the understanding of the poor prognosis of patients with high risk karyotype and fludarabine-refractory CLL. In contradistinction, an inability to obtain a remission in order to proceed to transplantation was the second most common reason transplantation was deferred. These data reflect the difficulty in some cases of determining the appropriate timing for allogeneic transplantation in CLL and the risk of delaying a transplant referral in patients with fludarabine refractory disease. Appropriate timing of transplant referral and choice of effective salvage therapy are critical to the successful long-term management of patients with fludarabine-refractory CLL.

Disclosures:

No relevant conflicts of interest to declare.