bluuebunny/crossref_metadata_2025 huggingface dataset:
MDR-1 Recognition by Cytotoxic T Cells.
Authors: Niethammer Andreas G. , Wodrich Harald , Loeffler Markus , Lode Holger , Krempien Robert , Huber Peter E. , Debus Juergen , Reisfeld Ralph A.
Published: October 2019

Acquired multidrug resistance (MDR) remains a major challenge in the treatment of cancer with chemotherapeutic drugs. It can be mediated by the upregulated expression of different proteins within the tumor cell membrane. Here we used murine MDR-1 as a target-antigen for the immunotherapy of cancer. We successfully demonstrated that peripheral T cell-tolerance can be broken by oral administration of a DNA vaccine encoding MDR-1 and carried by attenuated Salmonella typhimurium to secondary lymphoid organs. Thus, mice, immunized orally three times at 2-week intervals and challenged two weeks thereafter with either MDR-1 expressing CT-26 colon carcinoma cells or MDR-1 expressing Lewis lung carcinoma cells, revealed a significant increase in lifespan. This was evident, when compared to animals vaccinated with the empty control vaccine or to animals challenged with the maternal cell lines lacking overexpression of MDR-1. The immune response induced was antigen specific and CD8+ T cell-mediated. The presence of the target antigen led to upregulation of activation markers on CD8+ T cells and resulted in a strong cytotoxic T cell response as well as lysis of tumor target cells in vitro. We furthermore established the vaccine to be an effective treatment for established multidrug resistant tumor metastases resulting in a significantly increased lifespan of experimental animals. Absence of CD8+ T cells due to in vivo depletion led to abrogation of effectiveness. Taken together our results demonstrate, that T cell tolerance against the MDR-1 self antigen can be broken. It is anticipated that the combination of such an approach with chemotherapy could lead to more effective treatments of cancer.
Purification of c-Kit-Negative Hematopoietic Stem Cells from Runx1-GFP Knock-In Mice.
Authors: Lorsbach Robert B. , Ang Sonny O. , Sun Weili , Moore Jennifer , Downing James R.
Published: October 2019

The Runx1/Core Binding Factor- β (CBF β) transcriptional complex is required for the establishment of hematopoiesis during development. To permit the analysis of Runx1 expression in hematopoietic cell subsets, we have recently developed a novel murine line in which the expression of both full-length Runx1 and GFP is driven by the Runx1 promoter [Runx1-internal ribosomal entry site-green fluorescent protein (Runx1-GFP) knock-in mouse; Blood 103:2522]. Analysis of these mice has revealed that Runx1 is expressed in all hematopoietic lineages with the exception of erythroid cells. During our analysis, we identified in the bone marrow of these mice a cell population that expresses GFP at levels 2–5 fold higher than any other cell type (GFPhi). These cells have low forward and side scatter properties and do not express c-kit or several lineage-associated cell surface markers (lin−). In comparison to c-kit+lin− cells, these GFPhic-kit−lin− cells possess little colony forming activity in vitro. While they lack primary CFU-S activity in contrast to c-kit+lin− cells, GFPhic-kit−lin− cells possess secondary CFU-S activity. A c-kit−lin− hematopoietic stem cell (HSC) has been described by others that contributes to long-term, but not short-term, hematopoietic reconstitution in lethally irradiated recipients and may represent progenitors of c-kit+lin− HSCs in vivo (Ortiz et al. Immunity 10:173). The GFPhi bone marrow cells that we have identified share many of the properties of those c-kit−lin− cells identified by others; consequently, they likely represent the same cell population. Our ability to isolate these cells based on differential GFP expression should enable us to highly purify these GFPhic-kit−lin− cells and further characterize their immunophenotypic and biologic properties.
In Multiple Myeloma, Clonal Cells Are Unevenly Distributed among Peripheral Blood B Cell Subpopulations.
Authors: Papadhimitriou Stefanos I. , Gligori Ioanna , Kritikou-Griva Elpiniki , Gortzolidis Georgios , Davea Aggeliki , Paterakis Georgios S. , Goumakou Eleni , Savvidou Ioanna , Grigoraki Vassiliki , Anagnostopoulos Nikolaos I. , Georgiakaki Maria
Published: October 2019

BACKGROUND: Current evidence supports the existence of circulating clonal B cells in Multiple Myeloma (MM). However, attempts to enumerate and phenotypically characterise them have so far provided inconsistent results. Most investigators have studied unselected peripheral blood (PB) mononuclear cells, among which the clonal ones make only a small minority. Moreover, in most cases, numerical chromosomal changes were employed as a clonal marker, but aneuploidy is considered a late event in myelomagenesis. To overcome these difficulties, we have followed an alternative approach, by studying purified PB B cells and focusing on chromosomal translocations involving the immunoglobulin heavy chain gene (IGH) on region 14q32, a frequent, early and possibly crucial pathogenetic event in MM.

METHODS: The study included 33 MM patients with 14q32 rearrangements, detected by conventional cytogenetics or florescence in-situ hybridisation (FISH) in the bone marrow at diagnosis. PB CD19+ cells were immunomagnetically isolated (>99% purity) and cytocentrifuged on slides. The slides were studied with a FICTION technique, ie a combination of FISH using a “break-apart” IGH probe set (Vysis Inc, Downers Grove, Il, USA) and indirect immunofluorescence for CD34, CD5, CD10, CD23 and CD38. To avoid the possibility of contaminating plasma cells, isolates with >1% CD19+CD38+++ cells on flow cytometry were excluded from FICTION study.

RESULTS: “Positive” cells above the cutoff level of false positivity (4%) were detected in 25 cases (75.7%), ranging from 4% to 33% (median 9%) among the total CD19+ population. These cells were found to consistently express CD10 (83% to 100%, median 96%) and CD38 (79% to 100%, median 89%). They less commonly expressed CD23 (39% to 67%, median 46%) and very rarely CD34 (0% to 5%, median 0%) and CD5 (0% to 3%, median 0%).

CONCLUSIONS: Our data suggest that circulating cells bearing IGH rearrangements are the rule in MM, making a small but detectable fraction of CD19+ cells. There mmunophenotypic profile supports the concept that clonal B cells represent advanced ontogenetic rather than early stages in B lineage differentiation. Finally, the virtual absence of CD5+ clonal cells is in accord with the view that the high number of PB CD5+ B cells in MM reflects an immunoregulatory network and does not result from clonal expansion.
The Modulating Effect of Azacitidine (Aza C) on the Cytogenetically Tracked MDS Clone Impact Survival.
Authors: Najfeld Vesna , Scalise Angela , Odchimer-Reissig Rosalie , Silverman Lewis R.
Published: October 2019

MDS is a clonal hematopoietic stem cell disorder characterized by progressive bone marrow failure leading to death from infections and bleeding in the majority of patients. Transformation to acute leukemia occurs in 35-40%. Most treatments, other than allogeneic SCT, have been ineffective. Treatment with Aza C has been shown to be clinically effective and alters the natural history of disease (Silverman et al JCO 2002). We have previously demonstrated Aza C modulation of the MDS clone and established five different cytogenetic categories based on its modulating effect (Najfeld et al, ASH 2002). The goal of this extended study was to determine biological and clinical consequences of Aza C modulation on the five cytogenetic categories. Among 224 patients treated with AzaC, multiple sequential studies were performed in187 pts, while 41 pts without follow up studies, were excluded from this report. The initial karyotype prior to therapy with Aza C was normal in 99 pts (53%) and abnormal in 88 pts (47%). These latter patients were not further subdivided into good, intermediate or poor risk according to IPSS. The table below shows the Kaplan Meier median survival of the 5 subcategories based on the modulating effects of Aza C on the cytogenetically identified MDS clone.

Cyto Category No of Frequency of Hematological Survival Group Pts Emerging Response In No Chromosome Abnormalities Months 1 Normal->Normal 67 None CR:3, PR:10, IMP:31 29.3 2 Normal->Abnormal 32 +8:25%l+1q:25% CR:1, PR:6, IMP:18 28.1 +21:9% 3 Abnormal-> Abnormal 50 −7/del(7q):24%; CR:1, PR:4, IMP:30 11.7 −5/del(5q):20%,+1q:14% 4 Abnormal->Clonal 20 −7/del(7q):25%;+21:20% IMP:8 9.1 5 Abnormal->Normal 18 del(7q):33%,+8,28% CR:1, PR:1,IMP:8 22.7

When survival was compared between cytogenetic subgroups there was a statistical difference between some groups. Survival of patients with normal cytogenetics (group 1) was superior when compared to either group 3 (p=.0001) or group 4 (p=.00015). Even for pts who developed an abnormal clone while on AzaC (group 2) survival was significantly longer when compared to either group 3 (p=0004) or group 4 (p=00001). Moreover, prognostic significance was observed for pts who initially had an abnormal karyotype that was diminished or eradicated on AZA C therapy (group 5) when compared to groups 3 (p=0.009) and 4 (p-0.0006). The emergence of a cytogenetically abnormal clone while on treatment with Aza C is not associated with shortened survival compared to those who remain cytogenetically normal. Response to Aza C can occur even with the persistence of the MDS clone or with the emergence of a new clone. This suggest that AZA C can modulate the responsivness of MDS hematopoietic progenitors. Our findings demonstrate that AZA C modulation of the cytogenetically marked MDS clone is associated with distinct subgroups with differing survival characteristics.
The BCR-ABL1 Kinase Interferes with Pre-B Cell Receptor Signal Transduction in B Cell Precursor Leukemia Cells.
Authors: Klein Florian , Feldhahn Niklas , Wernet Peter , Müschen Markus
Published: October 2019

The BCR-ABL1 kinase expressed in B cell precursor leukemia drives malignant transformation of human pre-B cells. To identify novel target genes of BCR-ABL1-mediated transformation and to elucidate how the oncogenic BCR-ABL1 kinase may interfere with normal pre-B cell receptor signaling, we compared genome-wide gene expression of BCR-ABL1+ B cell precursor leukemia cells with their normal counterpart.

While pre-B cell receptor signals are critical for differentiation and survival of normal pre-B cells, nearly all components of pre-B cell receptor-dependent signal transduction were downregulated in the leukemia cells. Surprisingly, BCR-ABL1+ leukemia cells mostly even do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH gene rearrangement nonfunctional. However, even BCR-ABL1+ leukemia cells harboring a functional VH gene rearrangement are unresponsive to pre-B cell receptor engagement. Independently from engagement, BCR-ABL1 kinase activity induces activation of PLCg1, resulting in an autonomous oscillatory Ca2+ signaling. BCR-ABL1 kinase activity is also linked to the expression of a truncated isoform of the pre-B cell receptor-associated linker molecule SLP65, which acts as a tumor suppressor in mice. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 show responsiveness to antigen receptor engagement and express only full-length SLP65. These cells differentiate into immature B cells expressing Ig light chains.

We conclude that BCR-ABL1 interferes with pre-B cell receptor signal transduction in B cell precursor leukemia cells, while its inhibition reconstitutes the selection for functional (pre-)B cell receptor signaling. Hence, a functional pre-B cell receptor signal-transduction cascade may provide these cells with a potential mechanism to escape apoptotic cell death induced by inhibition of BCR-ABL1.

Figure Legend: BCR-ABL1 + leukemia cells were incubated for 48 hours in the presence or absence of 10 μmol/l STI571. Differentiating subclones among the STI571-treated cells replace their pre-B cell receptor by a B cell receptor and were enriched for surface IgM-expression by MACS.

Figure Figure
Growth Potential of Human Leukemia Blast Cells in SCID Mice and Association with Prognosis of Human Acute Leukemias.
Authors: Yan Ying , Steinherz Peter , Guan Xiuqin , Jakubowski Ann , McGuirk Joesph P. , O’Reilly Richard J.
Published: October 2019

We have described a severe combined immunodeficiency (SCID) mouse model that permits the subcutaneous growth of primary human acute leukemia blast cells into a measurable subcutaneous nodule which may be followed by the development of disseminated disease. Utilizing the SCID mouse model, we examined the ability of patient-derived leukemic blasts to generate leukemic growth in the animals after subcutaneous inoculation without conditioning treatment. Leukemia blasts derived from 133 patients with acute leukemias, (67 acute lymphoblastic leukemia (ALL) and 66 acute myeloid leukemia (AML)), were inoculated into SCID mice. The leukemias displayed three distinct growth patterns: "aggressive", "indolent", or "no tumor growth". Out of 133 patients, leukemia samples 46 (34.5%) displayed an aggressive growth pattern, 14 (10.5%) indolent growth and 73 (55.0%) did not grow in SCID mice. The growth probability of leukemias from relapsed and/or refractory disease was 3 fold higher than that from patients with newly diagnosed disease. Serial observations found that leukemic blasts from the same individual, which did not initiate tumor growth at initial presentation and/or at early relapse, may engraft and grow in the later stages of disease, suggesting that the ability of leukemia cells for engraftment and proliferation was gradually acquired following the process of leukemia progression. Nine autonomous growing leukemia cell lines were established in vitro. These displayed an aggressive proliferation pattern, suggesting a possible correlation between the capacity of human leukemia cells for autonomous proliferation in vitro and an aggressive growth potential in SCID mice. It was demonstrated that patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SClD mice had a poor clinical outcome in patients with ALL as well as AML. Patients whose leukemic blasts grew indolently or whose leukemia cells failed to induce growth had a significantly longer DFS and more favorable clinical course.
Different Isoforms of BSAP Protein Regulate the Expression of Activation-Induced Cytidine Deaminase (AID) in Normal and CLL B-Cells.
Authors: Oppezzo Pablo J. , Dumas Gerard M. , Ines Lalanne Ana , Pritsch Otto F. , Vuillier Francoise M. , Dighiero Guillaume J.
Published: October 2019

Background: In B-cells somatic hypermutation (SHM) and class switch recombination (CSR) depend of AID expression, a typical event in germinal centers upon CD40 ligand (CD40L) stimulation. We recently showed, that in contrast to normal B-cells, AID is constitutively expressed in a majority of unmutated CLL patients (Oppezzo et al, Blood, 2003). Pax-5 gene, encodes for the B-specific activator protein (BSAP) an essential protein in B-cell development. BSAP has been shown to up-regulate AID gene expression, through binding to its promoter region, whereas inhibitor of differentiation Id-2 down-regulates it. To study the molecular mechanisms underlying AID regulation, we analyzed expression of BSAP and Id-2 transcripts in normal and CLL B-cells and correlated these results to expression of AID and CSR process.

Results: B-cells from 6 healthy donors and 40 CLL patients were analyzed for Pax-5, Id-2, AID and CSR. Results show that CLL and normal B-cells not expressing AID exhibit diminished expression of BSAP and express a spliced variant of the Pax-5 gene (Pax-5 ΔEx8) displaying a deletion in the C-terminal-active domain. Upon CD40-L and IL-4 stimulation expression of AID transcripts and disappearance of the Pax-5 ΔEx8 transcripts was observed. Translation of complete Pax-5 and Pax-5/ΔEx8 isoforms and their binding to AID gene promoter have been demonstrated by Western Blot and EMSA assays, respectively. In contrast, CLL B-cells with constitutive AID expression and active CSR, constantly show increased levels of BSAP and absence of Pax-5ΔEx8 isoform. High expression of AID and BSAP is also correlated with a decrease of Id-2 transcripts.

Conclusion: These results suggest that the presence of BSAP isoform deleted in exon 8 may interfere with the binding of the complete BSAP protein to AID promoter and down-regulate the expression of Pax-5a. Thus, we show for the first time a subtle regulation mechanism controlling the expression of AID protein through the differential splicing of a transcription factor as BSAP.
Role of Phospholipids in the Activation of Factor X by Tissue Factor/VIIa.
Authors: Hathcock James J. , Rusinova Elena , Väänänen Heikki , Nemerson Yale
Published: October 2019

The activation of coagulation factors IX and X by tissue factor(TF)/VIIa is the key step in initiating coagulation. The mechanisms by which FX binds to phospholipid and interacts with TF/VIIa remains disputed; and the reported dissociation constants between FX and FXa to well-defined phospholipid vary by orders of magnitude. In accord with other investigators we have found that at low lipid to TF ratios, adding additional lipid vesicles lacking TF, can accelerate the activation of FX. This acceleration has previously been attributed to the physically unrealistic notion that bound FX, regardless of whether it be bound to vesicles containing TF or naked vesicles lacking TF, is the preferred substrate for TF/VIIa. We have explored an alternative approach, namely that this acceleration is due to relief of Xa-mediated inhibition. Whereas it is well known that FXa can bind to TF/VIIa thus inhibiting further activation of FX, the presence of additional lipid appears to segregate FXa away from the enzymatic complex and localize it to lipid far from the reactive site. Using classical initial velocity measurements of product formation (typically 30 pM TF), we found that as we increased the lipid to TF ratio from 1.1 to 15 to 110 (x103), the level of inhibition achieved by 40 nM Phe-Phe-Arg-chloromethylketone-inactivated Xa (Xai), dropped from 67% to 40% to 10%. This concept is supported further by our new observation, evaluated by ellipsometry, that at saturation 30% PS/PC surfaces (phosphotidylserine/phosphatidylcholine) bind exactly twice as much FXa as FX; The measured dissociation constants (Kd) for FX and FXa binding to our lipid were 34.5 ± 2.9 nM and 53.9 ± 2.7 nM respectively and have been verified using independent techniques. We also consider herein the likely possibility that lipid-bound FXa exists on the surface as an opposing dimer. At very high lipid to TF ratios (>2x106) we observed reaction velocities decreasing with increasing lipid concentration. This decrease in velocity was associated with an increase in the apparent Km, which we show is due to loss of available substrate to phospholipid surfaces far from the reactive site. The interactions between TF/VIIa, FX, FXa and phospholipid surfaces, although extensively studied, have not been well understood nor well-agreed upon. The true parameters describing these interactions are needed to correctly interpret typical TF/VIIa experimental results as well as to model and understand the roles of these proteins in coagulation. Results of a computational kinetic model addressing the interplay of these reaction components will be presented.
Three-Drug Induction and BFM-Type Intensive Consolidation Chemotherapy for Treatment of Standard Risk Childhood Acute Lymphoblastic Leukemia Defined Also by DNA index. The Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) Study ALL-9501.
Authors: Arico’ Maurizio , Conter Valentino , Valsecchi Maria Grazia , Boccalatte Marie France Pinta , Barisone Elena , Messina Chiara , De Rossi Giulio , Nigro Luca Lo , Pession Andrea , Locatelli Franco , Micalizzi Concetta , Basso Giuseppe , Masera Giuseppe
Published: October 2019

In this study, we tried to select a very small subset of children with acute lymphoblastic leukemia (ALL) at minimal risk of treatment failure - identified to not only by early response in vivo, one of the strongest predictors in the I-BFM-SG experience, but also by age, blood count and in particular high DNA content - which we treated with a reduced-intensity BFM schedule. The AIEOP-ALL 9501 study enrolled patients with standard-risk (SR) ALL, defined as: <20,000 WBC/mm3, age 1 to <6 years, non-T immunophenotype, DNA index between 1.16 and 1.6, non t(9;22), no extramedullary leukemia, good response to prednisone (PGR, defined as <1,000/mmc blasts in the peripheral blood after 7 days of prednisone and one injection of IT-MTX), CR at the end of induction therapy. Follow-up was updated at December,31st 2003 and median follow-up was 5.9 years. Treatment consisted of a modified BFM schedule: 3-drug (VCR + PDN + Erwinia ASP), 43-day induction, no phase IB, 4x2 g/m2 MTX, reinduction with protocol II followed by 6MP+MTX continuation therapy up to 2 years; CNS directed therapy consisted of IT-MTXx18. Between May 95 and August 2000, 137 patients were identified as SR (7.8% of the ALL-95 population), of whom 102 received the SR protocol and are here reported.Of them, 1 died in remission of septicemia, and 1 had developed a second malignant neoplasm (T-ALL after initial B-lineage ALL); 11 patients relapsed (bone marrow, n=8; central nervous system, n=1; marrow + testis, n=1; eye, n=1) and their re-treatment included chemotherapy only (n=3, 1 dead) or plus bone marrow transplant (n=8) either autologous (n=2) or allogeneic from matched (n=4, 1 dead) or partially matched (n=2) unrelated donor. The remaining 89 are in first CR; the probabilities (and related standard errors) of survival and event-free survival (EFS) were 97.0% (1.7) and 86.7% (3.5) at 5 years, 95.3% (2.4) and 86.7% (3.5) at 7 years, respectively. There was no difference in the outcome between the 56 females [7 events, 7-yrs EFS, 87.3 (4.5)] and the 46 males [6 events, 85.9 (5.4)]. Although most of the relapsed patients were rescued, the long-term EFS in this small, very selected group of patients remains inferior to expectance. Thus, alternative selection criteria, for instance related to minimal residual disease as in current AIEOP-ALL 2000, should be considered in order to address the issue of treatment reduction.
Genetic Polymorphisms and the Toxicity of Antileukemic Agents in Children with Acute Lymphoblastic Leukemia.
Authors: Kishi Shinji , Cheng Cheng , Pei Deqing , Das Soma , Cook Edwin H. , Hijiya Nobuko , Rizzari Carmelo , Rosner Gary , Pui Ching-Hon , Evans William E. , Relling Mary V.
Published: October 2019

Regimen-related toxicity, one of the main reasons for discontinuation of chemotherapy in acute lymphoblastic leukemia (ALL), can not only be life-threatening but may affect the risk of relapse. In 246 children with newly-diagnosed ALL who were treated using a single protocol (St. Jude Total XIIIB), we used a candidate-gene approach to determine whether acute toxicities [gastrointestinal (stomatitis or diarrhea), hyperbilirubinemia, infection, or neurotoxicity] were related to 16 common polymorphisms in genes plausibly linked to the pharmacodynamics of the drug therapy. During the high-dose methotrexate consolidation therapy, only genotypes related to methotrexate disposition were associated with grade 3–4 gastrointestinal toxicity, adjusting for demographic factors and ALL risk group. Patients having the RFC AA/AG (p = 0.025) genotypes or those having the MTHFR 677 TT (p = 0.048) genotypes had a higher risk of the toxicity (OR = 10.4 and 3.2 respectively), than those with RFC GG or MTHFR CT/CC. In addition, the risk of hyperbilirubinemia during consolidation was higher among those with the lower activity UGT1A1 7/7 genotype than those with other UGT1A1 genotypes (OR = 12.2, p < 0.0001). Adjusting for time at risk over the entire therapeutic period, RFC AA/AG was again prognostic for gastrointestinal toxicity (p < 0.0001); the GSTP1 AA/AG genotypes (p = 0.021) and TPMT heterozygous genotypes (p = 0.047) were also risk factors. The RFC AA/AG genotype was also a modest risk factor for infection (p = 0.05). There were only weak predictors of neurotoxicity, with the MDR1 exon 26 TT genotype a risk factor (p = 0.025) for presumed methotrexate-related neurotoxicity over the entire therapeutic period. We conclude that germline polymorphisms influence the toxicity of antileukemic therapy and their identification may provide a tool for tailoring therapy in childhood ALL.
Identical Novel C-Kit Transcripts in Two Patients with Mast Cell Leukemia.
Authors: Ozer Ozden , Anastasi John , Vardiman James W. , Godley Lucy A.
Published: October 2019

Background: Mutations of c-kit have been well documented in mast cell neoplasms. The most common alterations found in human cutaneous and systemic mastocytosis are V560G and D816V. Mast cell leukemia is the most aggressive mast cell neoplasm, characterized by diffuse bone marrow and peripheral blood involvement. It has not been well studied due to its rarity. To date, no C-KIT mutational analysis of primary mast cell leukemia cells has been reported, although a mast cell leukemia cell line expresses the V560G and D816V mutations. Here we report on C-KIT cDNA analysis on two patients diagnosed with mast cell leukemia at the University of Chicago Hospitals. Both patients had peripheral blood involvement. Their diagnosis was confirmed on bone marrow biopsies with tryptase stains. Patient 1, diagnosed in 1989, died shortly after diagnosis. Patient 2 achieved a complete remission with allogeneic stem cell transplant. Upon relapse, treatment with Gleevec, resulted in an incomplete but significant response. This patient eventually died of sepsis following refractory cytopenias.

Methodology: We retrieved liquid nitrogen frozen, pre-treatment bone marrow aspirate samples from both patients. We performed RT-PCR analysis of the entire coding region of C-KIT by amplifying 21 exons in smaller fragments. Each PCR product was cloned and sequenced. A thorough sequencing analysis was performed by screening several clones of each amplification product, with the purpose of being able to identify low abundance transcripts.

Results: In both patients, the most abundant transcript was alternatively spliced and lacked exons 12 and 13. In addition, coexisting on the same allele, there were novel but not identical single amino acid deletions: deletion of L521 in patient 1 and of S715 in patient 2. A second novel alteration detected in both patients was the duplication of Q252. Patient 2 also demonstrated two independent, low abundance transcripts, one encoding the point mutation V559G and the other containing a deletion of exon 12.

Discussion: Here we describe two novel and identical C-KIT alterations in two patients with mast cell leukemia (deletion of exons 12 and 13 and duplication of Q252). We hypothesize that both are key molecular changes underlying mast cell leukemia. Exons 12 and 13 encode the N-terminus of the kinase domain, which is thought to have modulatory effect on the kinase active site. Amino acid 252 is located in the fourth immunoglobulin-like domain of the extracellular region, which is thought to be critical for receptor dimerization and phosphorylation. We predict that these mutations result in the superactivation of C-KIT, based on the aggressive behaviour of mast cell leukemia. The response to Gleeevec seen in patient 2 indicates that at least some of these transcripts are Gleevec sensitive. Functional assays to test if the encoded C-KIT proteins are constitutively activated and whether Gleevec inhibits the novel C-KIT proteins are in progress.
Prognostic Impact of C-Kit Mutations in Core Binding Factor-Leukemia.
Authors: Cairoli Roberto , Beghini Alessandro , Nadali Gianpaolo , Elice Francesca , Lunghi Maria , Cuneo Antonio , Grillo Giovanni , Nichelatti Michele , Ripamonti Carla B. , Lazzarino Mario , Rodeghiero Francesco , Pizzolo Giovanni , Larizza Lidia , Morra Enrica
Published: October 2019

C-KIT gene mutations are not a rare event in Core Binding Factor Leukemia (CBFL). To investigate their prognostic impact in this setting , we attempted at correlating the KIT mutational status with the WBC count, the level of the WBC-index, the presence of extramedullary disease (EMD) and the outcome, in t(8;21)(n=30) and inv(16) (n=20) AML patients. On the basis of genomic DNA analysis of c-KIT gene exons 2, 8, 10, 11, 17 for mutation detection, we categorized 21 patients (42%) in the “KIT mutated group” (KIT+) and 29 patients (58%) in the “KIT unmutated group” (KIT−). The median age at diagnosis was 46.4 and 45.6 years for the KIT(+) and KIT(−) groups, respectively (P=0.872). Among the KIT(+) patients, we found mutations in exon 8 (n = 4), exon 10 (n =1), exon 11 (n = 1) and exon 17 (n = 15). We recorded mean WBC counts of 28.6 x 109/L vs 34.5 x 109/L (P=0.051), and a mean WBC-index, expressed as WBC x (% Bone Marrow blasts/100), of 37.2 vs 15.5 (P=0.0395), for KIT(+) and KIT(−), respectively. Seven out of 50 patients experienced an EMD: in 6 cases (28.57%) this event occurred in the KIT(+) group. 40 patients (age < 60 years) were evaluable for clinical response; 18 out of them were KIT mutated. At a median follow-up of 24 months (range 10–107), we recorded for KIT(+) and KIT(−), respectively: CR incidence of 88.8 % (16/18) vs 100% (22/22) (P=0.38); Relapse Incidence 81.3% (13/16) vs 31.81% (7/22), P=0.0072; OS 27.7% (5/18) vs 77.3% (17/22), P=0.0049; DFS 16.7% (3/18) vs 77.3% (17/22), P=0.0005. Using a log-it linear model for univariate and multivariate regression analysis, we found a significant contribution to death (P=0.048) and relapse (P= 0.045) exerted by mutation in the whole group of patients; this effect was more evident when the analysis was restricted to patients <60 years (P=0.003 and P=0.014, respectively). In conclusion, this study confirms the correlation between c-KIT mutational status and high WBC-index in CBFL patients. Furthermore, our data indicate a statistical correlation between the presence of c-KIT mutations and both the OS and the DFS.
Id1 and Id2 Are Retinoic Acid Responsive Genes and Induce a G0/G1 Arrest in Acute Promyelocytic Leukemia Cells.
Authors: Nigten Jeannet , Nikoloski Gorica , De Witte Theo , Van der Reijden Bert A. , Jansen Joop H.
Published: October 2019

Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which overcomes the differentiation arrest and induces terminal granulocytic differentiation of the leukemic blasts. In 98% of the cases of APL, the leukemic cells express a promyelocytic leukemia (PML)- retinoic acid receptor a (RARa) fusion protein as a result of a t(15;17) chromosome translocation. Previously, we have identified Id1 and Id2 as direct retinoic acid target genes being upregulated after ATRA stimulation in the APL cell line NB4 as well as in primary leukemic cells from APL patients. Id (inhibitor of DNA-binding) proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors by trapping them in heterodimeric complexes, thereby inhibiting DNA-binding and gene transactivation. Various bHLH proteins are pivotal in the control of differentiation and proliferation in various tissues (like muscle and nerve). We have studied the expression pattern of E2A, an ubiquitously expressed bHLH protein, which is generally considered as a promiscuous heterodimerization partner of other, more tissue restricted bHLH proteins. The expression of E2A was high in untreated APL cells and strongly downregulated upon ATRA stimulation. The simultaneous upregulation of Id1 and Id2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells upon treatment with ATRA. To assess the importance of Id1 and Id2 induction for the neutrophilic differentiation of the cells, we have overexpressed both proteins in the APL cell line NB4 using amphotropic retroviral transduction. Ectopic expression of Id1 and Id2 resulted in respectively 27% (n=3, SD= 9%) and 48% (n=3, SD=25%) inhibition of clonogenic growth in semi-solid medium, compared to vector-transduced control cells. Apart from the reduction in the number of colonies, overexpression of Id1 and Id2 did not alter the ATRA sensitivity of APL cells. NB4 liquid cultures revealed that Id1 and Id2 overexpression resulted in inhibition of proliferation and an increase of the percentage of cells in G0/G1, without having an effect on differentiation or apoptosis. These results indicate that Id1 and Id2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.
Proteomic Identification of the C/EBPalpha Multiprotein Complex Reveals That JNK1, an Activator of C/EBPalpha, Is Inactive in Acute Myeloid Leukemia.
Authors: Trivedi A. , Bararia D. , PeerZada A. , Hiddemann W. , Behre G.
Published: October 2019

CCAAT Enhancer Binding Protein Alpha (C/EBPalpha) is one of the key myeloid transcription factors whose function is impaired in Acute Myeloid Leukemia (AML). This is either due to mutation or inhibition by other proteins via protein-protein interaction. Here, we hypothesised that protein-protein interaction of C/EBPalpha with other proteins might be important for its activation. Based on our hypothesis, we analysed the multiprotein complex of C/EBPalpha. After GST-pull down from U937 nuclear extract and using a proteomic approach we were able to identify JNK1, PAK6, MADP, ZNF45 and others as interacting proteins of C/EBPalpha. Following confirmation of physical interaction of JNK1 with C/EBPalpha, we were able to show that JNK1 phosphorylates C/EBPalpha, increases its protein half-life and thereby enhances its transactivation and DNA binding capacity. However, in various AML subtypes, JNK1 mRNA and Phospho-JNK1 protein expression is downregulated as compared with normal bone marrow which is one of the possible reasons why C/EBPalpha is inactive in AML. Thus, here we report that C/EBPalpha is impaired in AML due to inactivation of JNK1.
Identification of Distinct inv(16) Subclasses in Adult Acute Myeloid Leukemia Based on Gene Expression Profiling.
Authors: Bullinger Lars , Scholl Claudia , Bair Eric , Dohner Konstanze , Frohling Stefan , Schlenk Richard F. , Tibshirani Robert , Dohner Hartmut , Pollack Jonathan R.
Published: October 2019

Recurrent cytogenetic aberrations have been shown to constitute markers of diagnostic and prognostic value in acute myeloid leukemia (AML). However, even within the well-defined cytogenetic AML subgroup with an inv(16) we see substantial biological and clinical heterogeneity which is not fully reflected by the current classification system. To better characterize this cytogenetic group on the molecular level we profiled gene expression in a series of adult AML patients (n=26) with inv(16) using 42k cDNA microarrays.

By unsupervised hierarchical clustering we observed that samples with inv(16) separated primarily into two different subgroups. These showed no significant differences regarding known risk factors like age, WBC, LDH, etc. However, these newly defined inv(16)-subgroups were characterized by distinct clinical behavior. There was a strong trend towards unfavorable outcome with shorter overall survival times in one group (P=0.09, log rank test). Since the primary translocation/inversion events themselves are not sufficient for leukemogenesis, distinct patterns of gene expression found within each of these cytogenetic groups may suggest alternative cooperating mutations and deregulated pathways leading to transformation. Therefore, we performed a supervised analysis to determine the characteristic gene expression patterns underlying the cluster-defined subgroups. This Significance Analysis of Microarrays (SAM) method identified 260 genes significantly differentially expressed between the two newly defined inv(16)-subgroups (false discovery rate = 0.002). High expression levels of JUN, JUNB, JUND, FOS and FOSB characterized the first inv(16) subgroup (having less favorable prognosis). FOS gene family members can dimerize with proteins of the JUN family, forming the transcription factor complex AP-1 which has been implicated in the regulation of cell proliferation, differentiation, and transformation. Among the second subgroup, the proto-oncogene ETS1,displayed elevated expression, possibly resulting from aberrant MEK/ERK pathway activation as these cases also showed an over-expression of MAP3K1 and MAP3K2.

In conclusion, both supervised and unsupervised methods provide numerous insights into the pathogenesis of AML with inv(16), identifying clinically significant patterns of gene expression, as well as candidate target genes involved in leukemogenesis.
Arginine Methylation of Runx1 Regulates Its Biological and Transcriptional Activities.
Authors: Zhao Xinyang , Parkanani Animesh , Zhang Jin , Dunne Richard , Xiao Andrew , Allis C. David , Nimer Stephen
Published: October 2019

The AML1/Runx1 protein is required for definitive hematopoiesis and for the maturation of adult megakaryocytic cells. Alterations of the Runx1 gene by mutations, deletions and chromosome translocations are associated with several types of acute leukemia. Runx1 functions as both an activator and repressor of gene transcription with promoter and cell type context dependency. Likely, this relates to the ability of Runx1 to interact with a variety of transcription factors such as MEF, C/EBPa, Ets-1 and GATA-1 and also with repressor proteins such as Groucho, mSin3 and HDACs. Runx1 is post-translationally modified through acetylation and phosphorylation, and the acetylable and phosphorylable forms of Runx1 can activate transcription to higher level in Runx1 dependent reporter assays when HATs or Erk2 are coexpressed. Runx1 has also been shown to be methylated on lysine residues by SUV39H1 methyltransferase in fibroblasts. Based on the presence of a SGRGK motif in the runt domain of Runx1, we have been examining whether Runx1 is methylated on arginine residues by the protein arginine methyltransferases (PRMT). We have found that PRMT1 and PRMT5 are associated with Runx1 in AML cells by co-immunoprecipitation assays and using in vitro by GST-pulldown assays with in vitro translated PRMT(s) have shown that the interactions are direct. Using a luciferase gene reporter assay, we show that PRMT1 acts synergistically with p300 to activate Runx1 mediated transcription in response to cell proliferation signals. We have mapped the arginine methylation sites in Runx1 using GST-Runx1 fusion proteins, site-specific mutagenesis and mass spectrometry analysis. We have found three potential arginine methylation sites, one in the Runt domain, and two in the Runx1 carboxy-terminal region. Interestingly, one of these sites is in the region shown to interact with both the mSIN3A transcriptional repression complex and with p300. This suggests that arginine methylation of Runx1 may affects its transcriptional activating and repressing functions. Chromatin immunoprecipitation assays are underway to show how arginine methylation of Runx1 affects its activities in hematopoietic cells. Additional studies examining the effects of cross-talk between arginine methylation, lysine acetylation and serine phosphorylation has on Runx1 functions (biological and biochemical) will be presented.
Impaired Expression of the DNA Repair Factor DDB2 in Human Myeloid Leukemias.
Authors: Takimoto Rishu , Kato Junji , Takada Koichi , Matsunaga Takuya , Niitsu Yoshiro
Published: October 2019

Recent studies have suggested that chromosomal deletions might represent a mechanism of inactivation of DNA repair system in various hematological malignancies, including myeloid leukemias. However, the precise mechanisms remain unclear. Damaged DNA binding protein-2 (DDB2), a DNA repair factor induced by tumor suppressor p53, plays an important role in the nucleotide excision repair of UV-damaged DNA. Despite frequent mutations of p53 in human leukemic cells, the role of DDB2 on the leukemogenesis is unkown. In this study, we examined expression of DDB2 mRNA in four human myeloid leukemia cell lines (K562, KG1, HL60, and MEG01) and in fresh leukemic cells obtained from 4 patients with myeloid leukemias. In all leukemia cells, expression of DDB2 mRNA was remarkably decreased as compared to that of CD34 cells We then assayed DDB2-dependent DNA repairing activity in the leukemic cells using specific antibodies against photoproducts, and found that DNA repairing activity was reduced. When a plasmid encoding DDB2 gene (pCV-DDB2) was transduced into K562 cells, the DNA repairing activity was significantly restored. Finally we tested the expression of DDB2 mRNA in five myeloid leukemias obtained from patients, and found loss of DDB2 expression in four patients. These results suggested that in human myeloid leukemias, suppression of DDB2 expression may contribute to accumulation of gene mutation through the dysfunction of DNA repair.
An Alternative Growth Factor for Hematopoietic Stem Cells and Megakaryocytopoiesis.
Authors: Yang Mo , Li Karen Kwar Har , Chuen C.K.Y. , Chick Ki Wai , Pong Nga Hin , Fok Tai Fai
Published: October 2019

The role of serotonin (5-hydroxytryptamine, 5-HT) on the regulation of blood stem cell proliferation and thrombopoiesis has not been recognized until 1996, when we reported that serotonin has a mitogenic effect on murine megakaryocytopoiesis via 5-HT2 receptors (Yang et al, Blood Coagul Fibrin 1996). Our study also indicated that the uptake ability of serotonin is well established in human megakaryoblasts (Yang et al, Int J Hematol, 1996). 5-HT 2A, 2B and 2C receptors were identified on human megakaryocytes and serotonin also promoted human megakaryocytopoiesis via these receptors (Yang et al, Blood, 2001; 2002 suppl). Thus, we established a new concept that serotonin is a growth factor for megakaryocytopoiesis (Yang et al, Blood, 2003 suppl). We further investigated the role of serotonin on human hematopoietic stem cells, bone marrow stromal cells and platelet formation. Serotonin (200 nM) significantly enhanced TPO, SCF plus FL -induced the ex vivo expansion of CD34+ cells, CD34+38- cells, CD41+61+ cells, CFU-GEMM and CFU-MK from cord blood CD34+ cells (MACS) (n=25) at day 8 (P<0.001). More significantly, serotonin enhanced the engraftment of human CD45+ cells, and their myeloid subsets CD33+ (p=0.05) in NOD/SCID mice. The expression of 5-HT 2A, 2B and 2C receptors was detectable in fresh CD34+ cells by RT PCR. These 5-HT receptors were further demonstrated in 1% of CD34+ cells by FACS. Our data also demonstrated that serotonin significantly stimulated the proliferation of bone marrow stromal cells in a dose-dependent manner (10–500 nM) in human CFU-F assay. A maximum stimulation was obtained at 200 nM of serotonin (n=5, p=0.03). The effect of serotonin was similar to that of PDGF and VEGF, but weaker than that of FGF-2 at their optimal dose. Serotonin also significantly enhanced FGF-2, PDGF or VEGF -induced CFU-F formation (p<0.05). 5-HT 2A, 2B and 2C receptors were also demonstrated in bone marrow stromal cells by RT-PCR and around 1% positive confirmed by FACS. Results on thrombopoiesis showed that 5-HT2A, 2B and 2C receptors were expressed strongly (80–99%) on MB megakaryocytes and MK cell lines. Serotonin also increased the size of cultured megakaryocyte suggesting it has a promoting effect on megakaryocyte maturation. Ketanserin, a 5-HT2B receptor antagonist, was showed in the same study to block the mitogenic effect on megakaryocyte differentiation. Serotonin also has an effect on actin re-organization in Meg-01 cells. We have provided the evidence of serotonin as a growth factor for blood stem cells and MK cells. Based on this concept, alternative drug could be developed for the treatment of thrombocytopenia.
Frequency of Heparin-Platelet Factor 4 Antibodies among Patients with Acute Coronary Syndromes Presenting to the Emergency Department.
Authors: Francis John L. , Drexler Alane , Walker Jamie , Duncan Mary Kathryn , Ahmad Sarfraz
Published: October 2019

Heparin is widely used to treat patients that present with acute coronary syndromes (ACS). A significant, albeit under-recognized complication of heparin therapy is heparin-induced thrombocytopenia (HIT), which is estimated to occur in 1–3% of patients. A significantly higher proportion (20–50%) of patients undergoing cardiac surgery and other invasive cardiology procedures develop an antibody against the heparin-platelet factor 4 (HPF4) complex. Most patients remain asymptomatic, but are nevertheless at risk for HIT with subsequent heparin exposure. Patients with severe coronary artery disease frequently have multiple hospital admissions, and heparin exposures. HPF4 antibodies are transient, and most will become undetectable three months after their initial formation. Therefore, patients with a recent history of hospitalization would be at greater risk of HIT on heparin re-exposure. We tested this hypothesis in a prospective study of patients with ACS presenting to the Emergency Department. We studied 230 patients presenting to the ED with ACS. Patients were stratified into Group 1 (having a recent [<6 months] history of hospital admission), or Group 2 (having a more remote [or no] history). Plasma samples were tested for HPF4 antibodies with a commercially available ELISA and positive samples were re-tested with a serotonin release assay (SRA). Sixteen patients (7.0%) tested positive for HPF4 antibodies by ELISA. Group 1 subjects had a higher incidence of HPF4 antibodies (10.7%) than Group 2 patients (3.2%). Of the 16 patients positive by ELISA, 14 were also tested by SRA. Of these, 7 were positive, 6 were negative and 2 gave borderline results. We conclude that approximately 7% of all patients presenting with acute coronary syndromes have a pre-existing HPF4 antibody. The incidence of a positive test is tripled in patients that have been recently hospitalized with high probability of recent heparin exposure. A significant proportion of these patients has a functionally active HPF4 antibody, which would presumably carry a higher risk of clinical HIT if re-exposed to heparin therapy for ACS and subsequent cardiovascular procedures. Our data suggest that high-risk individuals can be identified on a history of recent hospital admissions, and should receive a non-heparin anticoagulant, e.g. a direct thrombin inhibitor, until their HPF4 antibody status can be established.