bluuebunny/crossref_metadata_2025 huggingface dataset:
Framingham Score in Congenital Hemophilia Patients (HP) with Cardiovascular Disease (CVD).
Authors: Parameswaran Rekha , Greist Anne , Miller-Rice Lorrie , Roberson Chris , Murry Renee , Sulkin Matthew , Lynn Henry , Shapiro Amy
Published: April 2020

CVD risk factors of age, hypertension, high cholesterol (HC), smoking, diabetes are expressed in the Framingham coronary risk score. Previous data suggest that hemophilia may protect against CVD. However, CVD risk factors may place HP at risk as they age. HIV infected patients are a rapidly developing group with CVD. No data address the effect of established risk factors on CVD prevalence in HP. We report an extensive analysis of a single hemophilia treatment center (HTC) database for CVD risk factors and prevalence in HP. Framingham coronary risk scores were calculated for HP patients with documented CVD events, using age at time of event to calculate risk. HTC population ≥40 yrs, used as denominator, comprised 156 with FVIII deficiency (63 severe, 16 moderate, 77 mild);109 with FIX deficiency (24 severe, 53 moderate, 31 mild). Forty-eight HP had HIV infection, 113 had hepatitis C, and 25 had HIV/hepatitis C coinfection. Twenty-one HP, 10 with viral infection, ranging 49 to75 years, experienced 23 CVD events. They included 11 FVIII (0 severe, 1 moderate, 10 mild),10 FIX (2 severe,6 moderate, 2 mild). CVD events included 13 myocardial infarction (MI); 5 cerebrovascular accidents (CVA); 1 patient with carotid stenosis and 4 HP with coronary artery disease on catheterization. Two patients each had both MI and CVA. Four patients underwent coronary artery bypass graft surgery. Of 8 patients on antiplatelet therapy, 2 had major bleeds (CNS and GI bleed); one had a minor bleed (epistaxis). Of these 21 HP with CVD, one patient had HIV disease; 8 had hepatitis C, 1 had co-infection with HIV and hepatitis C. One patient was on Vioxx® at the time of his CVD event.Six are deceased, all due to CVD. Overall, CVD affected 7.9% of HP aged >40 years in this population. Of all HP with viral infection in this HTC population, 40% experienced CVD. Ten-year CVD risk per Framingham model at time of event ranged from 10% to 37% (mean 20.1%; median 16%). All HP patients with CVD met the definition of high coronary risk per AHA guidelines with 10-year risk of CVD>10%. Management of established CVD in HP is difficult due to need for invasive procedures and bleeding risk with antiplatelet therapy. Primary prevention of CVD is thus crucial. Aggressive risk factor modification is indicated in all HP with 10-year risk score >10% for primary prevention of CVD. To our knowledge, this is the first application of the well validated Framingham score in the HP population.We conclude that the easily calculable Framingham score should be assessed in all HP over age 40, especially those with viral infections, and incorporated into the comprehensive care model.
Microarray Analysis of Non- Classical Haemophilia A Patients.
Authors: Cutler Jacky , Mitchell Mike , Savidge Geoffrey F.
Published: April 2020

Haemophilia A is classically due to a defect in the factor 8 gene. Mutations are heterogeneous, and located throughout the gene. Other than the IVS 22 inversion mutation, causative of approximately 46% of severe haemophilias, there are few mutation hot spots. It has been established that approximately 2% of patients with confirmed Haemophilia A have no detectable genetic abnormality in the factor 8 gene. Genetic analysis at the RNA level has generally failed to identify mutations not seen at the genomic level. Splicing abnormalities appear to be restricted to the splice junctions as no cryptic splice sites have yet been identified deep within introns.

One possible approach in the investigation of these non-F8 associated Haemophilia patients, is to look at up/down regulation of other genes via genome wide gene expression analysis. To this end we have subjected a small number of confirmed Haemophilia A patients with no identifiable mutation to microarray analysis, utilising an Affymetrix GeneChip® array system comprising 54676 genes. Their gene expression pattern was then compared to that of patients with similar phenotype in whom mutations have been defined. Initially, data analysis has focussed on the X chromosome, as the sex distribution in this group of patients does not appear to differ from classical F8-associated Haemophilia.

Preliminary results suggest a significant down regulation of ARHGAP6, a Rho GTPase activating protein. This gene is located at Xp22, comprises 14 exons, and encodes a 974 amino acid protein, with multiple splice variants. Interestingly, ARHGAP shares with factor 8 the possession of an embedded gene within a large intron. The F8A gene located within intron 22 of F8 is involved in the recombination event which results in the IVS22 inversion mutation. Intron 1of the ARHGAP gene spans more than 280kb and contains the amelogenin gene (AMELX). Like F8A, AMELX is transcribed from the opposite strand to the ’host gene’. This unusual phenomenon has only been reported in a very small number of human genes.

ARHGAP6 is known to have two independent functions, one as a GTPase-activating protein (GAP) involved in signal transduction, and another as a GTPase activator, regulating the interaction of signalling molecules with the actin cytoskeleton. It is possible that such interactions affect the secretion of the factor VIII protein from hepatocytes.

The genetic basis of Haemophilia A becomes more complex as the patient base investigated increases. It is likely that the number of genes that have an affect on plasma FVIII levels will continue to grow. ARHGAP6, and other GTPase activating proteins, may be worthy of closer examination in the search for genetic modifiers of FVIII levels.
The Pharmacogenetics and Inhibitor Risk (PIR) Study: Establishing the Spectrum of Factor (F)VIII Gene (F8) Mutations in African-American Hemophilia A Patients.
Authors: Howard Tom E. , Machiah Deepa K. , Viel Kevin R. , Channel Cynthia , Ameri Afshin , Iyer Rathi V. , Watts Ray G. , Lutcher Charles , Davis Joanna , Abshire Thomas C. , Almasy Laura
Published: April 2020

The development of neutralizing antibodies against plasma-derived or recombinant (r) molecules of wildtype (wt) factor (F)VIII protein is the most serious complication of replacement therapy for hemophilia A (hA) patients. Although the pathogenesis of these antibodies, termed inhibitors, is complex and poorly understood, ethnicity, a recently established risk determinant, is clearly involved since African-American (AA) patients experience this complication ~2-fold more often than Caucasians. In a previous study -- in which the FVIII genes (F8) from 137 unrelated healthy people, representing 7 ethnic groups, were resequenced -- we identified 4 common nonsynonymous single nucleotide polymorphisms (nsSNPs) and thereby demonstrated that FVIII is not a monomorphic protein in non-hemophiliacs. Interestingly, 5 of the 6 distinct wt proteins encoded by the allelic combinations or haplotypes (H) of these nsSNPs, designated H1, H2, ..., H5, are expressed by AA’s compared to only 2 in Caucasians (H1 and H2). Because H3, H4 and H5 are 1) partially defined by AA-restricted minor alleles of R484H and M2238V, 2 nsSNPs that substitute amino acids in the A2 and C2 major B-cell inhibitor epitopes, 2) represent the wt FVIII protein in ~27% of AA’s, and 3) differ from the rFVIII proteins used clinically, we designed the PIR study -- the 1st cross-sectional study of AA hA patients -- to determine if pharmacogenetic factors contribute to their greater inhibitor risk. Because the strongest known risk factor for inhibitors is the F8 mutation type, we are resequencing the known functional regions of F8 in the 1st 50 enrolled PIR subjects, out of the 223 total AA hA patients treated at the six participating Region IV South hemophilia treatment centers, to test the plausible alternative hypothesis that the higher inhibitor incidence is due to a different spectrum of molecular abnormalities than has been found in other ethnic subgroups of patients already examined at the DNA sequence level. We have 1) determined the plasma FVIII-activity and -antigen levels, 2) identified the causative mutation in 18 patients, several of which are novel, and 3) are currently performing RT-PCR analyses for detecting the common inversions in introns 1 and 22. Once completed, we will statistically compare the prevalence of AA F8 mutation subtypes to that in hA patients from other ethnic subgroups (detailed in the HAMSTeRS database), and the proportion of AA FVIII deficiencies that are 1) severe, moderate and mild, and 2) CRM-positive and -negative.
Human Metapneumovirus Infection in Adult Patients after Allogeneic Hemopoietic Stem Cell Transplantation.
Authors: Colombo Anna A. , Algarotti Alessandra , Debiaggi Maurizia , Parea Maurizio , Bragotti Letizia Zenone , Bernasconi Paolo , Caldera Daniela , Canducci Filippo , Lazzarino Mario , Alessandrino Emilio P.
Published: April 2020

The human metapneumovirus (hMPV) has been described as possible cause of respiratory disease in pediatric and adult subjects. There are not, however, data concerning the epidemiology and or the clinical impact of hMPV infection in adults receiving allogeneic hemopoietic stem cell (HSC) transplant. We report the results of a prospective study aimed to asses the incidence of hMPV infection in patients undergoing allogeneic HSC transplant. We analyzed by reverse transcription PCR assay, the presence of hMPV in nasopharingeal aspirate (NPA) collected before transplant and on day 0, +15, +30, +60, +90, +180, +360 from transplant. Detection of hMPV RNA was based on two set primers pair located in the L and N genes. Amplified hMPV RNA was sequenced for genotyping in region N.

Twenty patients with a median age of 37 yrs (range 25–58), receiving allogeneic HSC for hematological disorders (AML 11 pts, ALL 5 pts, MDS 2 pts, NHL and MM 1 pt each), entered the study. Six pts received transplant from an HLA identical sibling and 14 from an unrelated donor (MUD). As preparative regimen, 15 pts had a standard myeloablative conditioning regimen, and 5 received reduced intensity conditioning with Thiotepa and Fludarabine. Acute graft-versus-host-disease prophylaxis consisted of Cyclosporine A and Methotrexate. In 6 pts receiving MUD transplant, in vivo T depletion by ATG was performed. At transplant, all patients were in good performance status and none had signs of active acute respiratory disease. Of 20 patients, four had a prior history of pulmonary infection from agents different from hMPV.

Of 49 NPA samples analyzed so far, 29 (59%) resulted hMPV-positive. Sequence analysis performed in 12 patents detected a virus genotype-A. Of 12 pts in whom 3 or more NPA samples were collected, 9 (45%) resulted positive for hMPV RT-PCR, with persistence of infection for a period ranging from 14 to 84 days. No patient, however, showed clinical signs of respiratory disease. These data indicate an high prevalence and a long persistence of asymptomatic hMPV infection in adults receiving allogeneic HSC transplant.
A Randomized Crossover Study of High- and Standard-Dose Recombinant Factor VIIa for Treatment of Hemarthroses in Hemophiliacs with Inhibitors.
Authors: Santagostino Elena , Mancuso Maria E. , Rocino Angiola , Mancuso Giacomo , Scaraggi Francesco A. , Morfini Massimo , Daniele Filomena , Barillari Giovanni , Mannucci Pier M.
Published: April 2020

A multicenter randomized crossover trial was designed to compare efficacy, safety, feasibility and cost associated with standard- and high-dosages of rFVIIa for home treatment of hemarthroses in hemophiliacs with inhibitors. Enrolled patients were instructed to treat within 6 hours from the onset of bleeding 4 consecutive haemarthroses of ankles, knees or elbows either with the rFVIIa standard dose of 90 mcg/kg (repeated as necessary every 3 hours) or with a single high dose of 270 mcg/kg. Patients who did not achieve a success within 9 hours continued rFVIIa treatment with repeated standard doses. The response to treatment was assessed for up to 48 hours by patients/caregivers who reported on a visual analogic scale (VAS) graded from 0 to 100 the improvement in symptoms and also rated the response as effective, partially effective or ineffective. Success was defined a treatment rated as effective and VAS score ≥70 and failure a treatment rated as ineffective and VAS score ≤30, responses that did not fulfil these criteria being considered a partial response. Of 20 hemophiliacs with inhibitors enrolled (median age: 27 years), 18 treated 32 hemarthroses assigned to the standard- and 36 to the high-dose regimen during the study period of 18 months. Forty-eight hemarthroses (71%) occurred in target joints. Success rates for standard- and high-dose regimens were similar: 31% and 25% at 9 hours, 53% and 50% at 24 hours, 66% and 64% at 48 hours. Recurrence rates were identical (3%). The median number of rFVIIa boluses needed to achieve a successful course was 3 for the standard-dose and 1 for the high-dose regimen, and the median amount of rFVIIa used per successful course was identical (270 mcg/kg). Our results indicate that the use of a high-dose regimen with rFVIIa for home treatment of hemarthroses is effective, safe, does not imply an increased consumption of rFVIIa and requires the infusion of a smaller number of boluses. Its convenience is particularly relevant in cases with difficult venous access and in hemorrhages into target joints.
Quantum Dot Based Duplex In Situ Hybridisation for Gene Expression Profiling.
Authors: Tholouli Eleni , Di Vizio Dolores , O’Connell Fionnuala , Loda Massimo , Twomey David , Golub Todd , Levenson Richard , Hoyland Judith A. , Yin John A.L. , Byers Richard
Published: April 2020

Quantum dots (QDs) are fluorescent semiconductor nanocrystals (2–10-nm core diameter) possessing the unique properties of extremely high fluorescence efficiency, lack of photobleaching and long fluorescence lifetime, making them an ideal tool for bioimaging. We have developed a novel technique for in situ hybridisation (ISH) using biotinylated oligonucleotides conjugated to streptavidin coated QD, and used them in this study to label bone marrow trephine samples.

50-mer long oligonucleotide probes were conjugated to QDs prior to ISH and conjugation efficiency was demonstrated by gel electrophopresis. ISH conditions and molar ratio of QDs to probe were optimised using a polyT probe. Images were captured using a CRI Nuance spectral imaging system and signal intensity was semi-quantitated using IPLab software. Specific oligonucleotide hybridisation was demonstrated using a probe for myeloperoxidase (MPO) in 40 bone marrow sections infiltrated by acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) as well as reactive bone marrow. In each case hybridisation signal was consistent with the distribution of MPO by standard immunohistochemistry - MPO was strongly expressed by myeloid blasts and absent in lymphoid blasts; in CML, most, but not all, cells were positive for MPO, in comparison to many fewer positive cells in reactive marrow. Duplex ISH was demonstrated using a probe for bcl-2 together with MPO in 5 bone marrow sections infiltrated by follicular lymphoma (FL). Strong hybridisation signal for bcl-2 was detected in all cells of the paratrabecular aggregates of FL but showed only scattered positivity in the remainder of the bone marrow. Conversely, MPO was absent in the paratrabecular aggregates and present in the myeloid cells in the remainder of the marrow. This pattern was present in both single and duplex ISH for MPO and bcl-2 in the marrow infiltrated by FL. Duplex ISH was performed both by sequential hybridisation with bcl-2 followed by MPO, and simultaneously with a mix of bcl-2 and MPO probes. As negative controls, scrambled oligonucleotide probes for the corresponding genes were used in each case and did not show hybridisation.

In summary, we have developed a generic method for QD labelling and semi-quantitative detection of oligonucleotide ISH in routinely processed clinical tissue samples. Although, in this study we primarily used bone marrow trephine samples, this technique can be applied to any tissue. It has the potential to facilitate transfer of microarray-identified gene signatures to clinical research and diagnostics, whilst the ability of spectral imaging to resolve multiple signals offers the possibility of multiplexed probe detection.
Prepared and Application of New Microarray-Based Method for Detecting SNP in the Risk of Hematological Malignancy.
Authors: Chen Bao-An , Jiang Ni , Ji Mei-Ju , Hou Peng , Lu Zu-Hong , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Cheng Jian , Zhao Gang , Fei Fei , Chen Ning-Na
Published: April 2020

Objective To investigate a new microarray-based method for single nucleotide polymorphisms (SNP) genotyping, and to study the role of 5,10-methylenetetrahydrofolate reductase(MTHFR) SNP in hematological malignancy pathogenesis in the Chinese population.

Methods Prepared a aimed gene microarray based on cDNA microarray theory, dual-color fluorescenece hybridization was used for detecting SNP loci, sequencing was performed to confirm the results. In order to prepare the function SNP detection microarray, three different functionalized glass slides were selected, which were modified with 3-aminopropyltriethoxysilane (APTES), poly-l-lysine and glutaraldehyde, respectively. In order to obtain satisfied signal intensity, the ultraviolet crosslinking energy and the immobilized DNA concentration were optimized. By using the above SNP microarray, MTHFR C677T and A1298C SNP loci of 157 controls and 127 patients with hematological malignancy[30 multiple myeloma(MM),28 non-Hodgkin’s lymphoma(NHL), 22 acute lymphoblastic leukemia(ALL),40 acute myeloid leukemia(AML),7 chronic myeloid leukemia (CML)] from Jiangsu province were detected.

Results Based on the immobilization efficiency, APTES modified glass slides were selected to fabricate DNA microarrays. The optimal ultraviolet crosslinking energy was 400 mJ and the optimal concentration of immobilized DNA was 300 mg/ml. Homozygous wild type, heterozygote type and homozygous mutant type yielded green, yellow and red fluorescence, respectively. Sequencing validated the results.The allele frequencies of MTHFR677C, 677T were 58.7%, 41.3% in patients and 66.9%, 33.1% in controls, respectively; the frequencies of 677CC, 677CT, 677TT genotype were 37.8%, 41.7%, 20.5% in patiens and 45.9%, 42.0%, 12.1% in controls, respectively. The allele frequencies of MTHFR1298A, 1298C were 83.1%, 16.9% in patients and 84.1%, 15.9% in controls, respectively; the frequencies of 1298AA, 1298AC, 1298CC genotype were 70.9%, 24.4%, 4.7% in patiens and 69.4%, 29.3%, 1.3% in controls, respectively. In multivariate analysis, significant difference was observed in the allele frequencies of C677T(χ2=4.08, P=.043) and in the genotype frequency of 677TT(OR 2.09, 95%CI 1.04–4.20, P=.039). The frequencies of 677TT (33.3%) and 1298CC(6.7%) in MM group were found significantly higher than in control group(OR 8.92, 95%CI 1.06–75.24, P=.006; OR=4.80, 95%CI 1.56–14.73, P=.044), resectively. No associations were found between any polymorphisms and susceptibilities to ALL, AML and NHL.

Conclusions This microarray-based method is accurate, high-throughput and cheap, suitable for SNP genotyping in a large of individuals. MTHFRC677T polymorphisms influence the risk of hematological malignancy in Chinese population. Both MTHFR677TT and MTHFR1298CC genotype are susceptive to MM.
AF1q Is a Novel Poor Prognostic Marker for Adult Acute Myelogenous Leukemia (AML) Patients with Normal Cytogenetics.
Authors: Strunk Crawford J. , Platzbecker Uwe , Thiede Christian , Schaich Markus Andreas , Illmer Thomas , Kang Zizhen , Leahy Patrick , Li Chunbiao , Reiner Sunee , Laughlin Mary J. , Lazarus Hillard M. , Bunting Kevin D. , Gerson Stanton L. , Ehninger Gerhard , Tse William W.
Published: April 2020

Adult AML with normal cytogenetics accounts for 45–55% of de novo AML patients, who usually are categorized as intermediate-risk but have wide 5-yr overall survival (OS) rates between 24–42%. The optimal post-remission treatment strategies, especially hematopoietic stem cell transplantation (HSCT), are yet to be defined. Recent molecular studies suggested that clinical outcome may be predicted by presence or absence of mutation or changes in expression of specific genes such as FMS-like tyrosine 3 (FLT3). The AF1q gene, an MLL fusion partner, originally was identified in an AML patient’s leukemic cells with t(1; 11) (q21; q23) abnormalities. We previously found that high AF1q expression is an independent poor-prognosticator for OS and disease-free survival (DFS) in pediatric AML patients. Data indicated that allogeneic HSCT in AML patients with high AF1q expression might improve survival. To test this hypothesis in adults, we studied leukemia marrow AF1q expression by real-time polymerase chain reaction (Q-PCR) in 290 normal cytogenetic adult AML patients treated within a trial of the German AML Study Initiative Leukemia (DSIL). Patients were induced with two cytarabine-based cycles followed by consolidation therapy. Forty-eight patients received allogeneic and 58 patients received autologous HSCT after initial induction. Over 95% of AML patients had elevated AF1q expression compared to control with highest up to 409-fold. Quartile analysis of the study population showed that the highest AF1q expression trended toward worse OS (p=0.09) and DFS (p=0.1) compared to low AF1q expression despite similar complete remission rates after induction (low 66% vs. high 69%). High AF1q expression was significantly associated with the presence of an FLT3 ITD mutation (p=0.013). Patients with high AF1q expression given allogeneic HSCT had a similar OS and DFS compared to pts with low AF1q expression (62% & 56% vs 64% & 62% at 3 yr, respectively). In fact, autologous HSCT showed an inferior OS (median 25 mo vs. median not reached, p=0.04) and DFS (p=0.09) in the high AF1q group compared to the low expression group. These data suggest that high AF1q expression may be a useful molecular prognostic marker for poor prognosis adult AML patients with normal cytogenetics and allogeneic HSCT may improve OS. Further studies are focusing on whether AF1q is interactive with FLT3 in leukemogenesis as well as serving as a simultaneous therapeutic target for AML with FLT3 mutation.
Distinct Characteristics of t(8;21) Acute Myeloid Leukemia with Active KIT Mutation.
Authors: Lorenzo Felipe , Nishii Kazuhiro , Monma Fumihiko , Katayama Naoyuki , Yagasaki Fumiharu , Miwa Hiroshi , Miyawaki Shuich , Shiku Hiroshi
Published: April 2020

The t(8;21) translocation, which generates a chimeric gene, AML1/ETO, is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML) and is associated with a good clinical outcome. However, the clinical features of t(8;21)AML with additional genetic aberrations have not been well elucidated. In this study, we screened for KIT mutations in 44 patients with t(8;21)AML. While 2 cases had a silent type mutation at codon 546 in the juxtamembrane domain, Lyn546Lyn, 8 cases (17.8%) had Asp816 mutations in the tyrosine kinase domain, Asp>Asn in 2 cases, Asp>Tyr in 4 cases, Asp>Val in 1 case and Asp>His in 1 case. Patients with Asp816 mutations showed leukocytosis (P = 0.001) and poor overall survival (P < 0.0001), when compared to those without Asp816 mutations. When the Asp816 mutation was transfected into a stem cell factor-dependent cell line, Mo7e, the cells grew rapidly and displayed stem cell factor-independent proliferation. These results suggest that t(8;21)AML with Asp816 mutations in KIT may be a distinctive subtype of t(8;21)AML and that this genetic aberration has an adverse effect on the clinical outcome for t(8;21)AML.
Differential Effects of HOXB4 Overexpression on Short and Long-Term Repopulating Cells in Nonhuman Primates.
Authors: Zhang Xiao-Bing , Beard Brian C. , Beebe Katherine , Humphries R. Keith , Kiem Hans-Peter
Published: April 2020

The inability to expand hematopoietic stem cells (HSCs) has been a significant limitation for clinical transplantation and gene therapy applications. Here we examined in a clinically relevant nonhuman primate model the ability of HOXB4 to expand HSCs and thus potentially overcome this limitation. Using a competitive repopulation assay we directly compared engraftment of HOXB4-transduced and control-transduced CD34+ cells. In 3 animals, cells were infused after a 3-day transduction and in 2 animals after an additional 6 to 9 days ex vivo expansion. Follow-up for these animals is up to 15-months. In the 3 animals that received HOXB4GFP-transduced cells without additional ex vivo culture, gene transfer efficiencies in CD34+ cells were similar between HOXB4GFP and YFP transduced cells: 45% (range 36–55%) vs. 38% (range 36– 40%). We observed a dramatic increase in HOXB4GFP marked cells from 20–30% to 52–62% during the early engraftment period, resulting in an up to 10-fold difference in granulocyte marking between HOXB4GFP and YFP marked cells at 5 weeks post-transplantation. Although gene-marking levels declined over time, HOXB4 marking was still about 2 to 3-fold higher than marking in control cells even at 15 months post-transplantation. A more pronounced effect was observed in the 2 animals that received HOXB4-overexpressing cells after an additional 6 to 9 days of ex vivo culture. Again, no difference in transduction efficiency was observed between YFP (range 34–49%) and HOXB4GFP (range 39–43%) marked cells before transplantation. However, HOXB4 marking was higher than YFP marking 1 week after transplantation, with up to a 34-fold difference in granulocyte marking at 2 weeks post-transplantation. Although the difference was decreased thereafter, a 4 to 10-fold difference was maintained in granulocyte marking after 3 months post-transplantation, suggesting a potential effect on the expansion of long-term repopulating cells. Subset analysis by flow cytometry and Taqman PCR showed HOXB4GFP and YFP marking in all subsets. Marking in CD13+ granulocytes and CD14+ monocytes was higher with HOXB4GFP-transduced cells and marking in CD3+ T cells was higher with YFP-transduced cells, suggesting that HOXB4 overexpression may have a more pronounced effect on engraftment and differentiation of myeloid than T-lymphoid precursors. LAM-PCR analysis demonstrated multiple clones of HOXB4GFP+ cells and control YFP+ cells. Our results demonstrate that HOXB4 overexpression in CD34+ cells has a very dramatic effect on expansion and engraftment of short-term repopulating cells with a less pronounced effect on long-term repopulating cells. These data should have important implications for the expansion and transplantation of HSCs, in particular for cord blood transplantations.
Isolation and Characterization of Human Antigen-Specific TCRαβ+ CD4−CD8− Double Negative Regulatory T Cells.
Authors: Fischer Karin , Voelkl Simon , Przybylski Grzegorz K. , Schmidt Christian A. , Andreesen Reinhard , Mackensen Andreas
Published: April 2020

Compelling evidence indicate that regulatory T (Treg) cells play an important role in the maintenance of immune tolerance to self and foreign antigens (Ag). Various subsets of T lymphocytes have been isolated in mice and humans that have the ability to down-regulate the proliferation of autoimmune effector cells. Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4−CD8− (double negative, DN) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of anti-graft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Furthermore, analysis of T-cell receptor excision circles (TRECs) indicate that DN T cells are not recent thymic emigrants, but rather an expanded T-cell subset. MHC multimer staining revelaed a distinct population of DN T cells recognizing common MHC class I-restricted CMV and EBV antigens. DN T cells exhibited a strong proliferative response upon stimulation with allogeneic antigen presenting cells (APC) and secreted high amounts of IFN-γ but no IL-2, with some IL-5, and marginal levels of IL-4 and IL-10. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide-HLA-A2 complexes from APCs by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2-restricted self peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A-HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizeable population of peripheral DN Treg cells exists in humans that are able to suppress Ag-specific T cells. DN Treg cells may serve to limit clonal expansion of allo-Ag-specific T cells after transplantation.
p38 MAPK Mediates Inhibition of Activation Induced Cell Death in T Lymphocytes Via Upregulation of Bcl-xL and FLIPshort upon Ligation of CD137/4-1BB or CD28.
Authors: Scholz Christian , Stärck Lilian , Lehmann Mario , Dörken Bernd , Daniel Peter T.
Published: April 2020

Costimulation is essential for the induction of proliferation in naive T cells and for the inhibition of activation induced cell death (AICD) in activated T lymphocytes. While costimulatory signals mediated through the immunglobulin family member CD28 play a prominent role during primary T cell activation, ligation of the tumor necrosis factor receptor family member CD137/4-1BB is more important during late primary and secondary T cell activation. Signals mediated through either costimulatory protein block AICD. Inhibition of AICD through ligation of CD137/4-1BB or CD28 involves upregulation of Bcl-xL and FLIPshort (Eur J Immunol 2005, 35: 1257–66). We further demonstrated that costimulatory signals mediated through CD137/4-1BB or CD28 depend on the activity of phosphatidylinositol 3-kinase and AKT/protein kinase B, two kinases that had formerly been implied in CD28-induced signaling, indicating that CD28- and CD137/4-1BB-mediated signals share downstream signaling pathways. Here, we demonstrate that p38 mitogen-activated protein kinase (MAPK) mediates CD137/4-1BB-induced as well as CD28-mediated costimulation of cell proliferation and inhibition of AICD. This coincides with upregulation of Bcl-xL and FLIPshort. Inhibition of p38 MAPK abrogates T cell receptor induced proliferation and antagonizes costimulation mediated survival. Thus, p38 MAPK, which was previously thought to be primarily involved in CD137/4-1BB-mediated signaling, is similarly important for CD28-induced costimulation and survival. This indicates that, while involving different protein families, signal transduction by CD28 and CD137/4-1BB depends on a common upstream and downstream network of survival kinases.
Phase I Trial of Siplizumab in CD2-Positive Lymphoproliferative Disease.
Authors: O’Mahony Deirdre , Morris John C. , Moses Leslie , O’Hagan Diana , Gao Wendy , Stetler-Stevenson Maryalice , Taylor Margaret , Hammershaimb Luz , Waldmann Thomas A. , Janik John E.
Published: April 2020

Siplizumab is a humanized IgG1κ class monoclonal antibody that binds to the CD2 receptor on human T- and NK-cells. In an animal model of adult T-cell leukemia/lymphoma (ATL), fifty percent of animals survived tumor challenge with 4 weeks of siplizumab treatment and the life-span of tumor bearing animals treated for six months was equivalent to that of animals not challenged with tumors. A single-center phase I dose escalation trial to assess maximum tolerated dose (MTD) and safety in patients (pts) with CD2-positive adult T-cell leukemia (ATL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and large granular lymphocyte leukemia (LGL) is ongoing. Eligibility includes Karnofsky performance status ≥70%; life expectancy >2 months; appropriate hematological status and normal organ function; prior treatment with chemotherapy and monoclonal antibodies is allowed. Cohorts of 3 pts each receive escalating i.v. doses of siplizumab ranging from 0.4 to 4.8 mg/kg administered over 2–3 days per treatment week every other week for 16 weeks or until unacceptable toxicity or disease progression (PD). 16 pts have been enrolled into 5 cohorts: 0.4 (3 pts), 0.6 (3 pts), 0.8 (3 pts), 1.2 (4 pts) and 2.4 (3 pts) mg/kg per week with planned dose escalation to 3.4 and 4.8 mg/kg. Median number of courses received is 4 (range 1–8). Diagnoses include ATL (9), LGL (4), PTCL (1) and CTCL (2). Grade 1/2 infusional reactions primarily manifested as fever and chills were confined to the first dose of treatment in the majority of patients. Meperidine pretreatment significantly ameliorated infusional reactions. The median (range) percentage decrease in CD4, CD8, NK and malignant cell numbers in peripheral blood from baseline are 98(71–100%), 93(50–100%), 89(55–96%) and 88 (0–100%), respectively. No DLTs have occurred at this time. To date, 3 confirmed PRs have been observed (2 ATL, 1 LGL), 6 pts had stable disease, and 7 pts had PD. 1 pt discontinued therapy due to a siplizumab-related adverse event of polymyalgia rheumatica and 3 pts (2 ATL, 1 LGL) were removed due to CMV antigenemia (which was an off-treatment criterion at the time of study drug discontinuation). 4 patients have died during the one-year follow up period due to PD and 4 patients have completed all 8 courses of treatment. In conclusion-siplizumab has been well tolerated in this trial and the maximum tolerated dose has not been reached. The protocol has been amended to allow for treatment of CMV antigen positive patients provided oral antivirals are effective in preventing CMV disease and rising CMV levels. Anti-tumor activity has been observed in some patients. Enrollment in this study continues.
CTGF (CCN2) Predicts OS and DFS in Adult Acute Lymphoblastic Leukemia.
Authors: Sala-Torra Olga , Gundacker Holly M. , Stirewalt Derek L. , Ladne Paula A. , Pogosova-Agadjanyan Era L. , Slovak Marilyn L. , Willman Cheryl L. , Heimfeld Shelly , Boldt David H. , Radich Jerald P.
Published: April 2020

Outcome of adult acute lymphoblastic leukemia (ALL) continues to be poor, and parameters to better discriminate patients with distinct prognosis are necessary. In studies comparing global gene expression differences between normal hematopoietic cells (whole bone marrow, peripheral blood, CD34+, and CD22+ sorted populations) and ALL cells using microarrays we found that the B-ALL cells showed evidence of increased expression of Connective Tissue Growth Factor (CTGF). The median log 2 transformed signal intensity of CTGF was 4.26 (range 3.95–4.76) in normal hematopoietic cells, and 6.59 (range: 3.85–10.62) in all leukemic samples; this difference in signal intensity is equivalent to a 5-fold increase in median expression of CTGF in leukemic cells. Therefore, we hypothesized that expression level of CTGF may have prognostic significance in adult ALL. Using real-time RT-PCR assays for CTGF we examined the expression of CTGF in 79 diagnostic ALL patients from SWOG protocol S9400 (28 bone marrow and 51 peripheral blood samples). Patients with L3 ALL were excluded from the study. The median age of patients was 35 (range 17–64), with the median WBC 23,400/ul (range 600–396,600), and peripheral blood blasts 56% (range: 0–98). Fifty patients had B-ALL (63%), 13 (16%) had T-ALL and lineage was unknown for 16 (20%). When treated as a continuous variable in a logistic regression model, the level of CTGF expression was significantly associated with inferior OS and DFS (p=0.007 and p=0.0012, respectively). When controlled for WBC and cell lineage, the association of CTGF with OS and DFS remained statistically significant. We then sub-grouped the ALL patients into three equal groups (tertiales) based on CTGF expression. This subgroup analysis found that the OS for patients in the highest tertile (highest CTGF expression) was approximately 11% (95% CI 0–24) at 5 years, as compared to 42% (95% CI 23–61) and 58% (95% CI 38–78) for patients with middle and low CTGF expression respectively (figure). In sub-analysis of patients with B-lineage ALL (n=50), the association of CTGF expression with OS and DFS was still statistically significant (p=0.009 and p=0.005) when treated as a continuous variable. This report is an example where a gene expression study detected a gene differentially expressed in leukemia, with clear clinical value. Moreover, this is the first report that correlates the level of expression of CTGF with outcome in ALL patients. We are actively pursuing the biological and clinical significance of CTGF in other ALL patients and model systems.

Figure Figure
Inhibition of Mutant FLT3 Activation by the Novel Tyrphostin, AGS324.
Authors: Williams Allen B. , Gazit Aviv , Nguyen Bao , Slavin Shimon , Small Don
Published: April 2020

FLT3 is a member of the class III tyrosine kinase family, consisting of five immunoglobin-like extracellular domains, a single transmembrane domain followed by a short juxtamembrane region and an interrupted kinase domain. Constitutive activation of FLT3 through mutation occurs in about 30% of AML cases. Studies show that targeted inhibition of FLT3 by small molecule tyrosine kinase inhibitors results in clinical responses in some patients. In light of the finding of resistant mutants selected by Gleevec treatment in CML, it is likely that similar mutants will arise in FLT3. Thus, the need for development of additional FLT3 inhibitors will be important to overcome this resistance. Here, we investigate a series of compounds derived from AG1295 (a FLT3 inhibitor with an IC50 of 300–500 nM) for activity against FLT3. Of these, AGS324 exhibited the most potent activity against FLT3 ITD in transfected BaF3 cells. FLT3 ITD phosphorylation was completely inhibited by treatment with AGS324 with an IC50 of ~40 nM. Likewise, downstream STAT5 and MAP kinase phosphorylation were reduced in parallel to inhibition of FLT3 activation. AGS324 inhibited proliferation of BaF3 FLT3 ITD expressing cells with an IC50 of 60 nM. IL-3 could completely rescue BaF3 ITD cells from AGS324 mediated cytotoxicity even at 10 uM of inhibitor, indicating no interference with IL-3 signaling pathways. There was also no activity against proliferation of BaF3 parental cells. Like AG1295, it showed greatly reduced activity against the D835Y point mutant for inhibition of FLT3 phosphorylation (IC50 >5 uM) and proliferation (IC50 > 10 uM). Not all cells expressing kinase domain point mutants were resistant to inhibition by AGS324. Cells expressing the D835G mutation were inhibited in a similar fashion to BaF3 ITD cells. In addition cells expressing a FLT3 Y842E activating mutation were also inhibited by AGS324. Proliferation of MV4-11 cells, derived from an AML patient, which express a FLT3 ITD mutation, was also inhibited with a similar IC50 (~80 nM). Interestingly, SEM-K2 cells, which express amplified wild-type FLT3 were relatively resistant to inhibition of proliferation and activation of FLT3 by AGS324. Several other compounds that resulted from modifications to AG1295 showed varying degrees of potency against FLT3. Taken together, these findings indicate that AGS324 is a potent inhibitor of FLT3 ITD and some kinase domain point mutations but has reduced activity against wtFLT3. Some structural modifications of AG1295 greatly increase potency against FLT3 ITD as well as some kinase domain mutations. We present some SAR data for this group of tyrphostins, which may be useful for more specific targeting of mutant FLT3.
The CCR1 Antagonist BX470 Inhibits the Growth of Multiple Myeloma (MM) Cells That Produce MIP-1α.
Authors: Lentzsch Suzanne , Anderson Gulsum , Cuiling Li , Richard Horuk , Mapara Markus Y. , Irene Ghobrial , Roodman David G.
Published: April 2020

Macrophage inflammatory protein-1alpha (MIP-1α) is a key factor in the pathogenesis of MM. MIP-1α is a growth and survival factor for MM (Lentzsch et al., Blood 2003), stimulates osteoclast formation, increases adhesion of MM cells to stromal cells and induces bone resorption and lytic lesions in patients with MM (Choi et al., JCI 2001). High levels of MIP-1α are present in the marrow of 70% of MM patients, and MIP-1α levels correlate with increased bone destruction, tumor burden and a poor prognosis in MM patients (Uneda et al., Br J Haematol 2003). MIP-1α binds to the chemokine receptors CCR1 and CCR5, which are expressed on myeloma cells as well as on osteoclasts. Therefore, targeting MIP-1α mediated effects on myeloma cells as well as on osteoclasts by blocking its receptors is a novel target for treating the majority of MM patients. BX471 is a potent and selective antagonist of CCR1 that inhibits the binding of MIP-1α to CCR1. BX471 inhibits OCL formation (Oba et al., Exp. Hematology 2005) and is currently being studied in phase II trials for multiple sclerosis. However it is unknown if BX471 effects all MM cells or only those MM cells that produce MIP-1α.

Secretion of MIP-1α of the MM cell lines (MM.1S, OPM 2, RPMI 8226) was measured by Bio-Plex Cytokine Assay. All cell lines were exposed to serial dilutions of BX 471 (10–1000 nM) for 48 hours and thymidine uptake was measured to assess inhibition of proliferation. CCR1 expression on MM cell lines was detected by PCR.

All MM cell lines expressed CCR1. OPM 2 and MM.1S secreted high amounts of MIP-1α within 48hours (2,032 pg/mL and 4,388 pg/mL, respectively). Secretion of MIP-1α by RPMI 8226 MM cells was not detectable. Significant dose-dependent inhibition of proliferation by BX471 was seen with MM.1S and OPM 2 MM cells (40% and 46%, respectively). In contrast RPMI 8226, which did not secret MIP-1α was not inhibited by BX 471 up to 1000 nM. These data show that blocking CCR1 induces significant growth inhibition of MIP-1α producing MM cells and underscore the relevance of MIP-1α as an important autocrine growth and survival factor in MM.

This study provides the framework for clinical evaluation of BX 471 in a phase II trial for patients with MM and supports the hypothesis that BX471 targets both the tumor and osteoclastic activity in MIP-1α producing MM cells and suggests that BX471 should be an effective treatment for the majority of MM patients.
Multiple Myeloma as Target for γδ T Cell Mediated Immunotherapy - Expression of the Putative Vγ9Vδ2-TCR Ligand F1-ATPase on Myeloma Cells.
Authors: Kunzmann Volker , Engert Judith , Kimmel Brigitte , Wilhelm Martin , Einsele Hermann
Published: April 2020

Activated Vγ9Vδ2 T cells, the major γδ T lymphocyte subset in humans, show cytolytic activity against various tumor cells. However, tumor antigens recognized by the TCR remained unkown so far. Recently, the ectopic surface expression of the F1-ATPase, normally expressed on the internal membrane of mitochondria, was implicated in tumor recognition of Vγ9Vδ2 T cells (Scotet E. et al., Immunity2005; 22:71–80). Surface expression of the a chain of the F1-ATPase (recognized by monoclonal antibody 7H10) strongly correlates with susceptibility of tumor cells against Vγ9Vδ2 T cell lysis. Different functions have been attributed to the ectopic expression of the F1-ATPase on the cell surface, including an immunoregulatory role induced by cell stress, receptor for angiostatin or regulation of lipoprotein transport through high-affinity apolipoprotein A-I binding. In this study we evaluated the surface expression of this F1-ATPase on hematopoetic tumor cell lines and on primary tumor cells from hematological malignancies. As already shown, the a subunit of F1-ATPase was clearly detected on several tumor cell lines which are consistently killed by activated Vγ9Vδ2 T cells (Daudi, K562, RPMI 8226), whereas the known Vγ9Vδ2 T cell resistant tumor cell lines (Raji, Jurkat) did not express detectable levels of the F1-ATPase. Analysis of 42 primary hematopoetic tumor cells (21 myeloma, 17 AML, 4 B-NHL) revealed frequent expression of F1-ATPase on primary myeloma cells (14/19 positive), whereas primary AML blasts (3/17 positive) and primary NHL cells (1/4 positive) expressed the putative Vγ9Vδ2-TCR ligand F1-ATPase less frequently. To further evaluate the functional role of F1-ATPase expression in Vγ9Vδ2 T cell mediated recognition of myeloma cells, cytotoxicity assays were performed. The mAb against the a subunit of F1-ATPase significantly decreased in vitro lysis of myeloma cells lines and primary myeloma cells by activated Vγ9Vδ2 T cells. These results suggests Vγ9Vδ2 TCR-dependent interactions between myeloma cells and Vγ9Vδ2 T cells and indicate that multiple myeloma should be considered as a major target for γδ T-cell mediated immunotherapy.
Stimulation with K562 Cells Transfected with 4-1BBL and IL-15 Expands and Activates Natural Killer (NK) Cells with Specific Cytotoxicity for Multiple Myeloma (MM).
Authors: Shi Jumei , Tricot Guido , Szmania Susann , Camet Cristyn , Rosen Nancy , Cottler-Fox Michele , Barlogie Bart , Campana Dario , van Rhee Frits
Published: April 2020

Introduction: Although stem cell transplant (PBSCT) supported high dose chemotherapy can induce remission in MM patients with high risk features (e.g. high CKS1-B expression and abnormal cytogenetics) there is a high rate of relapse. Immunologic therapies can augment high dose chemotherapy by eradicating chemo-resistant disease. We have previously established that KIR-ligand mismatched NK cells from haplo-identical donors are cytotoxic to primary MM cells. In this study we investigate whether co-incubation of NK cells with irradiated K562 cells transfected with 41BBL and membrane-bound IL15 (K562-41BBL-mIL15) could expand NK cells and augment cytotoxicity to primary MM cells.

Methods: Peripheral blood mononuclear cells (PBMC) of normal donors were co-cultured with irradiated K562-41BBL -mIL15 at a 1.5:1 ratio with varying concentrations of IL2 and re-stimulated on day 7. The cultures were then analyzed for proliferation, expansion, immunophenotype and cytotoxicity.

Results: By testing a range of IL2 concentrations (10–300 IU/ml) we first established that 100IU of IL2/ml induced optimal proliferation in H3 thymidine uptake assays and this concentration was used in all subsequent experiments. After two weeks PBMC co-cultured with K562-41BBL-mIL15 were enriched for NK cells (93%) and contained few T cells (0.8%) compared to PBMC cultured with IL2 alone (NK cells 11.9% and T cells 58.7% respectively). In absolute numbers there was as 60-fold (±0.6) expansion of NK cells in the K562-41BBL -IL15 cultures compared to only a 1.5-fold expansion of NK cells cultured with IL2 alone. NK cells were analyzed by flow cytometry for expression of KIR2DL1, KIR2DL2/3, and KIR3DL1 pre- and post-incubation with K562-41BBL-mIL15 and we observed no change in NK immunophenotype. 51Cr release assays of expanded NK cells showed specific killing of K562 cells (84±4%), the MM cell line U266 (88±3%), and primary MM cells (58±9%), without killing of non-myeloma patient cells (PHA-blasts) or autologous cells at a ratio of 7 NK effectors to 1 target cell. Expanded NK cells achieved much higher specific lysis and at lower E:T ratios of primary MM cells when compared side-by-side with non-expanded NK cells.

Conclusion: K562-41BBL-mIL15 transfectants stimulated vigorous expansion of NK cells without expanding T lymphocytes. Analysis of the KIR repertoire showed that there was no change in NK cell subpopulations after expansion. The expanded NK cells were highly active and killed patient MM cells better than non-expanded NK cells without significant lysis of normal cells. Expanded/activated NK cells may prove especially helpful in treating patients with relapsed/refractory MM who have a high tumor burden.