bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinctive Efficacy of IVIG in Ameliorating Thrombocytopenia Induced by Anti-Platelet GPIIbIIIa and GPIbα Antibodies.
Authors: Sayeh Ebrahim , Crow Min , Nieswandt Bernhard , Freedman John , Ni Heyu
Published: October 2019

Idiopathic autoimmune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibody-mediated platelet destruction. The major platelet antigens that autoantibodies are directed against in ITP are GPIIbIIIa and GPIbα. Intravenous immunoglobulin G (IVIG) is a treatment used in ITP; however, some patients are refractory to this treatment and the mechanisms of action of IVIG in this disease are not fully understood. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by GPIIbIIIa versus GPIbα anti-platelet antibodies. Thrombocytopenia was induced in Balb/c mice using three monoclonal antibodies against either mouse platelet GPIIbIIIa (JON1, 2, −3) or against GPIbα (p0p 3, 4, −5). Mouse platelet counts were performed daily from day one (pre-treatment) until day five using a flow cytometric assay. The efficacy of IVIG in inhibiting monoclonal antibody-induced platelet destruction in mice was assessed by intraperitoneal injection of IVIG (2 g/kg) 24-hours before anti-platelet antibody injections (7 mg/mouse, i.v.) on day two. We found that pre-treatment with IVIG resulted in a significantly higher platelet count on day three in all groups of anti-GPIIbIIIa-injected animals (n=3/antibody; for JON1, P=0.002; JON2, P=0.015; JON3, P=0.002) as compared to albumin-treated controls. In contrast, IVIG failed to prevent platelet clearance and thrombocytopenia on day three in two anti-GPIbα antibody-treated groups (p0p3 and p0p5, n=3/antibody), but did inhibit platelet destruction in mice that were injected with p0p4 antibody (P=0.0003). Our results indicate that the efficacy of IVIG treatment in preventing ITP induced by these anti-platelet antibodies is affected by their antigen specificities. These data also suggest that patients with ITP mediated by GPIbα may be less responsive to IVIG treatment than those with ITP mediated by GPIIbIIIa antibodies.
A 3′ UTR Single Nucleotide Polymorphism 829C→T in Dihydrofolate Reductase Gene Results in an Increase in DHFR Protein Level and MTX Resistance.
Authors: Mishra Prasunkumar J. , Longo Giuseppe S.A. , Menon Lata G. , Abali Emine , Banerjee Debabrata , Bertino Joseph
Published: October 2019

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF) required for the synthesis of thymidylate and purines. Methotrexate (MTX) acts as a tight-binding inhibitor of DHFR and remains an important chemotherapeutic agent for treatment of leukemias and lymphomas. Increased DHFR confers resistance to antifolates in target cells. A previously reported single nucleotide polymorphism (SNP) 829C/C→829T/T (829C→T) found in the 3′- untranslated region of DHFR gene transcript (between the first and second polyadenylation site) was associated with higher expression of the DHFR transcript. The SNP was identified in 5.4% of the cases and 6.0% in the controls of Japanese patients with childhood leukemia/lymphomas (Goto et al. 2001, Clinical Cancer Research, Vol. 7, 1952-1956). The objective of the present study was to determine the role of the 3′ UTR SNP 829C→T in DHFR gene expression, DHFR protein level and resistance to MTX. The mutation 829C→T in the 3′ UTR of wild type DHFR was introduced by site directed mutagenesis and the mutant cDNA expressed in DHFR deficient CHO cells (DG-44), wild type DHFR and vector alone constructs were also transfected into DG44 as controls. After two weeks of selection in G418 containing media, several well-isolated surviving colonies were picked and expanded as cell lines in media containing G418. Real-time quantitative PCR was used to compare mRNA and genomic DNA level of the clones while Western blotting was used to compare the protein levels. MTX cytotoxicity assay was carried out in media lacking thymidine. Clones expressing the mutant 829C→T showed greater than two fold enhanced expression of DHFR transcripts as compared to wild type clones. Corresponding to the high mRNA levels, an increase in DHFR protein level was observed in the mutant clones without an increase in DHFR gene copy number. Cytotoxicity studies showed that cell lines with increased levels of DHFR were significantly more resistant to MTX than cells with wild type 3′ UTR. Of interest clonogenic efficiency of the mutants in medium lacking thymidine was greater than wild type and was directly proportional to the level of DHFR expressed in the clones. This study demonstrates that when SNP 829C→T is introduced in the 3′ UTR of wild type DHFR, the expression of the DHFR mRNA is enhanced with a corresponding increase in the protein level. The presence of a SNP 829C→T in patients with ALL may contribute to treatment failure, as MTX is a key drug in curative regimen for this disease. Future studies are directed toward determining the abundance of this SNP in other populations, and the correlation between this SNP and clinical methotrexate resistance and or decreased MTX toxicity.
Heme Affects Sensitivity to Imatinib through Regulation of Nrf2 Activity in BCR/ABL-Positive Cell Lines.
Authors: Miyoshi Takuji , Nagai Tadashi , Nakamura Makiko , Ohmine Ken , Fujita Hiroyoshi , Taketani Shigeru , Komatsu Norio , Ozawa Keiya
Published: October 2019

Heme has been recognized as an important modulator of the functions of various cellular molecules. Recent work showed that heme binds to Bach1 transcription factor, resulting in the loss of the repressor activity of Bach1. Because Nrf2 transcription factor and Bach1 may competitively recognize the common gene regulatory elements, heme may have an effect on the Nrf2-madiated gene expressions. We recently reported that Nrf2 is involved in the resistance to imatinib in BCR/ABL-positive cells. Here we investigated the role of heme in the sensitivity to imatinib in human BCR/ABL-positive cell lines. Addition of hemin to KCL22 and KU812 cells resulted in significant increases in the IC50 values of imatinib in accordance with the abrogation of imatinib-mediated induction of apoptosis-related molecules, cleaved caspase 3 and cleaved PARP. The levels of phosphorylated BCR/ABL and phosphorylated ERK1/2 MAP kinase (active form) were both decreased by imatinib even when hemin was added, indicating that hemin does not interfere with imatinib-mediated inhibition of BCR/ABL function and that mechanisms of hemin-induced acquisition of imatinib-resistance is not related to BCR/ABL kinase activity. Hemin increased Nrf2/DNA complex formation at the antioxidant response element of the human γ-glutamylcystein synthetase (γ-GCS) light subunit gene promoter. Because γ-GCS is a rate-limiting enzyme of the synthesis of glutathione (GSH), which has been shown to be involved in resistance to imatinib, we next examined the level of GSH and found that the intracellular amount of GSH was increased by hemin treatment of these cells. In addition, the protein level of heme oxygenase-1, which is important for survival of chronic myeloid leukemia cells and the gene expression of which can be also regulated by Nrf2, was increased after hemin treatment. Addition of ascorbic acid, which suppresses Nrf2-dependent gene expression, partially restored the sensitivity to imatinib in hemin-treated cells. We also showed that inhibition of intracellular heme synthesis by succinyl acetone increased the sensitivity to imatinib in imatinib-resistant cell lines, KCL22/SR and KU812/SR. The results suggest that heme is critically involved in the sensitivity to imatinib, at least in part, through regulation of Nrf2 function.
Persistence of eGFP Marked Bone Marrow Cells in Long-Term Hematopoiesis.
Authors: Pilz Ingo H. , Schmidt Manfred , Ball Claudia , Glimm Hanno , von Weizsäcker Fritz , von Kalle Christof
Published: October 2019

To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.
The BCL6 Proto-Oncogene Suppresses p53 Expression in Germinal-Center B Cells.
Authors: Phan Ryan T. , Niu Huifeng , Saito Masumichi , Basso Katia , Cattoretti Giorgio , Dalla-Favera Riccardo
Published: October 2019

The proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is necessary for germinal center (GC) formation and is implicated in the pathogenesis of B-cell lymphoma. In ~50% diffuse large cell lymphoma and 10% follicular lymphoma, BCL6 gene expression is deregulated by chromosomal translocations or mutations that affect its 5′ regulatory region. The precise function of BCL6 in GC development and lymphomagenesis is unclear since very few BCL6 direct target genes have been identified. We report that BCL6 suppresses p53-dependent and p53-indepenent growth arrest and apoptosis responses in GC B cells. BCL6 directly suppresses the transcription of the p53 gene, as demonstrated by (1) chromatin immunoprecipitation (ChIP) assays showing that BCL6 binds the p53 promoter region in vivo; and (2) transient transfection/reporter assays identifying within the p53 promoter region two BCL6-binding sites that mediate BCL6-mediated suppression of p53 transcription.

Accordingly, suppression of BCL6 expression via specific siRNA leads to increased expression of p53 both under basal condition and in response to DNA damage. Consistent with a physiological role for BCL6-mediated p53 suppression, immunohistochemical analysis shows that p53 expression is absent in GC B cells where BCL6 is highly expressed. In addition, our data reveal that BCL6 inhibits the p53-independent activation of the p21/WAF1 cell cycle arrest gene by binding to Miz-1, a transcription factor involved in p21 activation. Consistent with a role of BCL6 in inhibiting p53-related cell cycle arrest and apoptotic responses, constitutive expression of BCL6 suppresses p53 expression and p53-target genes (P21 and PUMA) and protects B cell lines from apoptosis induced by DNA damage. These results indicate that one function of BCL6 is to allow GC B cells (centroblasts) to constitutively proliferate and to sustain the physiologic DNA breaks required for immunoglobulin switch recombination and somatic hypermutation without inducing p53-related responses. These findings also imply that B cell lymphoma with deregulated BCL6 expression are functionally p53-negative and impaired in apoptotic responses.
Risk Factors for Outcomes of Family Haploidentical Allogeneic Stem Cell Transplantation for Adults with Acute Leukemias: A Survey on Behalf of the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Ciceri Fabio , Labopin Myriam , Rocha Vanderson , Rowe Jacob M. , Bunjes Donald , Di Bartolomeo Paolo , Frassoni Francesco , Martelli Massimo F. , Aversa Franco
Published: October 2019

Results of family haploidentical allogeneic stem cell transplantation (Haplo-SCT) have been reported from single-center studies as a curative treatment option for patients with high risk acute leukemias. We analysed 273 adult patients with de novo acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) receiving an Haplo-SCT transplanted from 1995 to 2002 in 75 EBMT centers. Overall, 170 AML patients underwent transplantation in CR1 (n=39), CR2 (n=34) or in advanced disease (n=97); median age was 38y (16–70). Overall, 103 ALL patients underwent transplantation in CR1 (n=31), CR2 (n=22) or in advanced disease (n=50); median age was 26y (16–56). Graft composition was based on CD34+ cell selection of PBSC, with median CD34+ cells x106/kg of 7.3 (1.1–45.5) in AML and 8.3 (1.14–30) in ALL. Conditioning regimen was TBI-based in 69% and 87% in AML and ALL respectively. Primary engraftment was documented in 87% AML and in 83% of ALL patients, with ANC 0.5 x109/L in a median of 13 days (8–35) and a 2y probability of PLT > 50 x109/L of 80+/− 5%. The use of TBI was significantly associated with engraftment (90% vs 71%) when adjusting for CD34 dose. The cumulative incidence of acute GvHD >=II was 16% in AML and 15% in ALL. With a median follow-up of 19 months (1–85), the estimated leukemia-free survival (LFS) at 2 years in AML was 39±9%, 33 ±10% and 4±3% and in ALL was 28±9%, 16±8, and 0% for CR1, CR2 and advanced patients respectively. Cumulative incidence using competing risks, for relapse (RI) at 2 y in AML was 8 ±8%, 9 ±9%, 25±8% for CR1, CR2 and advanced; RI in ALL was 27±16% and 41 ±20% in CR1 and CR2. The non-relapse mortality (NRM) at 2 y in AML was 52±18%, 56 ±19%, 69±8% for CR1, CR2 and advanced patients; NRM in ALL was 44±18% and 41±21% in CR1 and CR2. In a multivariate analysis for LFS, advanced disease status at transplant (RR 0.4), ALL (RR 0.5), recipient’s age >33 years (RR 0.4), and absence of TBI in conditioning (RR 0.6) were factors associated with lower LFS. Factors associated with higher transplant-mortality were advanced disease status (RR 2.01), absence of TBI (RR 1.6) and year of transplant<2000 (RR 1.42). Predictive factors for RI were diagnosis of ALL (RR 2.39) and advanced disease (RR 1.96). In conclusion, outcomes of allogeneic transplantation from haploidentical family donors in adults with high risk acute leukaemia are promising and should be considered in the algorithm treatment for patients with an indication of allogeneic SCT lacking an HLA identical donor. Status of the disease, age, type of leukemia and conditioning are some factors associated with outcomes after Haplo-SCT.
Distinct Functions of Erythropoietin and Stem Cell Factor Are Linked to Activation of p70S6/mTOR Kinase Signaling Pathway in Primary Erythroid Progenitors.
Authors: Wickrema Amittha , Shahab Uddin , Ah-Kang Jeong , Zhou Ying , van Besien Koen , Ulaszek Jodie
Published: October 2019

Erythropoietin (Epo) and stem cell factor (SCF) guide erythroid cell maturation by exerting their effects at various stages of differentiation. Distinct and overlapping functions of these two growth factors have been well characterized. However, signaling pathways responsible for the antiapoptotic function of Epo and the proliferative function of SCF has not been fully characterized. Especially activation of common upstream signaling elements PI3-kinase, Akt kinase and phosphorylation/inactivation of forkhead transcription factors by both Epo and SCF bring about distinct functional outcomes have not been understood. In the present study we examined the activation of p70S6/mTOR pathway by Epo and SCF in CD34-derived primary erythroid progenitors. Our results provide evidence for activation of p70S6 kinase and mTOR by SCF but not by Epo or IGF-1 (insulin-like growth factor-1). We also show that only SCF phosphorylates protein translational regulatory proteins, 4E-BP1 and S6 ribosomal protein suggesting its involvement in promoting protein translation. Furthermore, we demonstrate that inhibition of mTOR by rapamycin results in reduction in erythroid cell proliferation and colony-formation under steady state culture conditions demonstrating the involvement of downstream signaling elements in the PI3/Akt kinase pathway in cell proliferation apart from its antiapoptotic signal. The reduction of both BFU-E and CFU-GM colony formation indicated that rapamycin also affects early hematopoietic cells. Examination of a parallel pathway involving signaling element Mnk1 showed that both Mnk1 and its downstream target eIF4E are not phosphorylated in response to SCF or Epo. However, these protein were constitutively phosphorylated in primary erythroid progenitors. Interestingly, we also found that during the proliferative phase of erythroid differentiation mTOR is mostly detected in the cytoplasmic fraction of the cells whereas during terminal phase of differentiation mTOR is detected in the nuclear fraction. These results suggest that mTOR may have additional functions associated with chromatin remodeling in erythroid progenitors, which occur prior to enucleation. Taken together, our data provide a mechanism for how distinct functions of Epo and SCF are accomplished through selective use of a common signaling pathway explaining in part how functional diversity is acheived.
Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.
Authors: Regan Baird T. , Gailani David , Furie Bruce , Furie Barbara C.
Published: October 2019

Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.
Thromboembolism in Hospitalized Neutropenic Cancer Patients.
Authors: Khorana Alok A. , Francis Charles W. , Culakova Eva , Fisher Richard I. , Kuderer Nicole , Lyman Gary H.
Published: October 2019

Background: Cancer is associated with thrombosis, but the frequency of thromboembolism in hospitalized cancer patients receiving contemporary chemotherapy regimens is not known. We investigated the frequency of arterial and venous thromboembolism in hospitalized cancer patients receiving active therapy (as identified by the presence of neutropenia) and characterized its association with in-hospital mortality.

Methods: We conducted a retrospective cohort study using the discharge database of the University HealthSystem Consortium. This included 66,106 adult neutropenic cancer patients with 88,074 hospitalizations between 1995 and 2002 at 115 academic medical centers. Patients were identified using ICD-9-CM codes that contained at least one diagnosis of malignant disease and agranulocytosis. Patients with thromboembolism were identified using codes for venous thrombosis, pulmonary embolism, arterial embolism, acute cerebrovascular disease, and acute coronary arterial disease. The association of VTE with clinical variables was studied in univariate analysis and in a multivariate logistic regression model. The chi-square test was used to compare categorical variables, and Cochran-Armitage test to determine trend.

Results: Thromboembolism was reported in 5,272 patients (8%), with 5.4% patients developing venous and 1.5% arterial thromboembolism during the first hospitalization. There was a significant association between the occurrence of venous and arterial thromboembolism (OR 1.73, 95%CI, 1.38–2.16). Venous thromboembolism was more frequent in patients with metastatic disease (OR, 1.23, 95% CI 1.13–1.34), but arterial thromboembolism was not (OR, 0.59, 95% CI, 0.51–0.69). In-hospital mortality was significantly greater in patients with venous (OR 2.01, 95% CI 1.83– 2.22) or arterial thromboembolism (OR 5.04, 95% CI, 4.38–5.79), even in patients without metastatic disease. Patients with lymphoma or leukemia accounted for one-third of venous events and one-half of arterial events. Clinical variables most frequently associated with thromboembolism in a multivariate logistic regression analysis were age ≥ 65 years, primary sites of cancer including lung, gastrointestinal, gynecologic and brain, length of stay ≥ 10 days, and comorbidities including infection, pulmonary and renal disease, and obesity. From 1995 to 2002, there was a 36% increase in venous and a 124 % increase in arterial events (P for trend <0.0001) (see Figure).

Conclusions: The incidence of thromboembolism is high across all cancer subgroups including patients with hematologic malignancies and with non-metastatic disease, commonly perceived to be at lower risk for thromboembolism. Of particular concern is the increasing frequency of venous and arterial thromboembolism. Prospective studies are necessary to characterize the risks and benefits of thromboprophylaxis in this patient population.

Figure Figure
Unreliability of Point of Care Devices for INR Monitoring of Oral Anticoagulant Therapy. A Re-Evaluation.
Authors: Sands Caryn O. , Padilla Vilma , Naraine Khrishan , Rand Jacob , Fox Amy S.
Published: October 2019

Oral anticoagulant treatment with vitamin K antagonists has been encumbered by the need for interval monitoring of INRs to adjust dosage. Point of Care (POC) testing of capillary blood has come into widespread use because of the advantages of rapid analysis and dose adjustment. To assess the reliability of this procedure, we compared INR results from 2 POC devices from different manufacturers - POC1 and POC2 - and compared these results to standard assays on venous samples with a BCS automated analyzer.

78 patients on warfarin for at least 3 months were studied. Results of 56 patients with POC2 and 76 patients with POC1 were compared to the BCS. Capillary blood samples were taken within 30 minutes of the venous draw. The therapeutic INR range was considered to be 2.0–3.5.

The bias plots for the 2 POC methods compared to the reference INR are shown in the 2 Figs, below. Within the therapeutic range, 71% and 85% of the results correlated with BCS for POC1 and the POC2, respectively. Both instruments showed the greatest variation from the reference measurement at INR values greater than 2.5 Both POC methods demonstrated clinically relevant overestimates of INR(see table).

Figure Figure Figure Figure

Agreement % of Reference INR Values to POC INR

Reference INR* POC1-subtherapeutic POC1-therapeutic POC1-supratherapeutic POC2-subtherapeutic POC2-therapeutic * INR = 2.0–3.5 BCS-subtherapeutic 77% 23% - 63% 37% BCS-therapeutic 4% 71% 25% 5% 85% BCS-supratherapeutic - - 100% - 33%

Conclusion: This study raises a significant question rearding the accuracy of POC methods for INR determinations. Despite ease of use and immediacy of results, the lack of correlation, even under study conditions, and the potentials for under-and overdosing makes relying on these devices problematic.
Development and Pilot testing of a Disease-Specific Quality of Life Questionnaire for Adult Patients with Haemophilia (Haem-A-QoL).
Authors: Mackensen Sylvia v. , Gringeri Alessandro ,   Haem-A-QoL Study Group
Published: October 2019

Background: Haemophilia and its treatment influence the every-day-life of patients and impact on their quality of life. For the adequate assessment of quality of life validated instruments are necessary. While haemophilia-specific questionnaires for children are existing (v. Mackensen et al., 2004; Young et al., 2004), up to now no validated instrument for adults is available. Currently some developmental works are on-going in order to develop such an instrument as well for adults. In a multi-centre validation study a health-related quality of life questionnaire for adults with haemophilia was developed and pilot tested in Italy.

Aim: This study was designed to develop and validate a haemophilia-specific quality of life measurement for adults (Haem-A-QoL) in Italy.

Methods: The study consisted of three phases: a) a qualitative study including focus groups (with patients and physicians), item formulation and expert ratings of a catalogue of items by patients and physicians, b) a psychometric study including cognitive debriefing, and feasibility testing and c) data analysis. The qualitative study has been performed by the University of Hamburg in cooperation with the University of Milan.

Results: Focus groups with semi-structured interviews were performed in 32 patients from 6 Italian haemophilia centres (Milan, Perugia, Genova, Bari, Castelfranco, Parma). 14 physicians and 8 nurses were asked the same questions. Patients and physicians/nurses consider similar dimensions important for quality of life. But the order of importance is different for patients (physical health, social interaction, dependence, future, work) and physicians/nurses (dependence, work, social interaction, treatment, physical health). Based on the focus group results a draft version of the questionnaire was formulated and given to patients and physicians in order to evaluate the items concerning importance for haemophilia and comprehensibility. The draft version consisted of 159 items. After expert evaluation the questionnaire was revised: several items could be eliminated and some were reworded. The revised version of the Haem-A-QoL consisting of 50 items and was psychometrically tested in 10 Italian centres. The pilot testing showed excellent psychometric characteristics in terms of reliability and validity (convergent, discriminant).

Conclusions: The development of a disease-specific quality of life instrument requires multiple steps and the involvement of different experts (patients, physicians, nurses) in order to have a standardised and valid questionnaire. The Haem-A-QoL is well-accepted by adult haemophiliacs and proved to be a valid and reliable disease-specific self-report measurement for the assessment of quality of life in haemophiliacs. In a next step the Haem-A-QoL will be applied in clinical trials to evaluate patient perceptions of different treatment regimens in haemophilia.
Targeted Pre-Emptive Anti-Aspergillus Therapy in Neutropenic Patients Not Receiving Empirical Antifungal Therapy.
Authors: Maertens Johan A. , Theunissen Koen , Verhoef Gregor , Verschaekelen Johnny , Lagrou Katrien , Verbeken Eric , Verhaegen Jan , Meersseman Wouter , Wilmer Alexander , Boogaerts Marc , Van Eldere Johan
Published: October 2019

Background. For several decades, empirical antifungal therapy has been the standard of care for the early treatment of invasive aspergillosis (IA) in neutropenic cancer patients. However, this approach results in the undue exposure to potentially toxic drugs, may select for resistant strains, and jeopardizes the hospital budget. We conducted a prospective study to evaluate the feasibility and safety of a targeted pre-emptive approach (while avoiding empirical therapy), based on the incorporation of sensitive, non-invasive diagnostic tests.

Methods. A total of 136 treatment episodes at high risk for IA (41 % ≥40 years of age; 26 % relapsed leukemia; 30 % high dose cytarabine; mean duration of neutropenia: 21.5 days; mean duration of broad-spectrum antibiotics: 15.9 days) were screened daily for circulating galactomannan by the Platelia (Bio-Rad) enzyme immunoassay (EIA) while receiving fluconazole prophylaxis. An index of ≥0.5 was considered positive if confirmed by the subsequent sample. In addition, well-defined clinical, radiological, and microbiological criteria triggered a diagnostics work-up for invasive fungal infection, including thoracic computed tomography scanning and bronchoscopy with bronchoalveolar lavage. Antifungal therapy (AmBisome 5 mg/kg/day) was withheld unless the patient became EIA-positive or had a positive culture or microscopy for molds with supportive radiological findings.

Results A total of 4170 serum samples (mean 30.6/episode) was analyzed. The combined EIA-CT-scan approach identified 19 episodes (14 percent) of IA. Less than one quarter of the episode that qualified for empirical therapy (unexplained persisting or relapsing fever) received antifungal therapy (6.6 percent versus 30 percent). Conversely, early antifungal therapy was started in 10 episodes (7.3 percent) not otherwise suspected for IA due to treatment with corticosteroids or due to the presence of confounding co-factors/co-infections. Undetected cases of IA were not found at autopsy. Unexpectedly, autopsy identified one patient with disseminated zymogycosis. At 12 weeks, a 77 percent survival rate was noted in case patients with controlled underlying hematological disease (versus 30 percent for patients with refractory underlying disease)

Conclusions. Replacing empirical therapy by a targeted, pre-emptive therapy offered effective antifungal control and spared patients from exposure to potentially toxic and expensive drugs without negative impact on mortality. This strategy should further be explored in a multicenter, randomized setting.
Augmentation of Post-Transplant Immunity: Antigen Encounter at Time of Hematopoietic Stem Cell Transplantation Enhances Antigen-Specific Donor T Cell Responses in the Post-Transplant Repertoire.
Authors: Mullen Craig A. , Kocak Ulker , Shaw Joanne L. , Mori Shahram
Published: October 2019

After transplant the immune system is reconstituted by cells derived from both hematopoietic stem cells and peripheral expansion of differentiated donor T cells. Immune function is poor despite transplantation of mature lymphocytes from immune competent donors. We tested the hypothesis that early antigen encounter at the time of cell transplant would enhance desired donor T cell responses in the post-transplant repertoire. 2 independent models of peptide-specific T cell responses were studied.

Model 1 : The model for CD4 cells employed T cells from transgenic DO11.11 mice that constitutively express the T cell receptor for the class II restricted ovalbumin (OVA) peptide 323–339. Fig 1: Early exposure to OVA antigen enhances clonal expansion of OVA specific transgenic T-cells following syngeneic BMT. Lethally irradiated BALB/c mice were injected with 300 μg of OVA peptide in CFA or CFA alone subcutaneously one day before transplantation (D-1). The transplanted mice received 2x106 transgenic OVA specific T-cells and 6x106 non-transgenic naive BALB/c bone marrow cells. At 2 days (A) and 7weeks (B) following BMT, draining lymph nodes were isolated and examined for the presence of OVA-specific T-cells using FITC-labeled KJ-126 antibody and PE-labeled anti mouse CD4 antibody. Naïve BALB/c animals were used as negative controls (C). The absolute number of antigen-specific T-cells was determined by multiplying the total cells recovered with the percentage of OVA-specific CD4+ T-cells identified by flow.

Figure Figure

Model 2: The model for CD8 cells employed nontransgenic H2-Db-restricted T cell responses to the influenza nucleoprotein peptide 366–374. Fig 2: Antigen specific CD8+ cells in antigen-exposed animals are functionally active. Donor SW mice were immunized three times by ip injection of virus-infected spleen cells. Recipient C57BL/6 animals underwent BMT using influenza-immune donors spleen cells and bone marrow (10x106 and 4x106 respectively). Some transplant recipients were exposed to influenza virus on D-1. Ten days following BMT, the animals were sacrificed and spleens were isolated and stimulated in vitro with 2 μg of NP peptide. After two rounds of stimulation, the splenocytes were assayed by intracellular cytokine assay for the secretion of IFNg by staining with PE-anti IFNγ and FITC-anti-CD8 antibodies. The results are representative of three experiments (total number n=4/experimental group).

Figure Figure

Encounter with specific antigen at the time of T cell transplantation led to clonal expansion of donor T cells and preservation of donor T cell function in the post-transplant immune environment. Antigen-specific donor T cell function was poor if antigen encounter was delayed or omitted. Severe parent>F1 graft versus host reactions blocked the effect of early antigen exposure.
Selected IL-21R Expression and Apoptosis Induction by IL-21 in Follicular Lymphoma.
Authors: Akamatsu Norihiko , Hasegawa Hiroo , Yamada Yasuaki , Makabe Koki , Asano Ryutaro , Kumagai Izumi , Murata Ken , Tsukasaki Kunihiro , Tomonaga Masao , Tsuruda Kazuto , Sugahara Kazuyuki , Kamihira Shimeru
Published: October 2019

IL-21 receptor (IL-21R), a member of the common gamma receptor family, is expressed on many kind cells including T-cells, B-cells, and NK cells. IL-21 promotes the proliferation and maturation of NK cells and shows anti-tumor activities in animal systems. Although these results suggest its clinical use in the treatment of malignancies, it has also been shown that IL-21 may conversely accelerate the proliferation of IL-21R-expressing tumor cells. Considering the potential for therapeutic application of recombinant IL-21, we examined IL-21R expression and response to IL-21 in hematological malignancies. Using flow cytometry, we examined IL-21R expression in primary samples from AML, ALL, B-CLL, B-NHL and adult T-cell leukemia/lymphoma (ATLL) caused by a retrovirus, HTLV-1. Among 35 primary samples, only 5 samples from 16 patients with B-NHL and one sample from 8 patients with ATL were IL-21R positive. Of note, all the IL-21R positive NHL also expressed CD10, one of the marker molecules of follicular center cells. Indeed, two of the five patients were diagnosed follicular lymphoma with t(14;18)(q32;q21). Corresponding to these results, the follicular lymphoma cell line SUDHL-4 and most ATLL and HTLV-1-infected cell lines were IL-21R positive. Interestingly, IL21 showed dual effects on these cell lines; IL-21 stimulated the proliferation of ATLL cell lines but suppressed the proliferation of SUDHL-4. Annexin V and propidium iodide staining for detection of early and late apoptosis and mitochondrial membrane potential analysis indicated that the suppression was the result of apoptosis. Exposure of SUDHL-4 to IL-21 resulted in significant time-dependent induction of caspase-8 and caspase-3 activation. More importantly, IL-21 decreased Bcl-2 expression but drastically increased Bax expression. Thus, selected expression of IL-21R in follicular lymphoma cells and induction of apoptosis by IL-21 provide a strong background for IL-21 as an anti-neoplastic agent at least in a subset of follicular lymphoma.
Cell Cycle Dysregulation Represents an Early Effect of the Proteasome Inhibitor Bortezomib in Mantle Cell Lymphoma.
Authors: Hutter Grit , Zimmermann Yvonne , Rieken Malte , Weinkauf Marc , Weigert Oliver , Hiddemann Wolfgang , Dreyling Martin H.
Published: October 2019

Mantle cell lymphoma (MCL) is a distinct subtype of malignant lymphoma with an especially poor clinical outcome, a median survival time of 3 years and virtually no long-term survivors. On the molecular level, MCL is characterized by the chromosomal translocation t(11;14)(q13;q32) resulting in the constitutive overexpression of cyclin D1. However, additional genetic alterations of cell cycle regulators, e.g. deletions of the INK4A gene cluster, are detectable in the majority of cases. In various phase II studies the proteasome inhibitor bortezomib (Velcade) has demonstrated a high clinical efficacy with up to 60% remission rates in relapsed MCL. Additionally, in a previous in vitro study, the inhibitor induced a downregulation of cyclin D1 expression and a concomitant G(1) cell cycle arrest. However, little is known which molecules represent the critical targets of proteosome inhibition and how different regulators of cell cycle and apoptosis (inhibitors of CDK/INK4: p15INK4A, p16INK4B -and p14ARF and other kinase inhibitor proteins/KIP: 21CIP1, p27KIP1 and p57KIP2) are affected.

4 MCL cell lines (HBL2, GRANTA 519, Jeko-1, NCEB-1) and 2 hematological control cell lines (Jurkat, Karpas 422) were exposed to bortezomib at the minimal cytotoxic concentration (25 nmol) which corresponds to clinically achieved drug levels and results in a significant cytolysis after 48 – 72 hours. Real-time RT-PCR and protein expression levels of various CDK inhibitors (INK4s, KIPs) and cyclin D1 were determined a 0, 4, 8 and 12 hours after treatment with bortezomib. In addition, RNA- and protein expression data were compared to functional cell cycle phase (FACS) and cell apoptosis. Prelimenary data indicate that downregulation of cyclin D1 RNA expression after 12 and 24h of treatment represents a rather late event whereas alterations of other cell cycle regulators (like p21CIP1) were detected siginificantly earlier in all four MCL cell lines. Thus, expression of cell cycle regulators may indicate early events of proteasome inhibition. A comparative analysis of the cell cycle regulation network is currently being performed and will be presented at the conference.
B-Lymphocyte Stimulator (BLyS) Is Highly Expressed in Waldenstrom’s Macroglobulinemia.
Authors: Ansell Stephen M. , Grote Deanna M. , Ziesmer Steven C. , Witzig Thomas E. , Kyle Robert A. , Novak Anne J.
Published: October 2019

Waldenstrom’s macroglobulinemia is a serious and frequently fatal illness, however many of the mechanisms leading to this disease are not yet known. It is clear, however, that there is dysregulation of the balance between cell proliferation and programmed cell death. BLyS (B-lymphocyte stimulator) is a newly identified TNF family member expressed by monocytes, macrophages, and dendritic cells. BLyS has been shown to be critical for maintenance of normal B cell development and homeostasis, and has been found to stimulate lymphocyte growth. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B-cells. Studies of the effects of BLyS on B cell physiology have shown that it also regulates immunoglobulin secretion.

To determine the relevance of the BLyS receptor-ligand system in Waldenstrom’s macroglobulinemia, we examined malignant B cells from 5 patients with Waldenstrom’s macroglobulinemia for their ability to bind soluble BLyS and for the expression of the known BLyS receptors, TACI, BAFF-R, or BCMA. The malignant B cells were found to bind BLyS and express BAFF-R and TACI. BCMA expression was undetectable. We then determined the expression of BLyS in bone marrow specimens from 5 patients with Waldenstrom’s macroglobulinemia by immunohistochemistry and compared it to the expression in 5 normal bone marrow specimens. The lymphoplasmacytic cell infiltrate in the bone marrow of patients with Waldenstrom’s macroglobulinemia showed significantly increased BLyS expression. We further determined the serum BLyS levels by ELISA in stored serum specimens from patients with Waldenstrom’s macroglobulinemia (n=20), and compared them to serum BLyS levels in other patients with lymphoplasmacytic lymphoma without elevated immunoglobulin levels (n=10) and to serum levels in normal controls (n=50). Serum BLyS levels in Waldenstrom’s patients (mean: 49.6ng/ml) as well as those in patients with lymphoplasmacytic lymphoma (mean; 46.7ng/ml) were significantly higher than normal controls (mean 12.6ng/ml).

In conclusion, we have demonstrated that malignant B cells from patients with Waldenstrom’s macroglobulinemia express the receptors for BLyS and can bind soluble BLyS. Furthermore, we have found that serum BLyS levels are significantly elevated in patients with Waldenstrom’s macroglobulinemia when compared to controls. Strategies to inhibit BLyS may potentially have significant therapeutic efficacy in Waldenstrom’s macroglobulinemia.
Postremission Treatment with Chemotherapy or Allogeneic Stem Cell Transplantation (Allo-SCT) in Adults with Acute Myeloid Leukemia (AML) -JALSG AML-97 Trial-.
Authors: Sakamaki Hisashi , Miyawaki Shuichi , Ohtake Shigeki , Yagasaki Fumiharu , Mitani Kinuko , Matsuda Shin , Kishimoto Yuji , Miyazaki Yasushi , Asou Norio , Ogawa Yoshiaki , Honda Sumihisa , Ohno Ryuzo
Published: October 2019

The aim of this study was to investigate the efficacy of allo-SCT as a postremission treatment in patients (pts) with intermediate/poor risk AML in first CR. Previously untreated pts aged15–64 years were eligible. Pts received standard induction therapy consisting of cytarabine (100 mg/m2 d1–7) and idarubicin (12 mg/m2 d1- 3). If the pts did not achieve remission after the first induction therapy, the same induction therapy was given once more. The pts who achieved CR were randomized into an intensified short-course postremission regimen group (arm A) and conventional JALSG postremission regimen group (arm B). Pts were also categorized into good, intermediate or poor risk groups by risk factors based on previous JALSG AML trials using univariate and multivariate analyses. The intermediate or poor risk pts <=50 years old with an HLA-matched sibling were assigned to the allo-SCT group. The overall survival rate (OS) and the disease-free survival rate (DFS) of the pts of this group were compared with those of intermediate or poor risk pts <=50 years old without an HLA-matched donor (non-transplant group), to adhere to the intention-to-treat principle.

Result: 809 pts were registered between December 1997 and July 2001. 789 pts were evaluable (median age: 45 years). 621 pts achieved CR (78.7%) after one or two courses of induction therapy. Os of 789 evaluable pts at five years and DFS of CR pts at five years were 40.8% and 35.5%, respectively. Of the 75 pts who were assigned to the allo-SCT group, 56 pts underwent allo-SCT (38 during CR1 and 18 after relapse). For the allo-SCT group, OS and DFS at 5 years were 51.8% and 43.1%, respectively. For the non-transplant group (n=95), OS and DSF at 5 years were 32.2% and 18.3%, respectively. DFS was significantly better in the allo-SCT group compared to the non-transplant group (p=0.005). OS was marginally better in the allo-SCT group (p=0.06).

In conclusion, our study showed that allo-SCT for pts with intermediate or poor risk is effective as a postremission treatment in adult AML.
Molecular Analysis of the VH Gene Usage in a Familial Case of Waldenstrom’s Macroglobulinemia.
Authors: Siakantaris Marina P. , Kyrtsonis Marie-Christine , Lilakos Konstantinos , Angelopoulou Maria K. , Vavourakis S. , Kontopidou Flora N. , Vassilakopoulos Theodoros P. , Kalpadakis Christina , Kokoris Styliani I. , Dimitriadou Evangelia M. , Kilindireas Constantin , Pangalis Gerassimos A.
Published: October 2019

Waldenstrom’s Macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone marrow and/or other tissues and the presence of a serum monoclonal IgM. The etiology of the disease is still unknown but the identification of family clusters may contribute to the understanding of its pathogenesis. In this study we present a family with 4 members affected by WM consisting of three generations; 2 sisters of the first generation were both suffering from WM. One of them presented to our section with mild anemia, a monoclonal serum IgM compound, 80% infiltration by lymphoplasmacytes in the bone marrow and the diagnosis of WM was made. Her sister was also suffering from WM but followed in another Centre. Our patient had two sons that at the age of 52 and 46 years presented a serum monoclonal IgM. At presentation, the first one had a monoclonal IgM value of 1680 mg/dl and a 40% BM lymphoplasmacytic infiltration with no other findings, signs or symptoms; he is regularly followed in our section for 30 months and his disease is stable. The second son, presented with a serum monoclonal IgM value of 886 mg/dl and a 10% BM infiltration by monoclonal lymphoplasmacytes and plasmacytes and was initially characterized as having an IgM-MGUS; while under follow-up examination only, he developed peripheral neuropathy with positive anti-Mag antibodies and improved with rituximab administration. Nor the mother or the sons had HCV antibodies. The first son has 3 children and the second two. Buccal and blood DNA was analysed for the IgH rearrangement by PCR. The PCR products after ligation and cloning were sequenced in an automated sequencer. Sequences obtained from each sample were aligned with germline sequences in the BLAST directory. No Ig rearrangements were detected in buccal DNA. Monoclonality was demonstrated in all affected members and in 2 healthy individuals of the third generation by PCR while immunophenotyping did not show evidence of disease in those two members. VH3 family was expressed at the highest frequency (80%). Different gene segments were recognized for the analysed sequences. D segments were attributed in all sequences. All affected and 1 healthy member had mutated VH genes. This is the first study of a family with WM consisting of three generations and reporting blood monoclonality of healthy individuals by molecular methods. The biologic value of the finding remains to be seen. Further study and a long follow-up is needed to clarify this issue.