bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloid Expansion after Insertional Activation of MDS1/EVI1, PRDM16 and SETBP1 in a Successful Chronic Granulomatous Gene Therapy Trial.
Authors: Schwarzwaelder Kerstin , Schmidt Manfred , Ott Marion G. , Stein Stefan , Glimm Hanno , Deichmann Annette , Fischer Marlene , Ball Claudia , Pilz Ingo , Schmidt Sonja , Prinz Claudia , Fessler Sylvia , Wissler Manuela , Klingenberg Silke , Hoelzer Dieter , Seger Reinhard , Grez Manuel , von Kalle Christof
Published: October 2019

Successful gene therapy trials of ADA-SCID and SCID-X1 demonstrated the curative potential of oncoretroviral gene transfer. Integration of the retroviral vectors used in these studies has been thought to be a random process but severe side effects in gene therapy and in vitro studies revealed preferred insertion of these vectors mainly around transcription start sites. In SCID patients proliferation advantage of gene corrected cells was one reason for the success of the trials, whereas in the most recent chronic granulomatous disease (CGD) gene therapy trial corrected cells do not have any selective advantage therefore the two patients received mild busulfan treatment before transplantation. High efficiency transduction and conditioning have helped in the successful correction of the patients. Peripheral blood granulocytes show a stable expression (>10%) of the transgene (gp91phox) in patient 1 (15 months post treatment) as well as in patient 2 (11 months post treatment). We reasoned that, unlike T cells, which have the capability to proliferate independent of their bone marrow progenitors, granulocytes more directly reflect the influence of retrovirus insertion, and should therefore allow to closely monitor clonal fate in vivo and its potential relation to vector insertion. To study the clonality of the corrected myelopoiesis, the long term activity of individual cell clones, and the distribution of integration sites in active cells we carried out high sensitive LAM-PCR. The highly polyclonal composition of transduced cells forming myelopoiesis caused the sustained expression of gp91phox. Individual clones carrying the transgene could be detected at multiple time points. To define whether corrected cells have a proliferation advantage due to their vector integration we started large-scale sequencing and mapping of involved insertion sites. We here present >700 unique mappable integration sites of the two treated patients. The distribution of the SFFV based retroviral vector integration sites in this trial turned non random 5 months after transplantation. Corrected long-term myelopoiesis expanded 3- to 5- fold in the two patients due to activating common integration sites (CIS) in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1, suggesting that these genes influence regulation of normal long-term hematopoiesis in humans. Our data indicate that the therapeutic benefit in this trial was activated through insertional side effects, therefore our findings have important implications in novel gene therapy approaches.
Variables Predicting for Poor Mobilization of Peripheral Blood Stem Cells (PBSC) in Newly Diagnosed Myeloma Patients, Treated on TT-2 Protocol.
Authors: Cavallo Frederica , Bolejak Vanessa , Cottler-Fox Michele , Hoering Antje , van Rhee Frits , Zangari Maurizio , Tricot Guido
Published: October 2019

Autotransplantation is now considered the standard of care for myeloma patients ≤ 65 years. Tandem autotransplants result in doubling of the percentage of patients alive and event-free at 7 years when compared to a single transplant (IFM-94). A minimum number of 2 x 106/kg CD34 cells is required per transplant for reliable engraftment. The aim of this study was to identify variables associated with the inability to collect sufficient numbers of PBSC for tandem transplants. 668 recently diagnosed patients were enrolled in our TT-2 protocol consisting of intensive induction with VAD, DCEP, CAD and DCEP followed by tandem transplants. PBSC were collected after CAD plus hematopoietic growth factors (either G-CSF or GM-CSF). 49 patients (7.5%) failed to collect ≥ 4 x 106/kg CD34 cells (poor mobilizers) and the characteristics of these patients were compared to those of 77 patients who collected > 30 x 106/kg CD34 cells (excellent mobilizers) using chi-square tests. A multivariate analysis was performed using a stepwise logistic regression. The median number of CD34 cells collected in the group of poor mobilizers was 2.1 x 106/kg (range 0–3.9) in a median of 3 days (range 1–9). In the group of excellent mobilizers the median number of CD34 cells was 38.7 x 106/kg (range 30.1–86) in a median of 2 days (range 1–4). Baseline and mobilization variables significantly different between these 2 groups are listed in Table 1. On multivariate analysis duration of cytopenia (platelets < 50,000/μl and/or neutrophils < 1,000/μl) > 7 days (OR: 27; p<0.0001), randomization to thalidomide (OR 4.5; p=0.007), age ≥ 65 years (OR: 4.96; p=0.01), female gender (OR: 3.62; p=0.02), and neutropenic infection requiring intraveneous antibiotics (OR 3.8; p=0.03) were independently associated with poor collection. No differences in event-free (p= .68) or overall survival (p=.73) were observed between the excellent and poor mobilizers. These data confirm our previous observations of inferior collection in older patients and in those receiving thalidomide as part of upfront therapy. The duration of cytopenia after chemotherapy is a good reflection of patient’s stem cell reserve, while administration of broad spectrum antibiotics prior to collection has been shown to be myelosuppressive. The negative impact of female gender on the ability to collect well is new.

Baseline variables Poor mobilizers (%) Excellent mobilizers (%) P-value Female gender 59 26 <0.001 Age ≥ 65 35 13 0.004 Hb<10g/dl 45 21 0.005 B2M ≥4mg/L 39 17 0.006 P1<150,000/ μl 20 8 0.04 Mobilization variables Cytopenia > 7 days 59 5 <0.001
Low-Level Expression of the Tumor Suppressor Bim in CML Cells: Role of BCR/ABL, Delineation of Underlying Signaling Pathways, and Re-Expression by Imatinib, AMN107, and Proteasome Inhibitors.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Vales Anna , Derdak Sophia , Pickl Winfried F. , Selzer Edgar , Deininger Michael , Druker Brian J. , Sillaber Christian , Valent Peter
Published: October 2019

Chronic myeloid leukemia (CML) is a myeloproliferative disease in which BCR/ABL enhances survival of leukemic cells through modulation of pro- and anti-apoptotic molecules. Recent data suggest that pro-apoptotic Bim plays a role as a tumor-suppressor in myeloid cells, and that leukemic cells express only low amounts of this death activator. In the current study, we have investigated expression of Bim in primary CML cells and in the CML cell lines K562 and KU812, and in Ba/F3 cells inducibly expressing BCR/ABL on exposure to doxycycline (TonB.210-X). As assessed by Northern- and Western blotting, primary CML cells were found to express significantly lower amounts of Bim mRNA and Bim protein compared to normal bone marrow cells. The BCR/ABL-inhibitors imatinib (Novartis Pharma AG) and AMN107 (Novartis Pharma AG) were found to promote Bim expression in CML cells at pharmacologic concentrations. Correspondingly, BCR/ABL was found to down-regulate expression of Bim in TonB.210-X cells. The BCR/ABL-induced decrease in expression of Bim in leukemic cells was found to be a post-transcriptional event that depended on signaling through MEK, and was abrogated by the proteasome-inhibitor MG132. Interestingly, MG132 was found to up-regulate Bim-expression and to suppress the growth of Ba/F3 cells containing either wild-type BCR/ABL or various imatinib-resistant mutants of BCR/ABL including the T315I mutant that is resistant to all currently available ATP-competitive tyrosine kinase inhibitors (IC50: 30–100 nM). To confirm the role of Bim as a tumor suppressor in CML, a Bim specific siRNA was transfected into K562 cells. This siRNA was found to counteract imatinib- and MG132-induced cell death. In conclusion, our data identify BCR/ABL as a Bim-suppressor in CML cells and suggest, that re-expression of Bim by proteasome inhibition or by targeting of signaling pathways downstream of BCR/ABL may be an attractive therapeutic approach in imatinib-resistant CML.
Evaluation of Ph+/CD34+ Residual Cells in Chronic Myeloid Leukemia Patients after Long Lasting Treatment with Imatinib Mesylate.
Authors: Bocchia Monica , Abruzzese Elisabetta , Ippoliti Micaela , Calabrese Simona , Gozzetti Alessandro , Pirrotta Maria Teresa , Crupi Rosaria , Tozzuoli Daniela , Lauria Francesco
Published: October 2019

Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES<1% false positive; bcr-abl Dual Fusion 8–10% false positive) The clinical significance of these data as well as the role of this cell target to monitor minimal residual disease in CML needs to be evaluated on a larger serie of patients.
Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.
Authors: Saito Masumichi , Phan Ryan T. , Morse Herbert C. , Pasqualucci Laura , Dalla-Favera Riccardo
Published: October 2019

Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.
Polymorphisms and Lack of or Aberrant Expression of HLA Class I and II May Influence Antigen Presentation in Classical Hodgkin Lymphoma.
Authors: Diepstra Arjan , Poppema Sibrand , te Meerman Gerard , Niens Marijke , Nolte Ilja , Vellenga Edo , van Imhoff Gustaaf , Kluiver Joost , van den Berg Anke
Published: October 2019

In a previously performed population-based genetic screening analysis we found that polymorphisms in the HLA class I region are specifically associated with EBV-positive classical Hodgkin lymphoma(cHL)(Diepstra et al., Lancet2005;365:2216). We now studied the expression of HLA class I and HLA class II in this population (n=450) by immunohistochemistry. HLA-B/C, HLA-G, beta-2-microglobulin and HLA-DP/DQ/DR staining was performed on formalin fixed paraffin embedded tissue sections, and HLA-DM and HLA-DO staining on frozen sections. Hodgkin-Reed Sternberg (HRS) cells showed cell surface expression of HLA class I in 37.5% (159/424) of cHL tumors and expression was observed predominantly in EBV-positive cases (73%; 103/141). Expression of HLA-G, a molecule known to enable evasion from natural killer cell responses, was found in 67% of the cases and was associated with absence of MHC class I and EBV. Only 58% of cHL cases showed HLA class II positive staining in more than 50% of the HRS cells (224/387). The non-classical HLA class II protein HLA-DM, essential for the loading of HLA class II with antigenic peptides was reduced or absent in HRS cells in 7 out of 10 HLA class II positive cases, whereas HLA-DO, the natural repressor of HLA-DM, was not or minimally expressed. Impaired expression of HLA-DM may prevent presentation of antigens at the level of individual HRS cells. Reanalysis of the genotyping data revealed an association of HLA class I positive cases with the known susceptibility polymorphisms in the HLA class I region. However, this association was stronger for the EBV-positive cases than for the HLA class I positive cases. Within the group of EBV-positive cHL patients, the same predisposing alleles were present in both HLA class I negative and positive cases, suggesting that the negative cases probably did express HLA class I during early pathogenesis. Finally, in cases with HLA class II expression on the HRS cells, a genetic association with the HLA class II region was found. These changes may influence (EBV) antigen presentation by HRS cells in cHL.
Prediction of Relapse Risk by Day 100 BCR-ABL Quantification after Allogeneic Stem Cell Transplantation for Chronic Myeloid Leukaemia.
Authors: Rubio Marie T. , Asnafi Vahid , Delabesse Eric , Damaj Gandhi , Dhedin Nathalie , Varet Bruno R. , Buzyn Agnes , Macintyre Elizabeth A.
Published: October 2019

CML relapse after allogeneic SCT is a relatively frequent situation and is clearly correlated to disease status, time from diagnosis to transplant and T cell depletion. Defining other risk factors could help modulate post transplant immunosuppression. We evaluated the value of early minimal residual disease (MRD) quantification to predict relapse of CML patients receiving standard allogeneic SCT. MRD was analysed by RQ-PCR at Day 100 in 38 patients with a follow up after transplant > 1 year and was expressed as BCR-ABL/ABL, qualified by objective evaluation of RNA amplifiability relative to local normal values for MRD samples (the Quality Index or QI). This QI allows objective evaluation of the degree of correction for positive results and modification of the limits of detection for negative results. Thirty six of 38 patients received conventional conditioning regimen with either BU/CY or TBI/CY and classical prevention of GVHD based on cyclosporin and methotrexate. The median time from diagnosis to transplant was 21 months and median follow up of the cohort was 76.8 months. Patients were allocated to two groups according to their Day 100 RQ PCR level. We compared the characteristics and evolution of the 14 patients with a high MRD level (Day 100 RQ PCR >10−4) to that of the 24 patients with a low MRD level (Day 100 RQ PCR <10−4). There were no significant differences in terms of disease status at transplant, median age at transplant, time from diagnosis to transplant, type of conditioning regimen, source of stem cells, sex mismatch, grade of acute GVHD or incidence of chronic GVHD between the two groups. We show that Day 100 BCR-ABL transcript levels >10−4 by RQ-PCR represents an independent risk factor of relapse after conventional non T cell depleted SCT. These data should favour risk-adapted post transplant immunosuppression based on a single time point early evaluation of MRD.
Transplant Outcomes and Stabilization of Neurological Function in Young Children with MPS III (Sanfilippo Syndrome) after Unrelated Donor Umbilical Cord Blood Transplantation.
Authors: Lakshminarayanan Sonali , Szabolcs Paul , Prasad Vinod K. , Parikh Suhag , Allison June , Wood Susan J. , Escolar Maria , Kurtzberg Joanne
Published: October 2019

Between September 1998 and June 2004, 15 children, ages 10 – 55 months, 60% males, 14 Caucasian, 1 mixed racial background, underwent unrelated donor umbilical cord blood transplantation for treatment MPS III (Sanfilippo Syndrome, types A or B) Grafts were mismatched at 0 (n = 1), 1 (n = 10) or 2 (n = 4) HLA loci. All children were prepared for transplant with busulfan 40mg/m2/dose PO or 1mg/kg/dose IV targeting a Css of 600–900ng/ml), cyclophosphamide 50mg/kg/dose daily x 4, and equine ATG 30mg/kg/dose daily x 3. Cyclosporine and solumedrol were used for prophylaxis against GvHD. Engraftment with donor cells occurred in 10/15 patients, although one additional patient engrafted after a second transplant. Five patients, including the one transplanted a second time, died within 8 months of transplant of graft failure with respiratory failure and viral infections (Adenovirus, HSV). Six patients developed mild to moderate acute GvHD and 4 patients had limited (2) or extensive (2) chronic GvHD. All children were tested with baseline neurophysiologic, imaging and neurocognitive tests. These were then followed in surviving children every 6 months for 2 years and yearly thereafter. All children demonstrated deficits in hearing and mild to moderate developmental delays at the time of initial evaluation. MRIs showed varying degrees of volume loss generally increasing with the age of the patient at initial evaluation. EEGs were normal in the younger patients, but showed slowing and signs of generalized encephalopathy in the older patients. Two patients developed hydrocephalus requiring VP shunt placement within 1 year of transplant and one patient experienced subdural hematomas after minor head trauma. MRIs of all surviving patients stabilized for the first 4 years post transplant. One patient, now almost 5 years post transplant showed volume loss on the latest study. Patients transplanted at <3 years of age appear to have better neurocognitive outcomes than those over 3 since the younger patients continue to learn, albeit at a slower pace than typically developing children, while the older patients appear to have stabilized at their pretransplant level of functioning. All patients, however, show improvement in adaptive behaviors and general well being. Parents report less aggression, autistic like behaviors and improved sleep. We conclude that UCBT has a role in the therapy of children with MPS III. Effects of transplantation under the age of 2 years should be explored in a larger group of patients.
Mutational Status of Chronic Lymphocytic Leukemia (CLL) Determines Clonlal Evolution in Classical Hodgkin’s Lymphoma Variant of Richter’s Transformation.
Authors: Chen Wayne W. , Raffeld Mark , Pittaluga Stefania , Sorbara Lynn , Jaffe Elaine S.
Published: October 2019

Approximately 5% of CLL patients transform during their clinical course into a high grade lymphoma, referred to as Richter’s transformation, most commonly DLBCL, rarely (0.4%) classical Hodgkin’s lymphoma (cHL). Most cases of cHL transformation are Epstein-Barr virus positive and are often related to fludarabine treatment. Only a handful of cases in the literature, most of them EBV positive, have been studied for their clonal relationship with the pre-existing CLL. EBV negative cHL cases in CLL patients are rare and have not been well investigated. We have studied 2 EBV positive and 8 EBV negative cHL cases using single cell laser microdissection combined with PCR analysis for the IgH gene rearrangement. ZAP-70 immunohistochemical staining was used as a surrogate for Ig gene mutational status. Three cases of EBV negative and 1 EBV positive cHL case arose in ZAP-70 negative (mutated) CLL while 5 EBV negative and 1 EBV positve cHL cases arose in ZAP-70 positive (unmutated) CLL. Surprisingly, regardless of the EBV status, all 4 cHL cases from ZAP-70 negative (mutated) CLL shared the same clonal origin with the pre-existing CLL. In contrast, all 6 cHL cases from ZAP-70 positive (unmutated) CLL exhibited a different clonal origin. These results demonstrate that the mutational status of the CLL, and not the EBV status of the cHL, determines the nature of the evolution to cHL. Additional EBV positive cHL cases are currently under investigation. Passage through the germinal center may predict the ability of a CLL B-cell to transform to a Hodgkin’s cell.
Highly Efficient Gene-Transfer into Chronic Lymphocytic Leukemia Cells Using Adenovirus Type 35 Genetic Vectors.
Authors: Whitacre David C. , Hedjran Farah , Schmidt-Wolf Ingo , Prussak Charles , Reid Tony , Kipps Thomas J. , Whitacre David
Published: October 2019

Adenovirus (Ad) vectors have been used to introduce genetic material into mammalian cells for gene-expression studies and/or gene therapy. Ad serotypes 2 and 5, the most widely used Ad virus vectors, belong to the group C adenoviruses, which bind to the coxsakie/adenovirus receptor (CAR) present on cells that are most susceptible to virus infection. Because lymphoid cells generally do not express CAR, high-titer virus and optimal conditions are required to infect lymphocytes, including the neoplastic cells of most lymphoid malignancies. Ad35, on the other hand, is a member of the group B adenoviruses that infect cells by binding CD46, a receptor expressed on most cell types. We found chronic lymphocytic leukemia B cells (n = 10) expressed high-levels of CD46 by flow cytometry, whereas none of the CLL samples expressed CAR by immunoblot analyses. We examined and compared the relative susceptibility of CLL cells to infection by Ad5 versus Ad35 vectors at various multiplicity of infection (MOI). These studies revealed that CLL cells were 100-fold more sensitive to infection with Ad35 than with Ad5. To examine whether this improved tropism of Ad35 for CLL cells was due to the adenovirus receptor for CD46, we examined the relative susceptibility of CLL cells to infection by Ad5 vectors that had been engineered to express the Ad35 knob fiber protein (Shayakhmetov DM, et al., J Virol 2000, vol 74, pp. 2567-83) responsible for binding CD46, designated Ad5F35. Titration studies evaluating for expression of a reporter transgene in infected CLL cells (e.g. the gene encoding green fluorescence protein (GFP)), found that Ad5F35 vectors also were > 100-fold more effective than Ad5 vectors at transducing CLL B cells. These studies reveal that Ad5F35 is a highly efficient vector for transducing CLL cells, a quality that should make these vectors better suited than Ad5 virus vectors for studies requiring gene transfer and/or gene therapy of this and related lymphoid malignancies.
Quality of Life in the LRF CLL4 Trial.
Authors: Else Monica , Smith Alastair G. , Hawkins Kim , Crofts Shirley , Course Margaret , Richards Susan , Catovsky Daniel
Published: October 2019

The UK CLL4 trial randomised 777 previously untreated patients between 1999–2004 to chlorambucil (Chl) or fludarabine +/− cyclophosphamide (F v. FC). Other trial endpoints are reported elsewhere in this meeting (Catovsky et al). The current study seeks to determine whether there is any difference in the quality of life (QoL) of patients according to treatment arm; and whether QoL correlates with baseline patient characteristics and/or with treatment outcomes. All patients were asked to complete the validated EORTC QLQ-C30 (version 2.0) at start of therapy (baseline), at 3, 6 and 12 months, and annually thereafter. Scores (0–100) within each of 15 different measures of QoL (5 functional scales, 3 symptom scales, 1 global health/QoL scale, and 6 single items) were calculated at each point in time (currently up to 1 year), and analysed according to the EORTC QLQ-C30 Scoring Manual. Statistical significance was determined by the use of ANOVA. Patient compliance was high: 599 questionnaires were completed at baseline, 557 at 3 months, 559 at 6 months and 470 at 12 months, respectively 77%, 74%, 77% and 78% of those who had so far reached the time-period and remained alive. Analysis of baseline and 12 month QoL assessment by treatment group and response is presented. QOL results were the same for each treatment group at baseline and at 12 months (Table 1). Quality of response to treatment correlated with QoL at 12 months (Table 2). With treatment approaches having potentially different risk and side effect profiles, and perhaps limited impact on overall survival, QOL forms an important component of treatment outcome assessment. Further analysis of QoL parameters (in particular their relationship to prognostic factors and further follow-up) will be forthcoming within the next year.

Table 1: Mean QoL Scores by Treatment Group* Functioning Scales Physical Role Emotional Cognitive Social Global Health No. B 12M B 12M B 12M B 12M B 12M B 12M Abbreviations: B = baseline; 12M = 12 months. Chl 175 81 78 74 75 75 78 83 82 79 77 66 69 F 92 82 82 72 74 80 81 82 82 80 79 64 68 FC 94 80 77 77 73 78 81 85 82 82 76 66 68 361 Table 2: Mean QoL Scores by Response to Treatment* Functioning Scales Physical Role Emotional Cognitive Social Global Health No. B 12M B 12M B 12M B 12M B 12M B 12M Abbreviations: B = baseline; 12M = 12 months; CR/NPR = complete/nodular partial response; PR = partial response; NR/PD = no response/disease progression. P-values compare response groups with respect to the change in scores from baseline to 12 months. (* Only those patients are shown in the Tables for whom data is available at both time-points.) CR/NPR 159 84 84 76 80 78 84 86 85 82 83 67 73 PR 125 79 76 73 73 78 78 82 79 80 77 63 68 NR/PD 56 77 74 71 61 74 74 81 79 74 65 65 60 340 p=0.02 p=0.03 p=0.002
Follow-Up Results of a Prospective, Multicenter, Open-Label Study of Oral Fludarabine Phosphate in Patients with Previously Untreated B-CLL.
Authors: Rossi Jean-Francois , Van Hoof A. , De Boeck K. , Johnson S. A. , Bron D. , Foussard C. , Lister T. A. , Kramer M. H.H. , Littlewood T. J. , Marcus R. E. , Deconinck E. , Guibon O. , Montillo M. , Tollerfield S. M.
Published: October 2019

The IV formulation of fludarabine phosphate is an effective treatment in patients with B-cell chronic lymphocytic leukemia (B-CLL), yielding overall response rates of 60% to 80%. An oral formulation of fludarabine phosphate has been developed. In a previously published multicenter, open-label, phase II clinical trial, 81 previously untreated B-CLL patients received 10-mg tablets of fludarabine phosphate (Fludara® oral) 40 mg/m2/day for 5 days, repeated every 4 weeks. The primary endpoint of the trial was response rate, and secondary endpoints included safety and quality of life assessments. Of 81 patients (mean age, 61.2 years; range, 30–75 years) with previously untreated B-CLL, 81.5% were classified as Binet stage B or C. The overall response rate (complete response [CR] + partial response [PR]) using National Cancer Institute (NCI) criteria was 80.2% (12.3% CR and 67.9% PR) and the median time to progression was 841 days (range, 28–1,146 days) (Rossi JF, et al. J Clin Oncol2004;22:1260–1267). The most frequently reported grade 3/4 adverse event was myelosuppression: WHO grade 3/4 hematologic toxicities included granulocytopenia (32.1%), anemia (9.9%), and thrombocytopenia (4.9%). This analysis reports on the long-term follow-up of this cohort during the period from November 2001 to November 2004. Of the 74 patients eligible for the survival analysis, 61 were also assessed for duration of response using NCI criteria: 9 CR (14.8%) and 52 PR (82.2%). During the 3-year follow-up period, 22 (29.7%) patients did not progress. For those who progressed, median time to progression was 29.7 months, and median duration of response was 22.9 months. In 41 (80.4%) of these patients, an increase in circulating lymphocytes was reported as evidence of disease progression. In 23 patients (45.1%), an increase in the sum of the products of at least 2 lymph nodes and/or appearance of new palpable nodes was reported as evidence of disease progression. During the indicated follow-up period, 37 patients (50%) received subsequent treatment. Twelve patients (16.2%) died during the follow-up period: 7 patients (58.3%) due to disease progression, 3 patients (25.0%) due to adverse events, and 2 patients (16.7%) due to other causes. Results from this study suggest that oral fludarabine phosphate is clinically effective and well tolerated by patients with previously untreated B-CLL. Moreover, these data demonstrate that oral fludarabine phosphate achieves response rates and duration of response comparable to those achieved with first-line fludarabine phosphate IV therapy.
Genetic Aberrations and Response to Fludarabine as First Line Treatment in a Serie of B-CLL Patients.
Authors: Giraldo Pilar , Rubio-Martinez Araceli , Recasens Valle , Valganon Mikel , Latre Paz , Rubio-Felix Daniel , Calasanz Maria J. , Odero Maria D.
Published: October 2019

Background: Genetic factors have been established in the recent years as the most important independent predictors of disease progression and survival in patients with B-chronic lymphocytic leukaemia (B-CLL). Fludarabine is an approved first-line treatment for B-CLL, which achieves superior remission rates than traditional therapies. The drug is equally effective in early and advanced disease and in younger and elderly patients with B-CLL but different results have been found in patients with cytogenetic aberrations as methylation in the TP53 promoter

Aims: To evaluate the response to Fludarabine as first-line therapy in B-CLL patients with advanced stages.

Patients and methods: In the study 55 B-CLL patients were enrolled. The diagnosis was established based on standard morphologic and immunophenotypic criteria, and classified on high clinical risk category Rai stage III/IV treated with Fludarabine (25 mg/m2 daily for 5 days IV in a 28-day cycle) as first-line therapy from January 00 to December 03. Response was evaluated after 4 cycles of therapy and in those patients which had not achieved complete remission (CR) two additional cycles were administrated. Criteria for response were established using the revised 1996 NCIWG. FISH studies were performed using the probes LSI p53 (17p13), LSI D13S25 (13q14), CEP 12 and ATM gene (11q22). The sequences of the IGVH genes were determined in cDNA of the patients. Responses to therapy, time to progression disease and overall survival were available to analyse in 41 patients. Association was studied using contingence tables followed by Fisher exact test and to evaluate survival by acturial method of Kaplan Meier and Cox regression.

Results: Mean age 64 (36.9–83.9), female 53.7%. 75% of patients presented genetic aberrations associated with worse prognosis: del (17p) and/or del (11q) (45%). No-mutated IGVH genes (57%). Response: 83.5% of patients achieved response (CR 47.8%). Related to genetic aberrations the responses were: 55.5% in patients with del(17p), 75.0% del(11q), 100% +12, 88.8% del(13q) and 100% of patients without genetic alterations showed response to Fludarabine. The 48.8 % of patients developed progression of disease with mean duration of the response of 15.1 months. Related to genetic aberrations the progression was over 81% of patients with genetic aberrations versus 55.5% without genetic alterations (p=0.044). The overall survival was 68.0 months. 34.1% of patients are alive in CR, 36.6% are alive with stable disease 7.3 % are alive with disease in progression and 22.0 % have died.

Conclusions: A high response to Fludarabine in first line therapy was observed, however only 55.5% of patients with del(17p) showed response. Patients with genetic aberrations had higher significant rate of progression in comparison with those containing normal genotypes.
A Reduced Capacity To Generate Activated Protein C and the Role of PAI-1 Deficiency on Coagulation Activation and Fibrin Formation during Murine Influenza Pneumonia.
Authors: Keller Tymen , van Wissen Matthijs , van der Sluijs Koen , van Gorp Eric , Meijers Joost , van der Poll Tom , Buller Harry , Brandjes Dees , Levi Marcel , Gerdes Victor
Published: October 2019

Background: Recent studies have shown that influenza activates coagulation in vitro. We aimed to study influenza and post-influenza pneumonia induced hemostatic changes in vitro and to study the role a reduced capacity to generate activated protein C (APC) and PAI-1 deficiency on the prothrombotic state.

Methods: C57BL/6 mice were inoculated with influenza or PBS (control). After 14 days these mice received S. pneumoniae. PAI-1−/− , TM pro/pro and control mice were inoculated with influenza. Controls for the influenza-infected PAI-1−/− and TM pro/pro mice were C57BL/6 mice and wild-type littermates, respectively. TATc, PAI-1, D-dimer levels, fibrin deposition and pro-inflammatory cytokines were studied on day 0, 2, 4, 8, 14, 15 and 16.

Results: Influenza-infected C57BL/6 mice showed a dose dependent increase in TATc, PAI-1 and fibrin deposition compared to non-infected controls. Median TATc levels increased from 5.3 at baseline to 12.8 ng/ml at day 4 and PAI-1 levels increased from 4.0 to 16.0 IU/ml (both p<0.05). TATc and PAI-1 levels remained unchanged in non infected controls. Mice recovered from influenza infection were highly susceptible to pneumococcal pneumonia as reflected by a 3-fold increase in cytokines compared to non-influenza infected controls. In addition, an increase in TATc and PAI-1 levels was seen (both p<0.05). Furthermore, influenza-infected TM pro/pro mice showed higher TATc levels (16.2 vs. 12.0 ng/ml, p<0.05) and higher D-dimer levels (2.6 vs. 2.0 μg/l, p<0.05) compared to infected controls 4 days after infection. In influenza-infected PAI-1−/− mice TATc levels were similar as in infected controls, but D-dimer levels were increased (4.0 vs. 2.2 μg/l, p< 0.05).

Conclusion: This study demonstrates that influenza and post-influenza pneumonia exert a prothrombotic state in vivo. Mice with a reduced capacity to generate APC show increased thrombin generation. PAI-1 deficiency does not effect coagulation activation but results in a hyperfibrinolytic state.
Toll-Like Receptor Triggering Modulates Factor VIII-Specific Immune Memory in Murine Hemophilia A with Factor VIII Inhibitors.
Authors: Allacher Peter , Hausl Christina , Ahmad Rafi U. , Schwarz Hans Peter , Turecek Peter L. , Reipert Birgit M.
Published: October 2019

The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in the treatment of hemophilia A patients with FVIII products. Immune Tolerance Induction (ITI) therapy using long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and induce long-lasting immune tolerance. It is a common observation that infections, particularly central venous catheter infections during ITI cause a rise in anti-FVIII antibody titers that can prolong the course of ITI or possibly even lead to failure of ITI. Based on this observation, we asked the question whether microbial components derived from viruses or bacteria modulate the re-stimulation of FVIII-specific immune memory and disturb the recently described inhibition of memory-B-cell-re-stimulation by high doses of FVIII (Hausl et al.: Blood2005; in press). Microbial components are recognized by toll-like receptors (TLRs) that serve as an important link between innate and adaptive immunity. TLRs can discriminate various microbial components such as lipopeptides derived from bacteria or zymosan derived from yeast (recognized by TLR1/2 or TLR2/6), double-stranded RNA derived from viruses (recognized by TLR3), lipopolysaccharide (LPS) derived from gram-negative bacteria (recognized by TLR4), flagellin derived from bacterial flagella (recognized by TLR5), single-stranded RNA derived from viruses (recognized by TLR7/8) or bacterial DNA containing the unmethylated CpG motif (recognized by TLR9). We analyzed the re-stimulation of FVIII-specific memory-B cells using a murine model of hemophilia A as described previously (Hausl et al.: Blood2004; 104:115–22; Hausl et al.: Blood2005, in press). The following TLR ligands were tested: zymosan for TLR2 (0.1–10,000 ng/ml), poly I:C for TLR3 (1.0–50,000 ng/ml), LPS for TLR4 (0.1–10,000 ng/ml), Flagellin for TLR5 (0.01–1,000 ng/ml), Loxoribine for TLR7 (1.0–50,000 ng/ml) and CpG oligonucleotides for TLR9 (0.1–10,000 ng/ml). Our results indicate that none of the TLR ligands at the concentrations tested induced a significant re-stimulation of FVIII-specific memory B cells in the complete absence of either FVIII or T cells. However, ligands for TLR3, TLR4, TLR7 and TLR9 were able to disturb the inhibition of memory-B-cell-re-stimulation by high doses of FVIII and amplified the re-stimulation induced by low doses of FVIII substantially.

We conclude that triggering of TLRs by microbial components that are present during infections amplify the re-stimulation of FVIII-specific memory B-cells induced by low doses of FVIII and disturb the inhibition induced by high doses of FVIII.
Plasma Levels of High Mobility Group Box Protein in Patients with Organ Failure, Disseminated Intravascular Coagulation or Poor Outcome.
Authors: Wada Hideo , Nobori Tsutomu , Yamada Shingo , Maruyama Ikuro
Published: October 2019

High Mobility Group Box chromosomal protein 1 (HMGB1) is a nuclear DNA-binding protein acting as a proinflammatory cytokine when released in the extracellular space from necrotic cells, activated macrophages and dendritic cells. HMGB1 acts on a specific receptor, RAGE (receptor for advanced glycation end-products), and induces prolonged inflammation, organ failure, septicaemia and death. The aim of the study was to determine the diagnostic value of plasma HMGB1 concentration and its role in the development of organ failure in patients with disseminated intravascular coagulation (DIC).

Plasma HMGB-1 levels were measured in patients with suspected DIC by ELISA and their relationships with DIC, organ failure and clinical outcome were determined. The study group included 201 patients suspected of having DIC (79 females and 122 males, age; 52.1±16.5 years, mean±SD).

The study group represented all such patients admitted to the Second Department of Internal Medicine and Intensive Care Unit (ICU) of Mie University Hospital between January 1, 2002 and December 31, 2003. The underlying diseases were infectious diseases (n=40), hematopoietic diseases (n=58), solid cancers (n=16), trauma (n=21), aneurysm (n=12), autoimmune diseases (n=8), liver diseases (n=7), post-operation (n=32) and other diseases (n=7).

Results: Plasma HMGB1 was below the detection limit in normal subjects, but moderately elevated in patients with infectious diseases (4.54 ± 8.18 ng/ml, mean±SD), malignancies (2.15 ± 5.34 ng/ml), and traumas (6.47 ± 13.13 ng/ml). DIC was associated with significantly high plasma HMGB1 (14.05 ± 12.56 ng/ml) in these patients. The highest HMGBI levels were in patients with organ failure (8.29 ± 10.99 ng/ml) and non-survivors (16.58 ± 11.01 ng/ml). HMGB1 plasma levels correlated with the DIC score and sepsis-related organ failure assessment (SOFA) score. Plasma levels of HMGB1 were significantly higher in patients with PAI-I of >50 ng/ml (3.66 ± 6.36 ng/ml) than in those with PAI-I <50 ng/ml (1.38 ± 2.97 ng/ml) (p< 0.05) but there was no significant difference in HMGB1 levels between patients with TNF-a of >1.5 (3.04 ± 5,56 ng/ml) and those with less than 1.5 pg/ml (2.44 ± 5.36 ng/ml).

Our data suggest that HMGB-1 is a potentially suitable prognostic marker of OF or DIC.
Thrombophilia in Patients with Occlusions of Vascular Bypass Grafts.
Authors: Klamroth Robert , Orlovic Marija , Rimpler Hartmut , Landgraf Helmut
Published: October 2019

Background: Causes of vascular bypass occlusions are surgical failure, hyperplastic intima, progression of arterial occlusive disease and thrombophilic disorders. The aim of this registry was to determine the prevalence of thrombophilic risk factors in patients with occlusions of arterial bypass grafts of the lower extremities.

Patients and Methods: From 2001 to 2004 we studied 81 consecutive patients (pts) with a mean age of 60,5 years. 64/81 pts showed occlusions of arterial prosthetic grafts and 17/81 pts occlusions of venous grafts of the lower extremities. Bypass occlusion occured 7,1 months (mean value) after primary operation. 68/81 patients had concomittent risk factors for arteriosclerosis like arterial hypertension, diabetes, hypercholesterinemia or smoking.

The laboratory testing for thrombophilia (performed at least 4 weeks after the bypass occlusion) included antithrombin, protein C, protein S, homocysteine, factor V-G1691A-mutation, prothrombin-G20210A-mutation, lupus anticoagulant, anti-cardiolipin-antibodies, lipoprotein (a), factor VIII, plasminogen activator inhibitor (PAI) and induced platelet aggregation in platelet rich plasma with different concentrations of ADP to evaluate hyperreactive platelets.

Results: Thrombophilia screening revealed abnormalities in 65/81 pts. 36/81 patients showed combined defects. Screening for antiphospholipid antibodies (LA and/or ACA) revealed pathologic values in 31/81 pts, heterozygous factor V- mutation was present in 8/81 pts, heterozygous prothrombin-G20210A-mutation in 2/81. 20/81 pts had elevated homocysteine levels, 16/81 pts elevated lipoprotein (a) levels, 19/81 pts had a persistent factor VIII elevation higher than 200% and 14/81 pts had PAI-levels more than two fold higher than the upper normal value. There was 1/81 patient with protein S deficiency and 20/81 pts had abnormal platelet aggregation fulfilling the criteria of hyperreactive platelets.

Conclusions: In patients with occlusions of arterial bypass grafts of the lower extremities we found a high prevalence of hereditary and acquired thrombophilic risk factors. There seems to be a causal relation between arterial bypass occlusions and especially antiphospholipid antibosies. The evaluation of thrombophilic risk factors in patients with arterial bypass occlusions could lead to an improved antithrombotic treatment after surgical revasculation.
Murine Severe Combined Anemia and Thrombocytopenia (SCAT), a Model for Human Juvenile Cyclic Congenital Amegakaryocytic Thrombocytopenia (cyCAMT).
Authors: Barker Jane E. , Peters Luanne L.
Published: October 2019

Congenital inherited thrombocytopenias (CTP) are rare and often misdiagnosed in patients. SCAT is a heritable, recessive murine bleeding disorder that shares characteristics with some of the human CTPs. The disease is: present at birth, episodic, lethal unless treated and not immune-mediated. A platelet-specific antibody present in plasma of scat/+ mothers and their affected scat/scat pups initiates a short-lived platelet decrease when injected into adult +/+ mice and binds a 110 kD platelet protein on Western blots. The antibody is not responsible for the phenotype. scat/scat pups are anemic and thrombocytopenic at birth regardless of whether they and/or their scat/+ mother are immunodeficient and antibody free. Hematopoietic stem cell transplantation transmits the SCAT disease to normal mice. scat/+ x scat/+ matings generate only ½ the expected number of scat/scat neonates. Despite the deficit, the total number of live newborns from scat/+ x scat/+ matings is the same as from +/+ x +/+ matings. These observations suggest that the disease is incompletely penatrant or that scat/scat fetuses die in utero. In utero analyses indicate that the expected number of bruised scat/scat fetuses die near birth, but also an additional 24% of the total fetuses present are resorbed after implantation. Identification of the resorbed fetuses is impossible by molecular analysis, so the normal, live born survivors (?/+) were genotyped as adults by mating to a known scat/+ adult of the opposite sex. Results are consistent with early in utero loss of the scat/+ pups. Surprisingly, the scat/+ mothers and all their fetuses are platelet deficient. Platelets are more severely decreased in the live, bruised fetuses. Clearly, the scat/+ mother is more fecund and the scat/+ as well as the scat/scat fetuses are at risk. We hypothesize there is transplacental transfer of a thrombocytopenia-initiating factor between the fetuses and their mother in both directions. The putative initiator is produced only when a wild-type gene is present since scat/scat parents surviving the cyclical platelet deficiency can mate but give birth to unaffected scat/scat pups. The early onset, recessive inheritance, cyclical nature, defective stem cells and rapidly progressing marrow hypoplasia in scat/scat mice are reminiscent of juvenile cyCAMT. c-mpl, responsible for non-cyclical CAMT, is not in the scat interval on mouse Chr. 8. The scat gene must play an unpredicted role in maintaining stem cells and their progeny and could be a candidate gene for cyCAMT.