bluuebunny/crossref_metadata_2025 huggingface dataset:
Prospective Clinical and Epidemiological Evaluation of rHuEPO in the Routine Care of a Network of Hematological Centers.
Authors: Barbui Tiziano , Romero Marilena , Delaini Federica , Sasso Elisa , Tognoni Gianni , Cazzola Mario
Published: October 2019

The expected effects of rHuEPO treatments on the quality of life of patients exposed to chemotherapeutic regimens have favored its widespread clinical utilization, even in the absence of a solid consensus on the criteria for assuring an optimal cost-effectiveness and safety profile.

A prospective multicenter outcomes-oriented observational protocol adopted by a network of 29 hematological centers has explored specifically the relationship between the yield of rHuEPO, therapy and the levels of endogenous EPO in a cohort of 478 consecutively diagnosed patients (55% males), with NHL (43%), CLL (16%), MM (41%), in first (287, 60%) and successive (191, 40%) chemotherapy line. Over the 12 months follow-up, 4/252 and 32/226 patients with Hb > 11 g/dl and < 11 g/dl died; up to 20% of those with inadequate EPO levels did not receive rHuEPO, while 16/35 (46%) of those with adequate EPO levels were prescribed rHuEPO. At a multivariate analysis, only patients with baseline Hb levels between 10 and 11 g/dl and in first line chemotherapy regimen, but not those with levels < 10 g/dl or in > 1st. line therapy benefited from rHuEPO.

Conclusions: While providing an informative epidemiological profile of the patients exposed to rHu EPO during chemotherapy and of the care they receive in the routine practice of specialized centers, the study documents some of the areas of uncertain practice, and confirms the need of prospective, controlled trials in the specific value of rHu EPO in well-characterized subpopulations on chemotherapy for hematological malignancies.
Recommandations of the Societe Francaise des Cancers de l’Enfant (SFCE) for the Management of Tumors Lysis Syndrom (TLS) : A Validation Survey.
Authors: Bertrand Y. , Auvrignon A. , Nelken B. , Mialou V. , Mechinaud F. , Brethon B. , Patte C. , Plantaz D. , Urbieta M. , Baruchel A. , Leverger G.
Published: October 2019

TLS is a frequent metabolic complication of hematologic malignant diseases which can generate important renal impairment. As described in previous studies (Goldman S.C. et al, Blood2001;97, 2998–3003 and Pui C.H. et al., J.Clin.Oncol.2001;19, 697–704), there are High Risk (HR) patients (pts) of TLS. Rasburicase a recombinant urate oxidase enzyme transforms uric acid into the highly soluble compound allantoin which is then excreted by the kidneys. Rasburicase is highly effective in prevention and treatment of TLS. Based on these studies, the SFCE made the following recommandations for the management of TLS in children. HR pts are defined as : B cell Acute Lymphoblastic Leukemia (ALL), ALL or Acute Myeloblastic Leukemia (AML) with initial leukocyte count of at least 50x109/L, Stage III and IV T or B Non Hodgkin Lymphoma (NHL), any leukemia or NHL with a plasma uric acid concentration of at least 300 mmoles/L if <10 years old or 350 mmoles/L if >10 years old, serum creatinine or lactate deshydrogenase concentration (LDH) exciding twice the upper limit of normal (N), hyperphosphatemia ≥2mmoles/L. These pts are treated by hyperhydratation (3L/m2) ±alkaline and Rasburicase 0,20 mg/kg/d x 5days. Rasburicase is carried on if serum creatinine > 1,5N or phosphatemia >3mmoles/L or plasma uric acid concentration ≥200mmoles/L and until normalization. Low Risk (LR) pts are defined as pts not HR. They are treated by hyperhydratation (3L/m2) and Rasburicase 0,20 mg/kg/d for one day only. Rasburicase is carried on during 4 days or more on the same biological basis as HR pts. In order to check the validity of those guidelines, we are conducting a survey looking at the evolution of the pts managed according to the SFCE recommandations.150 pts are planned for a 6 months study. On July 30th, 39 pts are registred : 25 ALL, 6 AML, 8 NHL (3 Burkitt). At that time there is no SLT complication. Results will be presented at the 2004 ASH Meeting.
Time and Cost Savings with Bio-Set® Device in Reconstituting FVIII Concentrate.
Authors: Brewster Jessica , Mannix Sally , Butler Regina , Lloyd Andrew , Rentz Anne M. , Larson Peter J.
Published: October 2019

Introduction: Bio-Set® (Biodome, Issoire France) is a new needleless device developed for the reconstitution of a factor VIII concentrate, Kogenate® FS (Bayer HealthCare, Elkhart IN).

Objectives: Quantitate time required to prepare FVIII concentrate for infusion and estimate the cost of medical waste produced using 3 reconstitution methods.

Methods: 161 subjects (35 patients; 67 caregivers; and 59 nurses) were recruited from the US and Canada following an IRB-approved protocol. Reconstitution methods were Bio-Set®, the conventional 2 vial transfer needle reconstitution method, and 2 vial Baxject method (Baxter Healthcare, Westlake Village CA). Video and interviewer demonstrations were conducted, then participants practiced each reconstitution method once before performing a timed round. Diluent volume for the conventional reconstitution method and Baxject were controlled at 5 mL. After each timed round, participants separated reconstitution refuse into either medical waste or regular trash. The weights of component pieces were added and a cost for disposal of the medical waste was determined.

Results: Participants completed preparation of the infusion with Bio-Set® in the shortest amount of time compared to the conventional method and Baxject (both p<0.0001). Results were similar across the 3 participant groups. The average weight of medical waste was lowest for Bio-Set® and highest for Baxject. The resulting disposal cost was significantly lower for Bio-Set® (p<0.0001).

Conclusions: The results of the time study showed a reduction of 33% in infusion preparation time with the Bio-Set® when compared to the conventional method and 29% when compared to the Baxject. The cost of disposal of medical waste should be reduced with the use of the Bio-Set®.
An Evaluation of the Effectiveness of Darbepoetin alfa 300 mcg Every 3 Weeks (Q3W) on Clinical Outcomesin Cancer Patients with Chemotherapy-Induced Anemia.
Authors: Boccia Ralph Vincent , Liu Delong , Silberstein Peter , Tchekmedyian Simon , Tomita Dianne , Rossi Greg , Otterson Greg
Published: October 2019

Darbepoetin alfa (Aranesp®) administered at 300 mcg Q3W appears to be an effective dose in patients with hemoglobin (Hb) 10–12 g/dL to maintain Hb, and in patients with Hb 8–10 g/dL to elevate Hb to within the National Comprehensive Cancer Network (NCCN)-recommended target range of 11–12 g/dL. The objective of this multicenter, open-label study of cancer patients with chemotherapy-induced anemia was to evaluate the effectiveness of darbepoetin alfa 300 mcg Q3W, administered over a 16 week treatment period, in achieving and maintaining the therapeutic objectives of anemia treatment consistent with NCCN guidelines. This objective was assessed by determining the percentage of patients who achieved a post baseline target Hb level ≥11.0 g/dL in the absence of a transfusion within the previous 28 days, and the percentage of patients maintaining an average Hb within the NCCN target range. Secondary hematologic endpoints included the incidence of transfusion and the proportion of patients achieving a hematopoietic response (increase in Hb ≥2 g/dL from baseline or achieving a Hb ≥12 g/dL). Patient-reported outcomes were assessed using the FACT-Fatigue subscale with four additional questions relating to fatigue, energy, activity, and overall health. The relationship between clinical endpoints and the ability to achieve and maintain Hb within the NCCN target range were also assessed. Anemic patients (baseline Hb levels < 11 g/dL) with nonmyeloid malignancies receiving chemotherapy were eligible for enrollment in this study. Doses could be escalated after 6 weeks (2 doses) to 500 mcg Q3W if Hb levels remained < 10 g/dL and the increase in Hb from baseline was < 1 g/dL, or, based on physician discretion if Hb levels were > 10g/dL. To date, 539 patients have been enrolled. The majority of patients were female (59%), white (82%) with a median age of 64 years. The predominant cancers in these patients were lung (24%), gastrointestinal (22%), and breast (21%). The majority of patients had stage IV disease (45%) and a Karnofsky performance status score of 80 to 90 (65%). The mean (SD) baseline Hb level was 10.1 g/dL (0.7); 32% of patients had a baseline Hb of < 10.0 g/dL [mean (SD) Hb 9.3 (0.6) g/dL] while 66% had baseline Hb levels ≥10.0 g/dL [10.6 (0.3) g/dL]; 2% of patients (n = 12) received a transfusion within 28 days of baseline Hb measurement and were excluded from analysis. Since the majority of chemotherapy regimens are delivered on a Q3W basis, the ability to administer darbepoetin alfa at a fixed dose, synchronized with the chemotherapy schedule may reduce the number of injection-centric visits. This provides important benefits for patients and their caregivers, and represents a simplification in the treatment of chemotherapy-induced anemia in oncology practice.
Enzymatic Replacement Therapy (ERT) with Low Doses in Gaucher Disease in the State of Sao Paulo (Brazil).
Authors: Sobreira Elisa A.P. , Bortolheiro Tereza C. , Martins Ana M. , Porta Gilda
Published: October 2019

INTRODUCTION: The authors, who are members of the Gaucher Disease (GD) Specialists Committee of Sao Paulo State, Brazil, provide demographic data and report the evolution of patients from this Brazilian State, who were treated according to the Clinical Protocol and Therapeutic Instructions for GD treatment created by the Health Department www.saude.gov.br, Notice SAS/MS 449 of 08/07/2002.

METHODS: In May 2004, 130 patients were analysed. One hundred and thirteen of the the130 received Enzymatic Replacement Therapy (ERT) with imiglucerase and 75% of them (85 /113) were > 12 years old. The GD symptoms were investigated in patients above this age who received 15–30 U/kg/2 weeks, according to the Health Department Protocol between August 2002 and November 2003.

OBJECTIVES: Present the evolution of patients > 12 years old having a dose reduction in ERT following said Protocol.

RESULTS: For the 85 patients > 12 years, the mean dose of ERT was 37,6 U/kg/2 weeks (13 to 62 U/kg/2weeks), 4 cases improved without sign of pain and maintained a low dose. Fourteen cases again suffered from bone pain. One of them developed bone infarct and 1 experienced a painless bone lytic lesion. The patients who were in a worse condition received a dose increase (up to 60 U/kg/2 weeks). Of the patients taking doses between 20 and 30 U/kg/2 weeks, 6 had bone pain again and 1 case experienced Parkinsons’ disease. The latter received a dose increase up to 60 U/kg/2 weeks

CONCLUSION: The authors concluded that the dose in ERT must be individualised and that in many times, low doses (15 U/kg/2 weeks) are not enough to avoid GD complications.
Maintenance Treatment with Thalidomide after Autologous Transplantation for Myeloma : First Analysis of a Prospective Randomized Study of the Intergroupe Francophone du Myelome (IFM 99 02).
Authors: Attal Michel , Harousseau Jean-Luc , Leyvraz Serge , Doyen Chantal , Hulin Cyrille , Benboubker Lofti , Facon Thierry , Bourhis Jean-Henri , Garderet Laurent , Sotto Jean-Jacques , Michallet Mauricette , Renaud Marc , Voillat Laurent , Berthou Christian , Marrit Gerald , Monconduit Mathieu , Coiffier Bertrand , Caillot Denis
Published: October 2019

High dose therapy (HDT) supported with autologous stem cell transplantation has been introduced in the management of aggressive myeloma. However, after single or tandem transplantation, almost all patients ultimately relapse. New strategies are required to control any residual disease after HDT. The Intergroupe Francophone du Myelome (IFM) initiated a trial designed to evaluate the impact of maintenance treatment with Thalidomide on the duration of response after HDT.

From April 2000 to October 2003, 1004 myeloma patients at diagnosis under the age of 65 years were enrolled in the IFM 99 protocol. 780 of them, without or with only one adverse prognostic factor (beta-2 microglobuline >3 mg/l or deletion of chromosome 13), were enrolled in the IFM 99 02 protocol. They were to receive the following treatment: 1) 3–4 cycles of the VAD regimen; 2) a first autologous transplant prepared with Melphalan 140 mg/m2; 3) a second autologous transplant prepared with Melphalan 200 mg/m2. Patients without progressive disease 2 months after the second transplant were randomized to receive: no maintenance treatment (arm A), maintenance treatment with Pamidronate (arm B) or maintenance treatment with Thalidomide and Pamidronate (arm C).

As of May 2004, 580 patients (74%) have been randomized: 195 in arm A, 190 in arm B and 195 in arm C. Clinical characteristics of each group were similar. The median follow-up from diagnosis was 26 months (range: 6–50)

Thalidomide was found to improve the Progression-free survival (PFS; p<0.007) and the Event-free-survival (EFS; p<0.01).

Indeed, the 40-months post-diagnosis probability of PFS was 70% (95% CI= 42–80) in the Thalidomide arm versus 53% (95% CI= 37–65) in arm A, and 52% (95% CI= 36–68) in arm B.

Most patients (60%) enrolled in arm A and B received Thalidomide at time of relapse. The overall survival was similar in the 3 treatment groups.

Among the 580 randomized patients, 2 factors were found to be associated with a longer EFS : low beta-2-microglobulin at diagnosis (p<0.01) and treatment arm (p<0.01). Deletion of chromosome 13 (FISH analysis) was not related to EFS.

In conclusion, the first analysis of the IFM9902 trial strongly suggests that Thalidomide is an effective maintenance treatment after high dose therapy for myeloma. The next analysis (November 2004) will be presented during the meeting.
Induction and Patterning of Pre-Hemato-Vascular Mesoderm by the Mouse Mix Homeodomain Protein.
Authors: Baron Margaret H. , Willey Stephen , Sahr Kenneth , Zhang Hailan , Balbi Kevin , Dyer Michael A. , Adlam Matthew
Published: October 2019

Members of the Xenopus and zebrafish Mix/Bix family of paired class homeodomain proteins play determining roles in both mesoderm and endoderm development and are induced by members of the TGFbeta/BMP family of signaling molecules. A single Mix gene has been identified in mouse, humans and chick. Prior to gastrulation, the mouse Mix (mMix) gene is expressed in the visceral endoderm and later in the primitive streak and nascent mesoderm, where it overlaps, in part, with T. Mix expression in ES-derived embryoid bodies is early and transient, overlapping partially with Flk1 activation around the time of formation of hemangioblasts. Both mMix mRNA and protein are found in a FACS-sorted population of T+Flk1+ cells from ES cell-derived embryoid bodies (EBs) which contains hemangioblasts. A complex embryonic lethal phenotype has been reported for Mix deficient embryos, including defects in allantoic (vascular) and cardiogenesis. Mesoderm forms in these embryos but is not patterned properly. Embryonic lethality occurs around E10.5–11, presumably as a result of the cardiovascular defects. We have generated inducible ES cell lines in which expression of Mix protein is responsive to doxycycline. Ectopic expression of Mix in EBs results in premature, enhanced expression of hemangioblast, angioblast and hematopoietic stem cell markers (mRNA and FACS analyses) and increased formation of stem/progenitor cells in clonogenic assays in methylcellulose. Together, the expression analyses, knockout phenotype, and gain-of-function studies in ES cells suggest that mMix functions early in induction and patterning of mesoderm, including formation of hematopoietic and endothelial lineages. Potential mMix target genes are being identified by microarray analyses of the inducible Mix ES lines. To examine mMix activities in vivo, we have generated null and conditional mMix knockout mice from several independently targeted ES cell lines. Analysis of these animals is in progress. Like Xenopus Mix.1, mouse Mix may represent an important connection between the TGFbeta/BMP pathway and hematopoietic/vascular development in the embryo.
Gene Expression Profiling Identifies a Prognostically Favorable Subgroup in AML Independent of Cytogenetic Stratification.
Authors: Schoch Claudia , Kern Wolfgang , Kohlmann Alexander , Hiddemann Wolfgang , Merk Sylvia , Schnittger Susanne , Haferlach Torsten
Published: October 2019

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with varying clinical outcome. So far the karyotype of the leukemic blasts as well as molecular genetic abnormalities - both abnormalities on the genomic level - have been proven to be strong prognostic markers. However, even in genetically well defined subgroups clinical outcome is not uniform and a large proportion of AML shows genetic abnormalities of yet unknown prognostic significance. Here we addressed the question whether gene expression profiles are associated with clinical outcome independent of the known genomic abnormalities. Therefore, gene expression analyses were performed using Affymetrix U133A+B oligonucleotide microarrays in a total of 403 AML treated uniformly in the AMLCG studies. This cohort was divided randomly into a training set (n=269) and a test set (n=134). The training set included 18 cases with t(15;17), 22 cases with t(8;21), 29 cases with inv(16), 14 cases with 11q23/MLL-rearrangement, 19 with complex aberrant karyotype and 167 cases with normal karyotype or “other” chromosome aberrations. The respective data for the test set were: 10 t(15;17), 8 t(8;21), 11 inv(16), 8 11q23/MLL, 19 cases with complex aberrant karyotype and 78 with normal karyotype or “other” chromosome aberrations. Based on the clinical outcome the training cohort was divided into 4 equally large subgroups. We trained support vector machines (SVM) with the training set and classified the cases of the test set with the respective most discriminating genes. Next a Kaplan-Meier analysis was performed with the test set cases assigned to prognostic groups 1 to 4 according to SVM classification. Based on the expression level of 100 genes group 1 showed an overall survival rate of 57% at 3 years. 31 of 134 (23%) patients were assigned to this favorable subgroup. They belonged to the following cytogenetic subgroups: t(15;17) n=6, t(8;21) n=4, inv(16) n=3, 11q23/MLL n=4, complex aberrant karyotype n=1 and normal karyotype or “other” chromosome aberration n=13. The overall survival rate of groups 2, 3, and 4 did not differ significantly (17%, 21%, and 19% at 3 years). Among the genes highly expressed in the favorable group were MPO and the transcription factor ATBF1, which regulates CCND1. The unfavorable groups were characterized by a higher expression of the transcription factors ETS2, RUNX1, TCF4, and FOXC1. Interestingly, 10 of the top 40 differentially expressed genes are involved in the TP53-CMYC-pathway with a higher expression of 9 of these in the unfavorable groups (SFRS1, TPD52, NRIP1, TFPI, UBL1, REC8L1, HSF2, ETS2 and RUNX1). In conclusion, gene expression profiling leads to the identification of prognostically important alterations of molecular pathways which have not yet been accounted for by use of cytogenetics. This approach is anticipated to help optimizing therapy for patients with AML.
New Treatment Options Have Changed the Natural History of Follicular Lymphoma.
Authors: Fisher Richard I. , LeBlanc Michael , Press Oliver W. , Maloney David G. , Miller Thomas P.
Published: October 2019

The natural history of follicular lymphoma has not changed over the last 30 years (Horning, S.J., Seminars in Oncology20: 1993, 75–88). Median survivals have ranged from 7 – 10 years and the disease is generally considered incurable as there has been no plateau in the survival curve. However, multiple new treatment options, including biologic agents, have been developed in the last decade and their impact on the natural history of follicular lymphoma remains unknown. In order to determine the cumulative effects of all these new treatment options, we identified all previously untreated, advanced stage, follicular lymphoma patients treated with three sequential treatment approaches: CHOP chemotherapy +/− nonspecific immunostimulants (SWOG 7426 and 7713: 1974 – 1978), ProMACE-MOPP +/− interferon (SWOG 8809: 1988 – 1994), and CHOP followed by monoclonal antibody therapy (SWOG 9800 and 9911: 1998 – 2000) and determined their Progression-Free Survival (PFS) and Overall Survival (OS). More specificially, the monoclonal antibody trials included CHOP followed by rituximab (SWOG 9800) and CHOP followed by 131I-Tositumomab (SWOG 9911). The PFS are shown below:

Progression-Free Survival by Treatment Strategy

TREATMENT N DEATH/PROGRESSION 4-YR PFS CHOP + MoAb 179 75 61% ProMACE 425 290 48% CHOP 356 257 46%

The results demonstrate that the PFS remained unchanged until the recent studies that utilized CHOP followed by a monoclonal antibody for initial treatment. The results of OS from these three groups are shown below.

Overall Survival by Treatment Strategy

TREATMENT N DEATH 4-YR OS CHOP + MoAb 179 18 91% ProMACE 425 189 79% CHOP 356 226 69%

In contrast to the PFS, OS has increased with each subsequent study.

These data are consistent with the hypotheses that initial therapy with chemotherapy followed by a monoclonal antibody has a significant impact on PFS (p= .005) and OS (p < .0001) and that, even in earlier studies where we could not demonstrate improved initial treatment, sequential new treatment options have also changed the OS (p < .0001) and thus the natural history of follicular lymphoma.
Evidence for Fcγ Receptor IIIA-Independent Rituximab Effector Mechanisms in Patients with Follicular Lymphoma Treated with Combined Immuno-Chemotherapy.
Authors: Boettcher Sebastian , Pott Christiane , Ritgen Matthias , Hiddemann Wolfgang , Unterhalt Michael , Kneba Michael
Published: October 2019

The response to the anti-CD20 antibody Rituximab used as a single agent in follicular lymphoma (FL) patients can be predicted by the valine/phenylalanine (V/F) dimorphism of amino acid 158 of Fcγ Receptor IIIA (Fcγ RIIIA) (Cartron et al., Blood99: 754, 2002; Weng and Levy, JCO21: 3940, 2003). This suggested important contributions for Fcγ RIIIA mediated mechanisms like antibody dependent cellular cytotoxicity to the efficacy of the antibody. Recently, the German Low Grade Lymphoma Study Group (GLSG) demonstrated a significant shorter time to treatment failure (TTF) in patients who received standard CHOP (cyclophosphamide d1, doxorubicin d1, vincristin d1, prednisone d1-5) chemotherapy only compared to patients who received Rituximab (d1) plus CHOP (R-CHOP). Estimated median TTF was 2.6 years in FL patients treated with CHOP and was not reached after 3 years observation time in R-CHOP treated FL patients (p<0.0007) (Blood 102, Abstract # 352, 2003). We examined the Fcγ RIIIA dimorphism in 75 FL patients from this GLSG trial who were treated with 6 to 8 cycles R-CHOP followed by interferon maintenance or high dose chemo-radiotherapy and autologous stem cell transplantation. Using a TaqMan assay with allele specific fluorochrome labelled probes we detected a V/V genotype in 8 patients (10.7%), a V/F genotype in 38 patients (50.6%), and an F/F genotype in 29 patients (38.7%). Patients with different Fcγ RIIIA status responded similarly to R-CHOP (CR rate: V/V 37.5%, V/F 36.8%, F/F 24.1%; overall response rate: V/V 100.0 %, V/F 97.4%, F/F 96.6%; ns). Moreover, TTF was not significantly different between the groups with V/V, V/F, and F/F Fcγ RIIIA(log rank p = 0.48) after a median observation time of 24 months. A correlation between the Fcγ RIIIA status and the efficacy of R-CHOP could not be demonstrated in this large patient cohort. On the other hand it is evident that R-CHOP treated patients significantly benefited from the addition of Rituximab to CHOP chemotherapy in comparison to the standard arm of this randomized trial. This observation supports the hypothesis that the anti-lymphoma effects of Rituximab when used in combination with chemotherapy might be mediated by Fcγ RIIIA-independent mechanisms such as complement dependent cytotoxicity or direct apoptosis induction.
Absence of Inducible Costimulator (ICOS) on Alloreactive T Cells Reduces Graft-versus-Host-Disease While Leaving Graft-Versus-Tumor Activity Intact.
Authors: Hubbard Vanessa M. , Eng Jeffrey M. , Tjoe Kartono H. , Ramirez-Montagut Teresa , Muriglan Stephanie J. , Kochman Adam A. , Terwey Theis H. , Schiro Raffaella , Alpdogan Onder , van den Brink Marcel R.M.
Published: October 2019

Inducible costimulator (ICOS) is a member of the B7 family that is expressed on activated and memory T cells and is involved in the regulation of TH1 and TH2 effector cytokine production after CD3/TCR stimulation. Studies with ICOS inhibition or ICOS−/− recipients have demonstrated prolonged allograft survival after heart or liver transplantation in animal models. To study the role of ICOS expression on alloreactive T cells in graft-versus-host disease (GVHD), we used allogeneic MHC class I and II disparate hematopoietic stem cell transplantation (allo-HSCT) models. We first analyzed the expression of ICOS by transferring CFSE-labeled donor T cells into irradiated allogeneic recipients and observed an increased expression of ICOS on alloreactive T cells compared to non-alloreactive T cells. We then studied B6-ICOS−/− alloreactive T cells and found intact proliferation in vivo (as determined by adoptive transfer of CFSE labeled T cells and donor T cell numbers in the spleen of allo-HSCT recipients), intact cytotoxicity, intact up regulation of activation markers, but decreased IFN-γ production in vitro. We then performed GVHD experiments in two models with full MHC class I and II disparity and observed significantly less GVHD morbidity and mortality in recipients of ICOS−/− donor T cells. Furthermore, histopathological analysis demonstrated less GVHD in all target organs (skin, liver, small bowel and large bowel) of recipients of ICOS−/− splenic T cells compared to recipients of wild type T cells. We harvested target organs (spleen, thymus, liver and gut) on days 7, 14, and 21 to examine donor T cell content (naïve and activated T cells) and found no significant difference in the total T cell numbers and subpopulations. Interestingly, in GVHD/graft-versus-tumor (GVT) experiments, ICOS−/− donor T cells displayed intact GVT activity, while their GVH activity was diminished. We then tested the levels of IFN-γ in the sera of mice undergoing GVHD and observed decreased serum levels in recipients of B6-ICOS−/− T cells. In conclusion, alloreactive ICOS−/− donor T cells display less GVHD morbidity and mortality due to decreased IFN-γ production, while proliferation, infiltration and GVT activity remain intact. These data suggests that strategies to inhibit ICOS could be useful for the prevention and/or treatment of GVHD in recipients of an allo-HSCT.
Molecular Mechanism of Nef-Mediated Inhibition of T Cell Migration in Response to SDF-1α.
Authors: Park In-Woo , Mitsuya Yumi , Groopman Jerome E.
Published: October 2019

Lymphocyte trafficking comprises an intricate multistep process, in which lymphocyte surface integrins, endothelial adhesion molecules, and chemokines produced in the local microenvironment play key roles. We previously reported that HIV-1 nef inhibits T cell chemotaxis in response to the physiological ligand SDF-1αwithout altering CXCR4 expression. Here, we explore the mechanism of this effect. HIV-1 nef significantly reduced the expression of LFA-1, inhibited the adhesion of nef-expressing Jurkat T cells to the HUVEC endothelium, and reduced their transendothelial migration. To gain further insight into how nef mediated these effects, substitution or deletion mutations were introduced into the nef gene, and transwell and transendothelial migration assays were performed. Deletion of the N-terminus proximal basic-rich domain did not change the nef-mediated inhibitory effects on T cell migration, whereas removal of the myristoylation site or the proline-rich amino acid sequence motif of Nef abolished these effects. Similarly, HIV-1 nef blocked the transendothelial migration of Jurkat T cells across the HUVEC monolayer. However, the observed inhibition of transendothelial migration conferred by the wild type nef expression was significantly but not completely reduced by introduction of mutations in the myristoylation site and proline-rich motif, indicating the involvement of other regions in the nef gene. Taken together, these results demonstrate that HIV-1 nef can impair the transendothelial migration of T cells in response to SDF-1αby disturbing different steps in the process, and that the myristoylation site and/or proline-rich motif are key to this effect. These data help to establish the molecular mechanisms of nef-mediated impairment of the T cell migratory response, an essential component of host defense, and provide the basis for targeted therapeutic interventions in patients with AIDS.
Treatment of Adult Patients with High Risk Acute Myeloblastic Leukemia up to 60 Years: Role of Consolidation Therapy within a Prospective Multicenter Trial.
Authors: Krauter Jürgen , Heil Gerhard , Hoelzer Dieter , Ottmann Oliver , Martin Hans , Lübbert Michael , Finke Jürgen , Heit Wolf , Fiedler Walter , Hossfeld Dieter , Kanz Lothar , Schlimok Günter , Kirchner Hartmut , Raghavachar Aruna , Brugger Wolfram , Ganser Arnold
Published: October 2019

In our prospective multicenter AML 01/99 trial for risk-adapted therapy of patients aged up to 60 years with acute myeloblastic leukemia, patients with CBF-leukemias [t(8;21) or inv(16)] or normal karyotype and good response (GR) to induction I (up to 5% blasts in day 15 BM) were considered standard risk (SR), all other patients as high risk (HR). Patients with acute promyelocytic leukemia were excluded. Between January 1999 and May 2004, 421 patients (328 with with de novo and 93 with secondary AML) were treated. Induction course I consisted of standard dose araC, idarubicine and etoposide (IVA-I). Patients with GR to IVA-I continued with IVA-II on day 21 as double induction. In patients with bad response (>5% bone marrow blasts on day 15), the second cycle consisted of either IVA-II or FlAG/Ida. Double induction was followed by an early consolidation with intermediate dose araC. As late consolidation, HR patients were allo- [HLA-matched sibling or matched unrelated donor (MUD)] or autotransplanted. Matched sibling allotransplantation was done in all participating centers whereas MUD transplantation was favoured by only two centers. The remaining centers performed autologous PBSCT if there was no sibling donor available. 199 patients were classified as HR, of whom 118 (59%) achieved CR, and 25 (13%) of the HR patients died during induction. After 55 months, overall survival was significantly worse for HR patients (25%) than for SR patients (49%). This was also true for the relapse-free survival (32% vs. 42%; p = 0.049). In the HR group, there was no difference in overall and relapse-free survival between those patients classified as HR because of bad response to IVA-1 (n = 65), unfavourable karyotype (n = 71) or both characteristics (n = 63). Regarding the different regimens for late consolidation, the “as treated” analysis of the HR patients revealed that overall (59% vs. 20%) and relapse-free survival (59% vs. 20%) was significantly better after an allogeneic HLA-matched sibling transplantation (n = 26) than after autoPBSCT (n=24). Overall and relapse-free survival for the 23 patients who underwent MUD transplantation were 59% and 45% respectively.

In conclusion, in HR patients with an HLA-identical sibling donor an allotransplantation should be performed. The results obtained with MUD transplantation in this particular patient group are encouraging. Therefore, in HR patients without an HLA-identical sibling allografting from unrelated donors should be considered in first CR. On the other hand, HR patients as defined by our risk stratification do not seem to benefit from autografting even after intensive induction and consolidation therapy.
Survival Difference of AML Patients Receiving HLA Matched Sibling Allogeneic Bone Marrow Transplants (ABMT) Correlates with HLA-Cw Ligand Groups for Killer Immunoglobulin-Like Receptors (KIRs).
Authors: Sobecks Ronald , Ball Edward , Rybicki Lisa , Brown Stacey , Kalaycio Matt , Pohlman Brad , Maciejewski Jaroslaw , Bolwell Brian
Published: October 2019

The reactivity of NK cells and some T cell populations is regulated by the interaction of KIRs with target cell HLA class I molecules. KIR interactions have been suggested to influence outcomes of haploidentical and HLA-identical allogeneic hematopoietic stem cell transplants, particularly for AML patients. We analyzed the KIR ligand phenotypes of 60 AML patients who received HLA-identical sibling myeloablative ABMT from 4/9/97-11/5/03. The median age was 45 yrs (range 8–62). At the time of transplant 24 patients (40%) were in CR. All patients received a busulfan/cyclophosphamide-based preparative regimen and all received bone marrow (T-cell replete) as their stem cell source. Patient HLA KIR ligands were categorized as: 1) HLA-Cw groups C1/C1, C2/C2, C1/C2; 2) HLA-Bw4 (+ or −); 3) HLA-A3 or A11 (+ or −) (as reviewed in Farag et al Blood100:1935,2002). Recursive partitioning analysis for post-transplant time-related outcomes (acute GVHD, grade 3/4 acute GVHD, chronic GVHD, extensive chronic GVHD, freedom from relapse and survival) was performed for each KIR ligand group. Patients with C1/C1 or C2/C2 (n=26) had improved survival compared to the C1/C2 group (n=34) (median survival 43.5 months vs. 5.8 months, respectively, p=0.018), as shown below:

Figure Figure

This survival difference was associated with more relapse in the C1/C2 group (p=0.048), but not with incidence or severity of acute or chronic GVHD, age, infection or pretransplant disease status. There were 26/34 (76%) deaths in the C1/C2 group with 15 (58%) due to relapse as compared to 13/26 (50%) deaths in the C1/C1 + C2/C2 group where 4/13 (31%) were due to relapse. The median follow up of survivors was 36.3 mos (range 7.8–72.4 mos). No significant differences in outcomes were observed when patients were analyzed for the presence or absence of HLA-Bw4 or A3/11. The majority of patients had KIR genotyping performed for those KIRs with established HLA ligands. Among those tested there were no cases in which the donor did not have at least one inhibitory KIR gene specific for a Cw ligand present in the patient or donor. This may suggest that KIR expression at the cellular level rather than KIR genotype alone should be investigated. In conclusion, AML patients undergoing matched sibling donor ABMT who were heterozygous for HLA-Cw KIR ligand groups (C1/C2) had reduced survival compared to patients homozygous for these groups. The higher relapse rate observed in the heterozygous ligand group may suggest a less effective graft-versus-leukemia (GVL) response. Since C1/C2 heterozygotes have a greater opportunity to engage inhibitory KIRs than do C1 or C2 homozygotes, they may more effectively inhibit KIR-positive NK and T cell populations involved in GVL responses.
Multi-Step Fibrinogen-αIIbβ3 Binding/Unbinding Revealed at the Single Molecule Level Using Laser Tweezers.
Authors: Litvinov Rustem I. , Shuman Henry , Bennett Joel S. , Weisel John W.
Published: October 2019

The regulated ability of the platelet integrin αIIbβ3 to bind fibrinogen plays a crucial role in platelet aggregation and primary hemostasis. We have developed a model system based on laser tweezers that enables us to measure directly the mechanical forces needed to separate single receptor-ligand complexes. In this system, a ligand-coated bead can be trapped by the laser and repeatedly brought into contact with a receptor-coated pedestal so that the forces required to separate the two can be measured and displayed as “rupture force” histograms. We have applied this system to the interaction of fibrinogen and αIIbβ3 by measuring the rupture force required to separate a laser-trapped bead coated with fibrinogen from an immobilized pedestal coated with purified αIIbβ3. These measurements revealed that bimolecular αIIbβ3-fibrinogen interactions are characterized by rupture forces ranging from 20 pN to 150 pN. We found that the yield strength of αIIbβ3-fibrinogen interactions was independent of the rate at which force was applied over a range of 160 to 16,000 pN/s, but the frequency of fibrinogen binding to αIIbβ3 during repeated contacts correlated strongly with the duration of contact between the αIIbβ3- and fibrinogen-coated surfaces. The observed rupture forces could be segregated into small (20–40 pN), intermediate (40–60 pN), and high (>60 pN) force regimes, each of which displayed unique kinetic behavior. Thus, the contact time needed to reach half maximal fibrinogen binding at a constant loading rate of 1,600 pN/s was ~ 1 ms for the 20–40 pN, 20 ms for the 40–60 pN, and 70 ms for the >60 pN force regimes. Increasing the surface density of immobilized fibrinogen changed the probability of αIIbβ3 binding to fibrinogen >3.5-fold, but did not affect the yield strength profile, implying that these measurements represent interactions between individual molecules. Each force regime also differed in its susceptibility to the inhibitory effect of αIIbβ3 antagonists and to the activating effect of Mn2+. Thus, the low molecular weight αIIbβ3 antagonists H12 peptide, tirofiban, and RGDS were most effective in inhibiting weak interactions, whereas the monoclonal antibodies A2A9 and abciximab were most effective in inhibiting the stronger ones. In the presence of Mn2+, the strongest component of the yield force distribution increased more than 2.5-fold while the moderate force component increased only 1.5-fold. In addition, rupture forces in the range of 60 to 150 pN disappeared gradually when αIIbβ3 preparations were stored at 4°C for 7 d, suggesting that the αIIbβ3 conformation corresponding to these rupture forces is labile. Taken together, these data suggest that fibrinogen binding to αIIbβ3 is a complex, time-dependent multi-step process during which the strength of the bond between αIIbβ3 and fibrinogen appears to progressively increase.
A Critical Role for Membrane Sulfatide in Platelet Aggregation.
Authors: Guchhait Prasenjit , Shrimpton Corie , Honke Kochi , Thiagarajan Perumal , Lopez Jose A.
Published: October 2019

Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.
PIP5Kγ-Null Mutation Induces Cytoskeletal Changes within Megakaryocytes.
Authors: Wang Yanfeng , Lian Lurong , Bach Tami L. , Chen Xinsheng , Chen Qing-Min , Abrams Charles S.
Published: October 2019

All eukaryotic cells contain the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) that serves multiple roles in signaling cascades critical for actin dynamics. Phosphatidylinositol-4-phosphate 5 kinase (PIP5K) catalyzes the synthesis of PIP2 by phosphorylating PI4P. Although the classical PIP5K isoforms (α, β, and γ) generate the same phospholipid product, the isoforms have different primary structures, are expressed at different levels in various tissues, and localize in different intracellular compartments. Therefore, it is likely that the functions of these isoforms are different as well. PIP5Kγ is unique because it associates directly with talin and consequently localizes in focal adhesion. Given the role of PIP2 in actin dynamics, we generated a murine line containing a null mutation in the PIP5Kγ gene. We found that the targeted disruption of PIP5Kγ results in early prenatal mortality that is associated with pleotropic effects involving the cardiovascular, neurologic, and hematopoietic systems. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. After 5 days in culture, many of the non-adherent yolk sac progenitor cells were multinucleated, and approximately 70% expressed CD41 (αIIb), a marker of the megakaryocyte lineage. We then examined their cytoskeletal content and dynamics. Absence of PIP5Kγ had no effect on the quantity of F-actin in unstimulated or thrombin-stimulated cells that were adherent to fibrinogen or were maintained in suspension in the absence of stimulation. Next, we examined membrane dynamics during cell adhesion in real time using spinning disk video confocal microscopy. Wild type megakaryocytes actively formed and contracted lamellipodia and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ-null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. It is also noteworthy that a similar phenotype has been described in cells with defective anchoring of the membrane to the cytoskeleton because of a lack of filamin. Accordingly, our data are consistent with the hypothesis that PIP5Kγ generates compartmentalized pools of PIP2 that contribute to the association of the membrane with the cytoskeleton.
Manipulation of the Notch Pathway by γ-Secretase Inhibitors as a Novel Therapeutic Approach in Multiple Myeloma.
Authors: Jundt Franziska , Acikgoez Oezlem , Preissner Robert , Dorken Bernd
Published: October 2019

Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells. Thereby they control cell fate decisions and survival. We recently demonstrated that Notch has a pathogenetic role in multiple myeloma (MM), where tight interactions between neoplastic plasma cells and their microenvironment are essential for tumor cell growth (Blood.2004; 103:3511–3515). Our data provided evidence that Notch1 and Notch2 were highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts showed low to undetectable levels of Notch. Furthermore, our functional data indicated that activating the Notch pathway in MM cells by the Notch ligand Jagged1 potently induced tumor cell growth and suggested that these interactions contribute to myelomagenesis in vivo. In this study, we blocked Notch by novel γ-secretase inhibitors in cultured MM cells. γ-secretase catalyzes the release of the intracellular domain of Notch that subsequently translocates to the nucleus to activate expression of downstream target genes. Inhibition of γ-secretase activity is currently investigated as a therapeutic strategy in Alzheimer’s disease, because γ-secretase similarly cleaves amyloid precursor proteins to release Aβ peptides, accumulation of which is causally related to Alzheimer’s disease. To identify novel γ-secretase inhibitors that might block Notch in MM cells, we analyzed two million compounds by 3D in silico screening. Thereby structurally known inhibitors were compared with compounds from data banks. Novel structurally related compounds (27) were tested by in vitro assays. Specific inhibition of Notch signaling was controlled by RT-PCR of the down-regulated Notch target gene Hes-1 in MM cells. In addition, tumor cell proliferation of MM cells was efficiently blocked by 3 out of 27 newly identified inhibitors in a dose-dependent manner. Currently, we investigate whether these compounds also have potent anti-tumor activity against MM cells in mouse models in vivo. If so, interruption of Notch signaling by newly identified inhibitors might be a novel therapeutic principle to control the proliferation capacity of MM.