bluuebunny/crossref_metadata_2025 huggingface dataset:
Rituximab® and High Dose Methylprednisolone (HDMP) as a First Line Treatment for Patients with Chronic Lymphocytic Leukemia.
Authors: Castro Januario E. , Bole Jan E. , Prada Carlos E. , Loria Olivier , Kipps Thomas J.
Published: April 2020

High-dose methylprednisolone (HDMP) and the anti-CD20 mAb Rituximab® each can effect partial responses in patients with chronic lymphocytic leukemia (CLL). Previously, we noted that these two agents used in combination were highly effective in treating CLL patients with relapse/refractory disease. We examined whether patients with CLL could respond to these agents when used in combination as a first line therapy. To mitigate the toxicity associated with HDMP, the dose of methylprednisolone was reduced to 60% of that used in salvage regimens. Sixteen patients with CLL were treated with three four-week cycles of methylprednisolone at 1 gm/m2 daily for 3 days and weekly Rituximab® at 375 mg/m2 for four weeks. All patients satisfied NCI-working group criteria for requiring treatment. The median age of the patients was 60 years, the male to female ratio was 4:1, the ECOG performance status was ? 2. Eighty-six percent of the patients had high-risk disease prior to therapy, as per the modified Rai classification. Fifty-six percent of the patients had CLL cells that expressed ZAP-70 and/or unmutated immunoglobulin variable region genes. Response assessment was performed at the end of each cycle, two months after completion of treatment, and each 3–6 months thereafter until the patient experienced disease progression and/or required further treatment. Objective responses were observed in 14 out of 16 patients (Overall response rate 93%), with 1 patient achieving a complete response (CR) without disease detectable in the marrow, 1 patient achieving a nodular PR, 12 patients obtaining an excellent PR only with minimal residual disease in the bone marrow, and 1 patient having stable disease, as per the NCI-working group criteria. We observed significant reductions in the white blood cell (WBC) counts, increases in hemoglobin, elevations in platelet counts, and dramatic reduction and resolution in lymphadenopathy and splenomegaly. The median time to progression (TTP) after 12 months of follow up has not been reached. Overall, the treatment was well tolerated and all but one patient completed 3 cycles of therapy. Most adverse events were Grade I-II (fluid retention, cough, transient hyperglycemia, fatigue). We also observed 2 transient episodes of grade III–IV toxicity secondary to infection, resulting in pneumonia or sinusitis that resolved completely after antibiotic treatment. Pharmacodynamic studies showed that leukemia cells from patients responding to this combination regimen were induced to express pro-apoptotic molecules, such as Bid, Apaf-1, Smac-Diablo, and to downregulate expression of anti-apoptotic molecules, such as XIAP and Mcl-1. These data indicate that modified-dose HDMP/Rituximab® may be an effective and well-tolerated first line treatment for patients with CLL.
Alemtuzumab Is Effective in Patients with Advanced, Pretreated B-Cell Chronic Lymphocytic Leukemia: A Nationwide Study of Routine Use in Austria.
Authors: Fiegl Michael , Falkner Andreas , Hopfinger Georg , Brugger Stefan , Zabernigg August , Haslbauer Ferdinand , Andel Johannes , Thaler Josef , Fridrik Michael , Bauer Franz , Mian Michael , Tatzreiter Georg , Zojer Niklas , Demirtas Dita , Postner Gerhard , Auberger Jutta , Gastl Günther , Greil Richard
Published: April 2020

It is often questionable as to whether or not therapeutic benefit documented in prospective studies reflects that which is observed in routine use. Four prospective studies have explored the efficacy of alemtuzumab in heavily pretreated B-CLL patients, reporting response rates (RR) between 33% to 55%, and median survival between 13–28 months. Here, we report clinical outcomes after therapy with alemtuzumab of 106 consecutive, unselected B-CLL patients treated in 25 centers in Austria. Seventy-two patients were male and 34 female, the median age was 66 years (range, 46–88 years). The majority of patients had advanced stage disease, with 58% having Rai stage IV disease. The patients had received a median of 3 prior therapies (range, 1–11). Alemtuzumab was administered according to guidelines, but in the majority of cases, dosing was individualized, according to tolerability, side effects, and economic considerations. Thus, the median duration of therapy was 7 weeks (range, 2–24 weeks), and median dose was only 390 mg (range, 11–1333 mg). Therapeutic response was determined, strictly adhering to the NCI criteria (duration of complete response [CR]/partial response [PR]/stable disease [SD] ≥ 2 months; adequate radiologic studies of all non-palpable manifestation; marrow for CR). CR, PR, and SD were achieved in 5%, 17%, and 34% of patients (RR 22%). Progressive disease occurred in 34%, and responses were not evaluable in 9%, due to early death. In the 59 patients with overall CR/PR/SD, peripheral blood responses were as follows: 63% CR, 25% PR, and 12% SD. Survival times were encouraging and comparable with other studies. The median survival for all patients was 19 months; 15 months for fludarabine-refractory patients (n = 67) and 31 months for fludarabine-sensitive patients (n = 24; P = 0.04). Survival was dependent on response (P = 0.0001), the number of previous therapy lines (0–3 vs >3; P = 0.0001), and the presence of bulky disease (P = 0.005). Route of administration (IV: 49%; IV to SC 38%; upfront SC 13%) did not influence outcome. Toxicities were within the expected range, with grade 3/4 infections in 37% of patients; grade 4 neutropenia and thrombocytopenia in 25% and 23% of patients, CMV reactivation in 14% (CMV disease in 3%) of patients, and early death (<2 months after last dose) in 12% of patients. We conclude that in the routine clinical setting, alemtuzumab is a safe and effective treatment with profound prolongation of survival in pretreated B-CLL patients. This retrospective study is an important contribution to continuous, nationwide quality control.
The Novel Cancer Drug Seliciclib Engages the Mitochondrial Apoptosis Pathway Via the Mcl-1/Noxa Axis in CLL.
Authors: Hallaert Delfine , Spijker Rene , Derks Ingrid A.M. , Jak Margot , de Goeij Bart , de Boer Jan Paul , de Jong Daphne , Green Simon R. , van Lier Rene , Eldering Eric , Van Oers Marinus H.J.
Published: April 2020

Seliciclib (cyc202, R-roscovitine) is a novel cyclin dependent kinase inhibitor currently in pre-clinical testing. It indirectly inhibits RNA polymerase II and has been shown to be effective against B-cell malignancies by inducing apoptosis and Mcl-1 degradation. The aim of this study was to dissect the underlying apoptotic pathway(s) of seliciclib in chronic lymphocytic leukemia (B-CLL). Treatment of CLL cells with >10 μM seliciclib in vitro induced apoptosis within 24 hours, irrespective of IgVH mutation status (n=20). Gene profiling by means of RT-MLPA, did not reveal involvement of p53 as indicated by the absence of Puma upregulation. None of the >30 other apoptosis genes tested were induced; instead signals for certain labile mRNAs (Mcl-1, A1/Bfl-1, PI-9) were clearly decreased upon seliciclib. Detailed investigation of B cell lines overexpressing various anti-apoptotic proteins (Bcl-2, caspase-9-DN, Flip, FADD-DN), indicated that seliciclib activated the mitochondrial but not the death receptor pathway. Neither mitochondrial activation nor the rapid degradation of Mcl-1 protein could be prevented by caspase inhibition (zVAD) or overexpression of inactive caspase-9 (DN). This indicated that Mcl-1 decline is an upstream, caspase-independent event. Immuno-precipitation demonstrated that pro-survival Mcl-1 is engaged by pro-apoptotic Noxa and Bim. The specific contribution of Noxa was confirmed by RNAi, resulting in inhibition of apoptosis after seliciclib, but not after staurosporin, CD95- or BCR triggering.

These findings demonstrate that seliciclib induces rapid, p53-independent apoptosis via Mcl-1 degradation and mitochondrial activation. The involvement of Noxa, which is highly expressed in CLL, suggests this BH3-only protein may be a novel therapeutic target.
Comparison of Viral Vectors for Gene Transfer into CLL Cells: Efficient Transduction with Adeno-Associated Virus-8 (AAV-8).
Authors: Jewell Andrew P. , Cochrane Melanie , McIntosh Jenny , Benjamin Reuben , Nathwani Amit
Published: April 2020

Chronic Lymphocytic Leukaemia (CLL) remains largely incurable despite recent advances in therapy, and therefore alternative strategies are of interest in treating this disease. One such alternative is the use of gene therapy, but this relies on developing efficient gene transfer technologies. We have compared several viral vectors coding for green fluorescent protein (GFP) for their ability to transduce CLL cells. Three serotypes of adeno-associated virus (AAV) were used, AAV-2, AAV-5 and a relatively new isolate AAV-8, an EI-EIII deleted adenoviral 5 based vector, AV-5, all with GFP regulated by the CMV promoter, and a VSVG pseudotyped lentiviral vector in which GFP expression is controlled by EF1a promotor/enhancer complex. AV-5 resulted in variable GFP expression, 24.1±3.4%, n=10 but caused cell death at high multiplicities of infection (MOI). The lentiviral vector resulted in GFP expression of 23.5±2.6%, n=12, at the highest titre used, and expression declined in a distinct dose-dependent manner as titres were reduced. Of the AAV vectors, AAV-8 was the most efficient with GFP expression at 41.3±1.0% n=14. We conclude that AAV-8 is a promising viral vector for efficient transduction of CLL cells.

Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions.
The Role of the cAMP-Response Element in the Bcl-2 Promoter in the Regulation of Endogenous Bcl-2 Expression and Apoptosis in Murine B Cells.
Authors: Xiang Hong , Boxer Linda M.
Published: April 2020

We have previously shown in B cell lines that the cAMP-response element (CRE) is a major positive regulatory site in the bcl-2 promoter. This element is not only essential for bcl-2 deregulation in t(14;18) cells, but it is also responsible for the positive regulation of bcl-2 expression during the activation of mature B cells and the rescue of immature B cells from calcium-dependent apoptosis in vitro. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutant CRE site was introduced into the bcl-2 promoter region. The mutant CRE reduced the expression of bcl-2 mRNA in several tissues, including thymus, kidney, lung, liver, brain and heart. The levels of bcl-2 mRNA and protein were also significantly lower in splenic B cells from the knock-in mice. Consistent with these results, the activation of B cells from the knock-in mice by anti-CD 40, lipopolysaccharide (LPS) or anti-IgM was reduced as compared to B cells from wild-type littermates. B cells with the mutant CRE were more susceptible to the induction of apoptosis with several different agents consistent with the decreased expression of bcl-2. Preliminary flow cytometric studies suggest that the number of B cells is decreased in the knock-in mice at 8 weeks of age. Quantitative chromatin immunoprecipitation assays revealed essentially no binding of CREB or ATF-2 and decreased binding of CBP and c-Rel to the mutant CRE site in the bcl-2 promoter. Our previous studies have shown that the CRE site in the bcl-2 promoter is linked to the mediation of signal transduction pathways in B cells, so we investigated the effect of forskolin, a cAMP-elevating agent. We found that treatment of the B cells from the knock-in mice with forskolin led to significantly more cell death than observed with wild-type B cells. Taken together, these findings indicate that the CRE site in the bcl-2 promoter has a functional role in the regulation of endogenous bcl-2 expression and plays an important role in the regulation of apoptosis in B cells.
Gene Expression Signature of Acute Myeloid Leukemia (AML) with T(8;16)(P11;P13) and MYST3-CREBBP Rearrangement: A Microarray Study Validated by Multiple Real-Time PCR.
Authors: Camos Mireia , Esteve Jordi , Colomer Dolors , Jares Pedro , Rozman Maria , Villamor Neus , Costa Dolors , Carrio Ana , Nomdedeu Josep F. , Montserrat Emili , Campo Elias
Published: April 2020

AML with t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinical-biological features. The t(8;16)(p11;p13) translocation leads to the fusion of MYST3 and CREBBP genes, probably resulting in a disturbed transcriptional program of a myelo-monocytic precursor. In this study, the genetic signature of MYST3-CREBBP AML was compared with other well-defined AML subtypes. Genotypic analyses using oligonucleotide U133A arrays (Affymetrix) were performed on RNA of 23 AML patients, including three MYST3-CREBBP cases, PML-RARa (n=3), RUNX1-CBF2T1 (n=3), CBFβ-MYH11 (n=3), t(9;11)/AF9-MLL (n=1), monocytic AML (FAB M4/M5), n=8, and two cases of AML with multilineage dysplasia. Forty-six genes differentially expressed in MYST3-CREBBP cases were analyzed by multiple real-time RT-PCR using low-density arrays in an additional series of 40 patients, which included 7 MYST3-CREBBP cases, 18 AML samples with well characterized rearrangements (PML-RARa, n=3; RUNX1-CBF2T1, n=3; CBFβ-MYH11, n=3, MLL-rearranged AML, n=9), and 15 patients with normal karyotype AML. After unsupervised analysis, MYST3-CREBBP cases clustered together, displaying a distinctive expression signature. The analysis by RT-PCR confirmed the gene expression pattern found in the high-density array study. Thus, overexpressed genes included oncogene RET, several homeobox (HOXA9, HOXA10), genes involved in apoptosis (DAP) and prolactin gene. In contrast, cyclinD2, STAT5A, STAT5B and WT1 were underexpressed. Interestingly, MYST3-CREBBP cases showed up-regulation of a subgroup of genes (HOXA9, MEIS1, AKR7A2, CHD3, FLT3 and APBA2) that were also found overexpressed in MLL-rearranged leukemias. In summary, this study showed the distinctive genetic signature of MYST3-CREBBP AML, which harbours some similarities with MLL-rearranged AML. In addition, the low-density array methodology validated the results of the microarray analysis and allowed to study a larger series of patients, including samples not suitable for microarray analysis.
Biological Differences in Peripheral T-Cell Lymphomas Identified by DNA Microarrays.
Authors: Martinez-Delgado Beatriz , Bahl Manisha , Cuadros Marta , Fernandez Victoria , Benitez Javier , Jaffe Elaine S. , Staudt Louis
Published: April 2020

Peripheral T-cell lymphomas (PTCL) constitute a heterogeneous and aggressive group of tumors whose pathogenic alterations remain largely unknown. They show great morphologic, immunophenotypic and clinical differences. Expression profiling has been demonstrated to be a useful tool for the molecular classification of tumours. We used DNA microarrays (Affymetrics, Lymph_DX) to look for molecular differences in peripheral T-cell lymphomas.

We analysed the expression of 19 pathologically confirmed PTCL (11 PTCL, unspecified (u) and 8 angioimmunoblastic, AILT). A time-course of stimulation with anti-CD3 and anti-CD28 was performed in CD4+ and CD8+ isolated peripheral blood lymphocytes, and the gene expression of tumors was compared with resting and stimulated normal T-cells. Cases of classical Hodgkin’s lymphoma, with which PTCL may be confused, were included.

Gene expression of PTCL revealed several clusters of genes with biological significance showing differential expression within the tumors. We defined a “Cytotoxic Response” cluster characterized by the expression of cytotoxic molecules PRF1 and GZMB and GZMH, a “B-cell-related genes” cluster which included B-cell specific genes PAX5 or CD19, and an interesting “Resting-cells genes” cluster. These three clusters were negatively correlated with a “Proliferation” signature. On the other hand, the “Proliferation” signature significantly correlated with the expression of TNFRSF8 (CD30) (r=0.63, p=0.003) and the expression of CD3G (r=0.51, p=0.022). Other interesting genes were also found associated specifically to the proliferation of PTCL.

Comparison between PTCLu and AILT showed differences in the expression of CD21 and other genes reflecting the higher presence of dendritic cells in AILT, and also revealed differences in expression of relevant chemokines, such us CXCL5, CXCL13 and CCL19.

Intriguingly, compared with normal CD4 and CD8 T-cells, PTCL showed lower expression of most of the typical genes associated with T-cell biology, suggesting that PTCL often lose the common genetic program of T-cells. PTCL expression profiles are difficult to interpret due to the significant proportion of other infiltrating cells accompanying the tumor. Our results showed that microarrays are a helpful tool to dissect the PTCL expression profile, identifying those genes expressed by infiltrating cells and those expressed by tumor cells.
Antigen-Specific T Lymphocyte Function Following Unrelated Cord Blood Transplantation (UCBT).
Authors: Parkman Robertson , Cohen Geoff , Carter Shelley L. , Weinberg Kenneth I. , Masinsin Bernadette , Guinan Eva C. , Kurtzberg Joanne , Wagner John E. , Kernan Nancy A.
Published: April 2020

The T lymphocytes contained in cord blood are naïve and do not have antigen-specific function. Since the antigen-specific T lymphocytes contained in other hematopoietic stem cell (HSC) source may contribute to protective cellular immunity following transplantation, it has been hypothesized that the recipients of cord blood transplantation (CBT) might be at increased risk of opportunistic infections. The development of antigen-specific T lymphocyte function was measured in 153 recipients of unrelated cord blood transplants (UCBT) by determining antigen-specific T lymphocyte proliferation to the herpes viruses (CMV, HSC, VZV) for 3 years following UCBT. The first antigen-specific response was detected 29 days following transplantation to HSV. Positive T lymphocyte proliferative responses were detected in 66 recipients: 40 to VZV, 36 to HSV and 22 to CMV. Recipients with and without documented herpes infection/reactivation were equally likely to develop positive proliferative responses. The development of antigen-specific T lymphocyte function following UCBT documents successful immune reconstitution since the antigen-specific T lymphocytes are derived from naïve T lymphocytes or HSC, but not donor-derived antigen-specific T lymphocytes. The assessment of antigen-specific function following CBT is a model for the evaluation of post-transplant immune reconstitution without the compounding influence of donor derived antigen-specific function.
Synergy between Nonmyeloablative Doses of Intravenous Busulfan and Post-Transplantation Cyclophosphamide for Induction of Tolerance to MHC-Compatible Stem Cell Allografts.
Authors: Zhou Xiaotao , Wang Jie , Levy Moshe Y. , Symons Heather , Fuchs Ephraim J.
Published: April 2020

The goal of conditioning for nonmyeloablative allogeneic stem cell transplantation, or NST, is to permit the stable engraftment of donor cells with a minimum of toxicity to the patient. We have previously developed a minimal conditioning regimen, consisting of low dose total body irradiation (TBI; 100–200 cGy) and post-transplantation cyclophosphamide (Cy), that permits the induction of stable mixed hematopoietic chimerism following the transplantation of modest doses of MHC-identical allogeneic stem cells in mice. The goal of the current study was to determine whether intravenous (IV) busulfan (Busulfex; ESP Pharma, Edison, NJ), an agent with myelotoxic but minimal immunosuppressive activity, could be used in place of TBI in this regimen and still promote allograft tolerance. TBI was more immunosuppressive than Busulfex, because engraftment of allogeneic stem cells could be achieved in mice conditioned with 700 cGy TBI alone but not in mice conditioned with an equivalently myelotoxic dose of 60 mg/kg Busulfex IV. However, when post-transplantation Cy was added, stable mixed hematopoietic chimerism was achieved in mice conditioned with doses as low as 100 cGy TBI or 20 mg/kg Busulfex. Busulfex and post-transplantation Cy were both required for the induction of allograft tolerance, because graft rejection occurred in transplanted mice treated with either agent alone. The capacity of Busulfex to promote allograft tolerance in this model could not be explained by effects of the drug on the host immune system, because the graft rejection response was not restored in Busulfex-conditioned mice reconstituted with mature host lymphocytes at the time of transplantation. These results demonstrate that Busulfex effectively substitutes for TBI in promoting allograft tolerance, and suggests that the creation of marrow “space” promotes alloengraftment in conditions of suboptimal host immunosuppression.
A Non-Human Primate Model To Study Anti-HIV Gene Therapy Strategies.
Authors: Trobridge Grant D. , Beagles Karen , Beard Brian , Li Ming-Jie , Yee Jiing-Kuan , Rossi John , Hu Shiu-lok , Kiem Hans-Peter
Published: April 2020

AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We are developing methylguanine-DNA-methyltransferase (MGMT) selection strategies for HSCs in the primate model to allow for high level marking with vectors containing anti-SHIV/HIV transgenes. We have obtained marking levels over 90% in granulocytes and over 30% in lymphocytes. To determine the effectiveness of an anti tat/rev shRNA to inhibit SHIV in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV. Expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR and replication of SHIV was inhibited as evidenced by inhibition of p27 production. Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (~45%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (~55%). Thus M. nemestrina monkeys provide a powerful model to test lenti and foamy virus mediated anti-HIV gene therapy strategies.
Risk of Relapse (REL) after Umbilical Cord Blood Transplantation (UCBT) in Patients with Acute Leukemia: Marked Reduction in Recipients of Two Units.
Authors: Verneris Michael R. , Brunstein Claudio , DeFor Todd E. , Barker Juliet , Weisdorf Daniel J. , Blazar Bruce R. , Miller Jeffrey S. , Wagner John E.
Published: April 2020

Relapse remains a major obstacle that limits the success of hematopoietic stem cell transplantation. Therefore, we analyzed the outcome of patients with acute leukemia transplanted with myeloablative conditioning at a single institution who had a minimum follow-up of 1 year to determine if there were any unique risk factors associated with relapse after UCBT. 96 consecutive patients were evaluated, 39 were >18 years of age, 50 were male; 46 were CMV seropositive; 50 had acute lymphoblastic leukemia (11 CR1, 27 CR2, 9 CR3+, 4 relapse), and 46 had acute myeloid leukemia (11 CR1, 20 CR2, 3 CR3+, 11 relapse). Patients received two different conditioning regimens. Regimen A consisted of cyclophosphamide 120 mg/kg, TBI 1320–1375 cGy, and ATG, followed by cyclosporine A (CSA)/methylprednisolone immunosuppression (n=53). Regimen B consisted of cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, TBI 1320–1375 cGy pre-transplant and CSA/mycophenolate mofetil immunosuppression (n=43). Patients received either one (n=67) or two (n=29) 4–6/6 HLA-matched UCB units, so that the total MNC count was >2.5 x 107/kg. Accordingly, the demographics for recipients of one or two UCB units were similar except for older age (median age 24 yrs. [range 13–42] vs. 8 yrs. [range 1–52 yrs], p<0.01) and greater weight (median wt. 70 kg [range 48–120 kg] vs. 32 kg [range 10–108 kg], p<0.01) for recipients of two UCB units. Potential risk factors for relapse that were evaluated included: age, gender, recipient CMV sero-status, diagnosis, disease risk, HLA disparity, regimen (A vs. B), and TNC (or CD34+) dose of the unit responsible for sustained engraftment. Notably, in Cox regression analysis two factors were associated with lower relapse risk: disease risk (CR1/CR2 vs CR3+/REL [RR 0.32, p=0.02]) and transplantation of two UCB units (RR 0.3, p=0.03). Importantly, the diagnosis, UCB graft cell dose, and presence of acute GVHD had no demonstrable impact. All recipients of regimen A received a single UCB unit (relapse was 28% (95% CI, 15–41%)), thus interactions between regimen A and number of UCB units could not be assessed. For patients who received regimen B and were in CR1/2, there was a significantly lower risk of relapse for recipients of two UCB units compared to one unit (11% (95% CI, 0–25%) vs. 54% (95% CI, 23–85%), p<0.01). No significant difference in relapse risk could be discerned for patients with advanced disease (CR3-REL) when comparing single vs double UCBT (p=0.48). This report is the first to suggest that the use of double unit UCB grafts may be associated with a reduced risk of relapse in patients with acute leukemia. Larger studies are needed to confirm this clinical experience and to investigate the potential mechanisms by which double unit grafts could mediate protection against relapse.
Gene Therapy for Tolerance to Factor VIII Induced with B Cells and Bone Marrow Transduced with FVIII Domains.
Authors: Scott David W. , Lei Tie Chi
Published: April 2020

Tolerance induction for hemophilia inhibitor formation has recently been achieved by gene therapy using B cells transduced with a fusion protein of C2 and A2 domains of factor VIII with an IgG heavy chain. We have shown that inhibitor titers can be reduced over 90% by this approach in both naïve and highly immunized FVIII knockout mice (FVIII−/−). This process requires the presence of CD25+ regulatory cells for induction, and is stable for at least three months (Lei, T-C. and Scott, D.W. 2005. Induction of tolerance to fVIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B-cells as Ig fusion proteins. Blood, 105: 4865–4870). Herein, we wished to establish whether retroviral transduction and integration of the fusion Ig was necessary for tolerance. In addition, we also tested whether transduction of bone marrow could lead to long-term tolerance. B cells (resting and LPS blasts) from FVIII sufficient C57Bl/6 and FVIII−/− mice were pulsed with FVIII and injected into naïve FVIII−/− recipients that were subsequently challenged with therapeutic doses of FVIII in an immunogenic protocol. We reasoned that C57Bl/6 mice were FVIII sufficient and could display FVIII processed epitopes within their MHC class II. Moreover, pulsing with FVIII should lead to presentation of processed epitopes transiently in class II MHC. We found that specific tolerance to FVIII was only induced with B cells transduced with a combination of A2-IgG and C2-IgG, indicating that transient expression of FVIII epitopes and presentation by B cells is not sufficient for tolerance induction.

Resting B-cells pulsed with fVIII in vitro are not tolerogenic Resting B-cells pulsed with fVIII in vitro are not tolerogenic

In further studies, we examined whether tolerance could be induced by bone marrow transplantation of C2-Ig and A2-Ig transduced hematopoietic stem cells under a non-myeloablative regimen with busulfan in immunized FVIII−/− mice. This protocol could lead to long-term tolerance if pluripotential stem cells were transduced and engrafted. Our results indicate that tolerance to FVIII domains could be achieved even though the level of expression of GFP positive cells in peripheral blood of busulfan-treated mice was less than 1% (compared to >50% positive cells in lethally irradiated recipients of similarly transduced bone marrow cells). These results have implications for long-term therapy of inhibitor formation in hemophilia A. (Supported by NIH grants HL061883, AI035622 and a Lab Grant from the National Hemophilia Foundation.)
E2f-2 Regulates Caspase-3 Expression and Mitotic Checkpoint Control during End-Stage Erythroid Maturation.
Authors: Dirlam Alexandra , Spike Benjamin T. , Macleod Kay F.
Published: April 2020

E2f-2 is a member of the E2F superfamily of transcription factors that plays multiple roles in regulating cell cycle progression, cell cycle exit and checkpoint control [1]. While over-expression studies have ascribed a role for E2f-2 as a transcriptional activator of cell cycle genes, our loss of function studies performed in vivo have revealed a novel role for E2f-2 in repressing cellular proliferation and mediating differentiation. Our expression analyses demonstrate that E2f-2 is up-regulated in hematopoietic tissues, and is most strongly expressed in differentiating erythroblasts. Furthermore, E2f-2 is the major pRb-associated E2F in hematopoietic tissues, suggesting E2f-2 can function as a co-repressor of cell cycle genes during end-stage differentiation. Compound Rb−/−;E2f2−/− mice were generated to understand how deregulated E2f-2 activity contributes to the erythroid defects in Rb null mice. Loss of E2f-2 restored end-stage erythroid differentiation and enucleation to Rb null erythroblasts and extended survival of Rb null mice. Loss of E2f-2 was sufficient to bypass the mitotic arrest observed in Rb null erythroblasts [2] and was associated with reduced expression of known E2f target genes involved in M phase checkpoint control, such as Aurora B and Survivin. In addition, we observed a significant increase in caspase-3 expression in E2f-2 null erythroblasts and demonstrate, for the first time, direct regulation of caspase-3 expression by E2f-2. Given known roles for caspases in promoting erythroid differentiation and enucleation, we have over-expressed caspase-3 in Rb null erythroblasts and are currently analyzing its effects on cell cycle and maturation. Thus, our work identifies caspase-3 as a key downstream target of E2f-2 during end-stage erythroid differentiation whose expression may alleviate enucleation and mitotic defects in Rb null erythroblasts. Future work aims to understand which E2f-2 target genes ensure mitotic checkpoint control, and the molecular mechanism by which caspase-3 coordinates cell cycle control with erythroid differentiation.
RAS and G-CSF Receptor Mutations Are Mutually Exclusive in Leukemogenesis in Severe Congenital Neutropenia.
Authors: Germeshausen Manuela , Kratz Chris , Ballmaier Matthias , Shannon Kevin M. , Welte Karl
Published: April 2020

Activating RAS mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) and were found in some patients with severe congenital neutropenia (CN) after transformation to MDS/AML (Kalra et al. Blood1995;86:4579). CN is a rare genetic disorder characterized by severe neutropenia leading to recurrent life-threatening bacterial infections due to the failure of myeloid cells to mature from promyelocytes and myelocytes to neutrophils. The risk for malignant transformation in CN is currently estimated at 14%. Based on earlier investigations one proposed model of the genetic events associated with progression of CN to MDS/leukemia starts with the acquirement of a somatic G-CSF Receptor gene (G-CSFR) mutation. In this model, RAS mutations and chromosomal aberrations such as monosomy 7 or trisomies represent late events in the transformation process. We have screened bone marrow specimens from 44 CN patients for mutations in the G-CSFR and in the KRAS2 and NRAS genes. Remarkably, we not only identified G-CSFR mutations in 10 out of 14 CN patients with MDS/AML, but also in 21 out of 30 patients without transformation. Only 1 out of 44 patients with CN harbored an activating RAS mutation (NRAS, c.37G>C; G13R). In this patient, who developed secondary chronic myelomonocytic leukemia at the age of 18, the NRAS mutation was not detected in a bone marrow sample obtained at age 2. Results of cytogenetic investigations were available for 10 out of the 14 CN/MDS/leukemia patients. Chromosomal abnormalities were detected in all 10 patients, and included monosomy 7 (n=3), trisomies (n=3), deletions (n=2) and translocations (n=5). In addition to the cohort of 44 patients mentioned above, we analyzed 3 of the 5 CN patients with secondary myeloid leukemia in whom a NRAS mutation, c.35G>A (G12D), was described previously (Kalra et al. Blood1995;86:4579). None of these patients carried a G-CSFR mutation. Combining our data with data available from the literature the frequency of RAS mutations in CN patients is now 6/55 (11%). Accordingly, RAS mutations are less common during leukemic progression in CN than suggested by earlier studies, which may reflect changes in demographics of the CN population and/or effects of early treatment with G-CSF. The sequential occurrence of G-CSFR and RAS mutations in the leukemogenesis is very rare. Instead, our genetic data suggest RAS and the G-CSFR mutations are largely mutually exclusive in CN and may deregulate common signaling pathways.
DOCK180 Is Induced with Differentiation from CD14 Positive Monocytes and It Regulates Phagocytosis in Both Macrophages and Dendritic Cells.
Authors: Fujimoto Katsuya , Nishio Mitsufumi , Nishihara Hiroshi , Sato Ikumi , Wang Lei , Tanaka Shinya , Koike Takao
Published: April 2020

A guanine nucleoside exchange factor (GEF), DOCK180, which is an archetype of the CDM family protein, plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of small GTPase Rac. The expression of DOCK180 is mostly ubiquitous except in hematopoietic cells, while another CDM family protein, DOCK2 is strictly found in hematopoietic cells. Although a knockout study against DOCK2 demonstrated the essential role of DOCK2 regarding rodent T- and B-cell motility and chemotaxisis, no involvement of DOCK2 in the action of phagocytes, such as macrophages or dendritic cells (DCs), has yet been observed. We, therefore, investigated the possible role of DOCK180 in the phagocytosis of hematopoietic cells. CD14 positive monocytes, as well as T- or B lymphocytes, isolated from healthy human donors did not express DOCK180 observed by Western blotting. Consistent with this finding, a real time PCR study revealed near negligible levels of DOCK180 mRNA in monocytes. On the other hand, macrophages or DCs, differentiated from monocytes in vitro in the presence of M-CSF or IL-4 and GM-CSF for seven days, were found to express extremely high levels of DOCK180 protein and mRNA. Time course studies showed that DOCK180 mRNA rapidly increased by 6 hours after the induction of CD14 positive monocytes into both macrophages and DCs and its protein expression was clearly detected by Western blotting after Day 2. While the expression of DOCK180 on DCs gradually increased until it reached a plateu level on Day 4, the expression of macrophages showed a linear increase up to Day 7. In contrast to DOCK180, the expression of DOCK2 remained unchanged throughout this study. Furthermore, a phagocytosis assay using Zymosan A particles demonstrated an increase in the phagocyte activity accompanying the expression levels of DOCK180 during both types of differentiation. Protein knockdown assays against DOCK180 using siRNA also confirmed the significance of DOCK180 in the phagocytosis of differentiated macrophages and DCs. Finally, an immunohistochemical study with anti-DOCK180 monoclonal antibody showed that macrophages exhibit the phagocytosis in the front edge of inflammatory granulation tissue, and expressed a high level of DOCK180. Taken together, DOCK180 was thus found to play a role in the induction of phagocyte activity regarding the differentiation from monocytes into either macrophages or DCs, thereby demonstrating the biological significance of DOCK180 in the phagocytosis of human hematopoietic cells.
hDectin-1 Is Involved in the Uptake of Apoptotic and Infected Cells and Mediates Cross-Presentation of Engulfed Antigens.
Authors: Weck Markus M. , Grünebach Frank , Werth Daniela , Kanz Lothar , Sinzger Christian , Brossart Peter
Published: April 2020

Dectin-1 is a member of the c-type-lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In the present study we identified the hDectin-1b splice variant as the major isoform expressed in in vitro generated mDC using a quantitative RT-PCR and western blot analysis. Interestingly, stimulation of immature DC with the toll like receptor ligands Poly I:C (TLR3) or LPS (TLR4) but not with TLR ligands 2 and 7 or TNF-a led to a dramatic down regulation of hDectin-1 protein expression. To further analyze the possible involvement of Dectin-1 in the phagocytosis of cellular material we recombinantly expressed the extracellular domain (ECD) of hDectin-1b and used it to stain tumor cells. The recombinant ECD showed a specific binding to several human tumor cell lines that could be increased by induction of apoptosis in malignant cells and inhibited by incubation of the extracellular domain with zymosan, a crude cell wall extract of saccharomyces cerevisiae. Furthermore, uptake of tumor cells by immature mDC was reduced by zymosan or the presence of the recombinant Dectin-1 in phagocytosis assays suggesting that Dectin-1 is involved in the engulfement of tumor cells by DC. In line with the expression analysis of hDectin-1, we found that phagocytosis of apoptotic cells was dramatically reduced upon stimulation of DCs with Poly I:C or LPS as compared to immature DC or DC activated with TLR2 or 7 ligands. We next analysed the role of Dectin-1 in the cross-presentation of cell derived antigens and used DC that were incubated with CMV infected fibroblasts and an autologous CTL line specific for the HLA-A2 binding pp65 peptide. Stimulation of CMV peptide specific CTL in ELIspot assays by DC that were incubated with CMV infected fibroblasts efficiently stimulated IFN-gamma secretion that could be inhibited by zymosan and the extracellular domain of Dectin-1. In line with the results from phagocytosis assays stimulation of CMV specific CTL was reduced by stimulation of DC with TLR2 and 4 ligands.

Our results identify hDectin-1 as a new receptor for endogenous ligands on mammalian cells and indicate an important role of this molecule in the clearing of apoptotic and infected cells and cross-presentation of cell derived antigens..
A Selective Small Molecule Factor VIIa Inhibitor, CRA-027483, Suppresses Acute Inflammation in Mice.
Authors: Dalrymple Stacie A. , San Pablo Florentino D. , Sukbuntherng Juthamas , Janc James W. , Young Wendy B. , Vijaykumar Dange , Scheerens Heleen
Published: April 2020

Factor VIIa (FVIIa) is a well known member of the extrinsic coagulation cascade and therefore an attractive therapeutic target for thrombosis. CRA-027483 is a potent, selective, reversible small molecule inhibitor of human FVIIa that displays anti-thrombotic activities in vitro and in vivo. In addition to the well-characterized role of the FVIIa/tissue factor (TF) complex in coagulation, FVIIa/TF complex has more recently been implicated in inflammation via intracellular signaling initiated by the cleavage of protease activated receptors (PARs) by the complex. Therefore, we assessed the ability of CRA-027483 to inhibit acute inflammation using a mouse model of endotoxemia. The compound was delivered subcutaneously one hour prior to LPS challenge and serum or plasma was harvested at varying time points post-challenge. Soluble mediators of coagulation and inflammation induced by LPS were measured by ELISA. Additionally, prothrombin time (PT) and compound exposure were determined. CRA-027483 dose-dependently increased PT in mice as expected. Increases in PT correlated with increases in compound plasma concentrations. Coagulation markers plasminogen activator inhibitor-1 (PAI-1), D-dimer, and thrombin antithrombin (TAT) complex were dose-dependently inhibited by CRA-027483. In addition to increasing PT and inhibiting coagulation markers as expected, CRA-027483 also dose-dependently inhibited several pro-inflammatory mediators including cytokines, chemokines, and acute phase proteins. Inhibitory effects correlated with extension of PT, suggesting that the anti-inflammatory action of CRA-027483 was likely the direct result of inhibiting FVIIa/TF signaling rather than an off-target effect. Thus, we have demonstrated that selective inhibition of FVIIa with CRA-027483 results in suppression of acute inflammation in mice.
Inhibition of mTOR Reverses the Ability of c-MYC To Block the Differentiation of Murine Granulocytes.
Authors: Wall Meaghan , Poortinga Gretchen , Pearson Richard B. , Hannan Ross D. , McArthur Grant A.
Published: April 2020

The proto-oncogene c-MYC encodes a bHLH leucine-zipper transcription factor that acts as a potent differentiation inhibitor and as a promoter of cell cycle progression. Additionally, c-MYC and mTOR are widely acknowledged as major regulators of cell growth, the process by which cells accumulate biomass. Both c-MYC overexpression and constitutive activation of the PI 3-kinase/mTOR pathway are frequent events in acute myeloid leukaemia; a haemopoietic malignancy characterised by failure to undergo terminal differentiation. In this study we used the MPRO system, an in vitro murine myeloid terminal differentiation model, to study the relationship between cell growth effectors and differentiation in more detail. Enforced expression of c-MYC by the addition of 4-hydoxytamoxifen (4-OHT) to MPRO cells carrying the inducible mycER fusion protein prevents downregulation of growth and impairs retinoid-induced terminal myeloid differentiation. Because downregulation of mTOR limits growth we hypothesised that inhibition of mTOR may reverse the phenotypic effects of c-MYC. Strikingly, pharmacological inhibition of mTOR by rapamycin, but not growth limitation by other means such as amino acid deprivation or cycloheximide treatment, restores differentiation in granulocytes with enforced c-MYC expression (74.0±2.0% mature granulocytes in the rapamycin+4-OHT-treated mycER MPROs vs 4.5±1.0% in the cells treated with 4-OHT alone and 92.9±1.1% in vehicle-treated controls). Rapamycin restores cell growth parameters to levels equivalent to those of vehicle-treated control cells undergoing physiological granulocytic differentiation (2–3 fold reduction in 35S methionine incorporation in rapamycin+4-OHT-treated and vehicle-treated controls compared to levels in 4-OHT-treated cells) and facilitates cell cycle exit (26.0±7.14% of rapamycin+4-OHT-treated cells and 10.7±2.48% of vehicle-treated control cells in S phase vs 51.9±1.72% of tamoxifen-treated cells in S phase as determined by BrdU incorporation). Interestingly, rapamycin attenuates c-MYC’s ability to activate transcription of its target genes without impairing activation of RARα target genes. c-mycER protein levels are reduced while levels of retrovirus-driven c-mycER mRNA do not fall in rapamycin-treated MPRO mycER cells. Furthermore, c-mycER protein levels decrease in rapamycin-treated MPRO cells expressing the ER fusion of the mycT58A point mutation known to increase protein stability. Overall, this data suggests that rapamycin restores the differentiation potential of granulocytes in the MPRO mycER system by regulating c-MYC at the level of protein translation, rather than at the level of gene transcription or protein degradation. These findings suggest that mTOR could be targeted to influence terminal differentiation regulated by MYC in haemopoietic malignancies.
Capturing the Financial Impact of Heparin-Induced Thrombocytopenia.
Authors: Smythe Maureen A. , Koerber John M. , Mattson Joan C.
Published: April 2020

Data evaluating the financial impact of heparin-induced thrombocytopenia (HIT) (a severe adverse drug reaction which can result in life threatening thrombosis) is lacking. The goal of this case-control study was to evaluate the financial impact of HIT. Case patients were those with a new diagnosis of HIT from April 2003 to March 2004 for whom matched controls were available. Controls for each case patient were matched for the DRG under which the hospital was reimbursed, the patient’s primary diagnosis code and their primary procedure code. Case patients required identification of >1 control for inclusion. The hospital’s financial database was queried for length of stay (LOS), total cost, and reimbursement. For each case patient, the cost and reimbursement were compared to the cost and reimbursement for each group of matched controls. In an effort to eliminate the impact of variable reimbursement, a subset of only Medicare case and control patients was also evaluated. Of 72 new HIT patients, matched controls were identified for 31. The mean LOS for the case and control patients was 22.8 and 11.6 days respectively (p=0.006). The mean hospital cost of case and control patients was $55,440 and $26,505 respectively. From reimbursement minus cost calculations, our institution lost an average of $13,429 per HIT patient compared to an average of $393 per control patient (p=0.005). The mean LOS for Medicare cases (n=21) and matched Medicare controls was 26 and 14.6 days respectively (p=0.041). The mean hospital cost of Medicare case and control patients was $58,842 and $30,210 respectively. From reimbursement minus cost calculations for the Medicare subset, our institution lost an average of $20,229 per HIT case compared to $1844 per control patient (p<0.0001). Assuming 72 new cases of HIT per year, our institution incurs a projected annual financial loss of $980,000 from HIT. The use of alternate anticoagulants, although having a higher acquisition cost, may offset this loss through HIT avoidance.