bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloma with AL Amyloidosis: 10 Years Experience with Stem Cell Transplant.
Authors: De Padua Silva Leandro , Weber Donna , Wang Michael , Han Eric , Mendoza Floralyn , de Lima Marcos , Qazilbash Muzaffar , Champlin Richard E. , Giralt Sergio
Published: October 2019

Background: (AL) Amyloidosis is a rare and potentially fatal disease that involves deposition of light chain protein by a clonal plasma cell population. Treatment with high dose melphalan and autologous stem cell transplant (ASCT) can improve survival and reverse organ damage, but this treatment is associated with toxicity and mortality. We reviewed the outcome of patients with amyloidosis, who received hematopoietic stem cell transplant (HSCT) at our institution.

Patients and Methods: The retrospective study analyzed 32 patients with AL Amyloidosis who underwent transplant between 1997–2006. There were 17 men and 15 women, with a medium age of 53 years (range 35–73 years). All patients had diagnostic criteria for multiple myeloma (MM) and had biopsy confirmed amyloidosis of at least one organ site. Six patients had at least 1 organ involved with AL amyloidosis (3 bone marrows, 2 subcutaneous and 1 kidney), 20 patients had 2 organs involved, while 6 patients had 3 or more organs affected. All received a median of 3 cycles of chemotherapy (range 0–13) before transplant. Twenty-eight patients underwent autologous transplant, while 4 had an allogeneic transplants (2 syngeneic and 2 siblings). The preparative regimens received in 28 patients were high dose melphalan (26 autologous and 2 syngeneic), 2 patients received Busulfan and Melphalan, 2 allogeneic transplant Fludarabine and Melphalan.

Results: The median Charlson Comorbidities Index (CCI) was 3 (range 0–8). The median number of CD34+ cells infused was 4.85 x 106 cells/kg (range 1.43–7.82 cells/kg). The median time to neutrophil and platelet engraftment were 11 (range 2–21days) and 14 days (range2–41), respectively. 56% of patients developed moderate to severe gastrointestinal effects. Four patients underwent allogeneic transplant and 1 had acute and chronic graft vs host disease (GVHD). Twenty-one patients (65%) achieved a partial hematological remission (PR), and 5 patients (15%) achieved complete hematological remission (CR), with a total response hematological rate of 80%. The 100 day NRM was 3.2%. The median overall survival was (OS) 41 months (range 1–108), 7 patients died (4 relapsed and 3 infections) all 7 patients had CCI greater or equal to 4.

Conclusion: Selected patients with MM and amyloidosis can benefit from high dose therapy with stem cell support including allogeneic transplantation. The high mortality seen in patients with CCI >4 suggests that this index could be useful in selecting patients for high dose chemotherapy.
Mesenchymal Stromal Cells Gene-Enhanced To Express Human Factor IX and Their In Vivo Use for Correction of Hemophilia B Phenotype in R333Q Mice.
Authors: Coutu Daniel L. , Cuerquis Jessica , Griffith May , Blostein Mark D. , Galipeau Jacques
Published: October 2019

Hemophilia B is considered an appropriate disease target for gene therapy because it is a well characterized monogenic disease with a large therapeutic index. Despite promising preclinical and clinical trials in the last decade, safety and efficacy concerns associated with the in vivo administration of viral vectors still need to be addressed before gene therapy becomes part of the standard arsenal for clinicians. Our laboratory has developed a cell therapy approach using gene-enhanced autologous Mesenchymal Stromal Cells (MSCs) to deliver a therapeutic plasmatic protein which addresses these safety concerns. In this study, we tested whether MSCs engineered to express human Factor IX (hFIX) can be used to reverse the bleeding phenotype of R333Q hemophilia B mice developed by Stafford et al. We retrovirally engineered MSCs harvested from normal C57Bl/6 to express hFIX. A gene enhanced polyclonal population of MSCs was capable of producing carboxylated and fully active hFIX by in vitro clotting assays. By ELISA, the cells were shown to produce approximately 250ng of hFIX per million cells per 24h. Ten million of these cells were embedded in a collagen I gel matrix and implanted subcutaneously in R333Q hemophilia B mice (n=10). hFIX activity in mouse plasma (test and control groups) were followed weekly by aPTT assays. hFIX activity reached levels as high as 20% normal activity in some animals with an average +/− SEM of 11.2 +/− 2.1 (FIX activity in controls is <1%). The hFIX activity returned to baseline within 4 weeks. In conclusion, we demonstrate that gene-enhanced autologous MSCs can serve as an effective delivery of functional FIX for temporary correction of the hemophilia B phenotype. We hypothesize the presence of GFP co-expression by the implanted MSCs caused their immune rejection and we are currently testing this hypothesis.
Insertion of the 1.2kb Chicken β-Globin 5′HS4 Insulator Element in the LTR Inhibits HIV-1 Vector Mediated Transduction at the Early Infection Step.
Authors: Hanawa Hideki , Shimada Takashi
Published: October 2019

Insertional oncogenesis is the major concern in gene therapy using integrating vectors such as gamma-retrovirus vectors and lentivirus vectors. One promising strategy to circumvent this unwanted event is the use of insulator elements which can block trans-activation of proto-oncogenes by enhancers of the integrated vectors. The 1.2kb fragment containing the chicken β-globin 5′ hypersensitivity site 4 (5′HS4) is the most well characterized insulator element. The insulator elements are usually inserted into the U3 region of the 3′ LTR of the vector construct, because the insulator element is duplicated and transferred to the 5′ LTR during viral replication. In the transduced cells, two insulator elements are located outside the retroviral transcriptional unit and this configuration is thought to be essential for efficient enhancer blocking. However, we have previously shown that insertion of the 1.2kb 5′HS4 element in the LTR of SIN-HIV-1 vector significantly reduced the transduction efficiency assayed by GFP expression in HeLa cells. In the present experiments, we examined the mechanism of reduced biological titer. We compared SIN-HIV-1 vectors with (INS) or without (PARENT) the 1.2 kb insulator element. The biological titer on HeLa cells (TU/ml), p24Ag concentration (ngp24/ml), and virus RNA genome content (vg copy/ngp24) of these vectors were shown in Table 1. As we previously reported, the biological titer of INS was significantly lower than that of PARENT, although p24 and RNA contents were not influenced by insertion of the 1.2 kb insulator element. In the next experiment, we performed reverse-transcription kinetic assay. Genomic DNA and low molecular weight DNA extracted by Hirt’s method were prepared from transduced HeLa cells and the amounts of reverse-transcribed vector sequence were determined by qPCR. The data showed that reverse-transcription of INS was remarkably reduced compared to PARENT. These results suggest that insertion of the 1.2 kb insulator does not inhibit production of virus particles and packaging of RNA genome. The responsible step for reduced titer of INS appears to be somewhere from the cellular entry step to the reverse-transcription step.

Characterization of HIV-1 vector with the 1.2 kb 5′HS4 insulator

x106 TU /ml ngp24 /ml x103 vg copy/ngp24 Values are mean ± sem from quadruplicate. a: p<0.005 PARENT 27 ± 4.3 361 ± 56 860 ± 16 INS 4.8 ± 0.4 a 348 ± 37 650 ± 81
Adenovirus-Mediated Gene Transfer to Mesenchymal Stem Cells: Comparison of the Cytomegalovirus- and Rous Sarcoma Virus-Promoter.
Authors: Deng Weiwen , Giambernardi Troy A. , Hay Rick V. , Pietryga Daniel W. , Abdel-Mageed Aly S.
Published: October 2019

We have compared the efficacy and toxicity of the cytomegalovirus (CMV) promoter and the Rous sarcoma virus (RSV) promoter for expressing genes for adenovirus-mediated gene transfer to mesenchymal stem cells (MSCs). Mouse MSCs (mMSCs) were isolated from the femurs and tibias of 6-week old, female BALB/c mice and ex vivo expanded in MEM-alpha containing 20% fetal bovine serum, 100 u/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, and 2 mM L-glutamine. To transduce mMSCs with adenovirus, the cells were plated at a density of 10,000 cells/cm2 in 6-well plates and cultured overnight. The cells were counted and then exposed to fresh culture medium containing adenovirus at 0 to 2,000 multiplicities of infection (MOI) for 48 h. Two adenoviral vectors with CMV promoter (Adv/CMV) and two adenoviral vectors with RSV promoter (Adv/RSV) were used in this study. Transduction efficiency and cell survival were then determined. The Adv/CMVs, i.e. AdvCMVlacZ and AdvCMVIL-10, were more potent in terms of transduction efficiency and transgene expression than their corresponding Adv/RSVs, i.e. AdvRSVlacZ and AdvRSVIL-10. However, both vectors exhibit a dose-dependent relationship between the adenoviral vector MOI and the percentage of transgene-expressing cells. Furthermore, no toxicity was observed in Adv/CMV or Adv/RSV transduced mMSCs.
Human Herpesvirus 8 K1 Blocks Fas in Cells and Mice by Binding and Blocking Fas.
Authors: Maeng Hoyoung , Wang Suizhao , Berkova Zuzuna , Wang Shu , Jung Jaehoon , Felipe Samaniego
Published: October 2019

Background: Kaposi’s sarcoma and primary effusion lymphoma are linked to human herpesvirus 8 (HHV-8 or Kaposi’s sarcoma-associated virus) infection. Key to the pathogenesis of these cancers is the HHV-8 transmembrane oncoprotein K1. This viral protein, which immortalizes cells, produces lymphoproliferation and lymphomas through unknown mechanisms when expressed in mice.

Results: We show here that K1 blocks Fas (CD95)-mediated apoptosis by binding the ectodomain of K1 to Fas in hematopoietic cancer cells (BJAB, U937 and THP-1). K1 bound to Fas in K1-transfected BJAB cells. To assess whether K1 and Fas formed complexes in cells, we chemically crosslinked proteins in transfected cells using 3,3′-dithiobissulfosuccinimidyl propionate in the presence of N-ethyl maleimide. Stabled K1-Fas complexes were present in intact cells despite blocking of free reactive cysteines. The selective ability of K1 to antagonize Fas-mediated apoptosis was strictly dependent on having its immunoreceptor tyrosine-based activation motif (ITAM). Using activating anti-Fas (CH-11) and nonactivating anti-Fas (B10) antibodies in a serial manner, we showed that K1 associated with Fas that was not activated, strongly suggesting that K1 prevents Fas from being activated. Mice transfected with K1 were protected from the apoptosis inducing effects of agonistic Jo2 anti-Fas antibody and tissues from these mice contained K1-Fas complexes. K1-transfected compared to vector-transfected mice had fewer terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells (13 ± 3% vs. 60 ± 7%, P=0.008), fewer activated caspase 3 positive cells (24 ± 5% vs. 88 ± 6%, P=0.006). Tissues had less hemorrhaging, and necrosis.

Conclusion: These data support a mechanism of K1 direct binding to and blockage by K1 to Fas in suppression of apoptosis in an ITAM-dependent manner, which may be a key step in lymphoma development.
A Cost-Utility Analysis of Nilotinib for CML Patients Who Have Become Resistant to Imatinib.
Authors: Pilgrim H. , Jewitt K. , Ward S.
Published: October 2019

Background: New generation BCR-ABL tyrosine kinase inhibitors are promising treatments for CML patients who are imatinib resistant or intolerant. The objective of this study was to consider the cost-utility of nilotinib in comparison to ‘high dose’ (800 mg) imatinib (the current standard comparator in the UK) for chronic-phase CML patients resistant to standard dose 400 mg imatinib using a lifetime horizon. The analysis is based on a UK NHS perspective.

Methods: A Markov model was developed using interim data from the phase II CAMN107A2101 registration study of nilotinib and data from the 39 patients who were dose-escalated, due to a sub-optimal response, to 800 mg imatinib within the IRIS phase III trial of imatinib versus interferon. Based on this evidence, the estimated 18-month survival has been extrapolated over a lifetime using parametric Weibull regression. Since the price of nilotinib in the UK was still to be determined at the time of preparation, the cost was assumed to be £106.94 per day, price parity with high dose imatinib. The costs and utilities associated with adverse events have been incorporated during the first six months following treatment. All costs and utilities were discounted at 3.5%.

Results: Nilotinib is estimated to cost an additional £49K and generate an additional 2.18 Quality-Adjusted Life Year’s (QALYs) in comparison to high dose imatinib over a patient’s lifetime. Therefore, the incremental cost-utility of nilotinib versus high dose imatinib is expected to be approximately £22K per QALY gained. This result is subject to uncertainty around data extrapolation. However availability of longer term data from the ongoing nilotinib trial will allow validation at a later date.

Conclusions: Our analysis suggests that nilotinib provides a clinically and cost-effective treatment (according to conventionally acceptable thresholds) for CML patients who have become imatinib resistant, in an indication where limited alternative treatment options exist.
Prognostic Significance of VEGF, VEGF Receptors, and Microvessel Density in Diffuse Large B Cell Lymphoma Treated with Anthracycline-Based Chemotherapy.
Authors: Gratzinger Dita , Zhao Shuchun , Tibshirani Robert J. , Hsi Eric D. , Hans Christine P. , Pohlman Brad , Bast Martin , Avigdor Abraham , Schiby Ginette , Nagler Arnon , Byrne Gerald E. , Lossos Izidore S. , Natkunam Yasodha
Published: October 2019

Vascular endothelial growth factor (VEGF)-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of proliferation and/or survival induced by autocrine VEGF receptor-mediated signaling by lymphoma cells. We have recently shown that diffuse large B cell lymphomas expressing high levels of VEGF protein also express high levels of VEGF receptors 1 and 2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether either tumor vascularity as assessed by microvessel density counting, or expression of VEGF protein and its receptors as assessed by immunohistochemistry, contribute to patient outcomes. Increased tumor vascularity (Figure 1a, b, c -- increasing microvessel density) is associated with poor overall survival (p=0.047), and is independent of the international prognostic index (Figure 1d).

In contrast, high expression of VEGFR1 by lymphoma cells is associated with improved overall survival (p=0.044) and a trend toward improved progression-free survival (p=0.054). High expression of VEGF by lymphoma cells is also associated with a trend toward improved overall survival (p=0.089). The concordant trend toward improved survival with increased lymphoma cell expression of VEGF and its receptor VEGFR1, together with their coordinate overexpression in a subset of diffuse large B cell lymphomas (p=0.00025, chi value 21.5), suggests the presence of an autocrine VEGF-VEGFR1-mediated feedback loop in these lymphomas. Indeed the combination of high VEGF and VEGFR1 protein expression identifies a subgroup of patients with improved overall (p=0.003) and progression-free (p=0.026) survival; these findings are independent of the international prognostic index (Figure 2).

The presence of improved survival in a cohort of patients whose lymphomas potentially depend on autocrine signaling via VEGFR1 suggests that dependence on this pathway may render patients susceptible to the effects of anthracycline-based therapy; indeed, downregulation of VEGF expression at the mRNA level by anthracyclines and other chemotherapeutic agents has been demonstrated in a variety of non-hematolymphoid neoplasms. The relative importance of autocrine VEGF-mediated signaling versus vascularity should certainly be taken into account in clinical trials of anti-VEGF/VEGF receptor therapy in diffuse large B cell lymphoma.

Figure Figure Figure Figure
SB1518: A Potent and Orally Active JAK2 Inhibitor for the Treatment of Myeloproliferative Disorders.
Authors: Goh Kee C. , Ong Wai C. , Hu Changyong , Hentze Hannes , Liang Ai L. , Stunkel Walter , Tan Yong C. , Sangthongpitag Kanda , Goh Siok K. , Sieh Shirly , Bonday Zahid Q. , William Anthony D. , Lee Angeline , Blanchard Stephanie , Liu Te C. , Sattler Martin , Griffin James D. , Dymock Brian W. , Kantharaj Ethirajulu , Wood Jeanette M.
Published: October 2019

JAK2 is the most common mutated gene in bcr-abl-negative chronic myeloproliferative disorders (MPDs) making it an attractive target for drug discovery. Pre-clinical studies have shown that inhibition of JAK2 signaling pathways confer therapeutic benefits in animal models of MPD. We describe here SB1518, a potent, selective and orally active inhibitor of JAK2 with therapeutic potential for the treatment of MPDs. SB1518 is a potent ATP-competitive inhibitor of both JAK2 kinase (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) without significant activity against 40 other representative kinases across the human kinome. SB1518 selectively inhibits the proliferation of cell lines driven by JAK2 or its mutants (e.g. IC50 = 81 nM for a murine BaF3 cell line stably transfected to express erythropoietin receptor and GFP-labeled JAK2V617F). It reduces basal phosphorylation of JAK2 and STAT5 in BaF3-JAK2V617F cells in a concentration-dependent manner. The excellent physicochemical, metabolic and pharmacokinetic properties of SB1518 render it amenable to oral dosing. SB1518 was investigated in a model of advanced MPD established by intravenous injection of BaF3-JAK2V617F cells in nude mice. In several studies using different initial tumor burden and/or drug regimen, we observed dose-dependent and statistically significant therapeutic effects including normalization of elevated white blood cell count and reduction of GFP-labeled BaF3 cells in the peripheral blood, resolution of hepatosplenomegaly, reduction of phospho-STAT5 in diseased organs, prolonged survival and alleviation of terminal-stage anemia and thrombocytopenia. These therapeutic effects were observed at doses well tolerated by the animals. In conclusion, our data demonstrate the therapeutic potential of SB1518 for the treatment of myeloproliferative disorders caused by aberrant JAK2 signaling.
Significant Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPα Mutations.
Authors: Pulikkan John Anto , Dengler Viola , Peer Zada Abdul A. , Gelutu Mulu , Tenen Daniel G. , Behre Gerhard
Published: October 2019

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is crucial for the differentiation of granulocytes. Experimental data from animal models as well as patient samples suggest that loss of function or expression of C/EBPα provides a platform on which acute myeloid leukemia (AML) develops. C/EBPα is mutated in around 9% of acute myeloid leukemia. The mutations reported in C/EBPα are frame shift mutations at N-terminal domain and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. Peptidyl-prolyl cis/trans isomerase, Pin1 binds to and isomerizes the peptidyl-prolyl bond in specific phosphorylated Ser/Thr-Pro motifs. A growing number of studies show that Pin1 is overexpressed in many cancers and has significant role in tumorigenesis. In the present study we investigated the role of Pin1 in acute myeloid leukemia with C/EBPα mutation. Here we report C/EBPα-p30 could induce Pin1 transcription as assessed by quantitative Real-Time RT-PCR analysis. Affymetrix mRNA expression analysis show that Pin1 is upregulated in patients with acute myeloid leukemia. Silencing of Pin1 could overcome the dominant negative action of the C/EBPα-p30 over the C/EBPα-p42 transactivation capacity as analyzed by promoter assay. By silencing Pin1 with inhibitor against Pin1 (PiB), we could show myeloid differentiation in Kasumi-6 cells by FACS analysis with granulocytic specific markers. Western blot analysis shows that Pin1 inhibition by PiB could upregulate wild type C/EBPα protein level. Luciferase promoter assay for the Pin1 promoter shows that C/EBPα-p30 induces Pin1 promoter activity in association with E2F1. Mean while, wild type C/EBPα interferes with transactivation of the Pin1 promoter and downregulates Pin1 mRNA expression. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We have previously shown that c-Jun expression is high in AML patients with C/EBPα mutation and c-Jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that overexpression of Pin1 induces c-Jun mRNA expression, while inhibition of Pin1 by the inhibitor PiB downregulates c-Jun mRNA expression. We show that c-Jun blocks the DNA binding and transactivation of wild type C/EBPα protein as assessed by gel shift assay and promoter assay respectively. In conclusion, inhibition of Pin1 leads to granulocytic differentiation of human myeloid cells. Our findings suggest inhibition of Pin1 as a novel strategy in treating AML patients with C/EBPα mutation.
Anti-DKK1 mAb (BHQ880) as a Potential Therapeutic for Multiple Myeloma.
Authors: Fulciniti Mariateresa , Tassone Pierfrancesco , Hideshima Teru , Vallet Sonia , Ettenberg Seth , Stover David , Blotta Simona , Shen Zhenxin , Dimartino Maria Teresa , Neri Paola , Prabhala Rao , Nopur Raje , Goldring Steven R. , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Decreased activity of osteoblasts (OB) contributes to osteolytic lesions in multiple myeloma (MM). Dickkopf-1 (DKK1) is a soluble Wnt inhibitor produced by MM cells that inhibits osteoblastogenesis in the bone marrow (BM) microenvironment. DKK1, also present in MM patient plasma, has been shown to inhibit the differentiation of osteoblast precursor cells in vitro, and its plasma level correlates with focal bone lesions in MM. Therefore, we have evaluated DKK1 as a target in MM in the context of the BM microenvironment. We first analyzed the effects of a DKK1 neutralizing antibody (BHQ880) on OB differentiation. Anti-DKK1 Ab was able to increase differentiation of mesenchymal stem cells (MSCs) to OB and reduce IL-6 levels following MSC differentiation to OB. While OB activity in MSCs cultured with osteoblast differentiation media was reduced in the presence of INA-6 MM cells, treatment with DKK1 neutralizing antibody was able to restore OB activity in a dose-dependent manner, overcoming the negative effect of MM cells. We did not observe a direct effect of this Ab on growth or survival of human MM cell lines. However, when a panel of MM cell lines was cultured with BM stromal cells (BMSC), the Ab induced significant growth inhibitory effects on MM cells, associated with downregulation of IL-6 produced by MSCs. To evaluate the in vivo bone and antitumor effects of anti-DKK1 Ab treatment on MM cells in the human microenvironment, we used a SCID-hu murine model, using INA-6 MM cells. In this model we observed a direct correlation between the level of soluble human DKK1 in murine blood and the tumor growth. Following treatment with BHQ880, by bone histology analysis, we observed increased trabecular bone and numbers of OBs in the retrieved bone chip from BHQ880 treated mice compared to control mice, one month after initial dose. We also found an increase in human osteocalcin in the serum of BHQ880-treated mice compared to the controls, suggesting an increase in OB activity. Interestingly, we observed suppression of myeloma growth, measured by changes in serum level of soluble huIL6sR, 4 weeks following BHQ880 treatment of the mice. These results support DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of this molecule in MM to improve bone disease and to inhibit MM growth.
The In Vitro and In Vivo Antioxidant Effects of Erythropoietin in Thalassemia.
Authors: Fibach Eitan , Amer Johnny
Published: October 2019

Erythropoietin (EPO), a hormone produced on hypoxia mainly in the kidneys, enhances red blood cell (RBC) production (erythropoiesis) by stimulating the proliferation of erythroid progenitors and precursors in the bone marrow. This effect is mediated by the homodimeric EPO receptor, a class 1 cytokine receptor. Recombinant human EPO is widely used for the treatment of anemia, e.g., in patients on dialysis, patients with myelodysplastic syndrome and oncology patients undergoing chemotherapy. Treatment with EPO was also tried experimentally in patients with thalassemia. In these patients, in spite the state of chronic anemia, the levels of EPO is usually low relative to the degree of anemia. Administration of EPO to thalassemic patients have been shown to increase erythropoiesis and in some cases to elevate fetal hemoglobin production. In addition, EPO has been suggested to have cardio- and neuro-protecting effects and to increase RBC survival in dialysis patients. We have previously shown that RBC and platelets derived from patients with beta-thalassemia and sickle cell disease are under oxidative stress; they have elevated potential to generate reactive oxygen species (ROS) and membrane lipid peroxides, and have lower content of reduced glutathione (GSH) than normal RBC. This oxidative stress resulted in high exposure of phosphatidylserine (PS) that is considered a major factor in shortening the life span of thalassemic RBC, and in the tendency of platelets to undergo activation and thus contributes to the high incidence of thromboembolic complications in thalassemia. In the present study, we investigated the in vitro and in vivo effects of EPO as an antioxidant on RBC and platelets from beta-thalassemic patients and mice. Using flow-cytometry methodology, we showed that in vitro treatment of blood cells from beta-thalassemic patients with 1–4 U/ml of EPO for 2 hours increased the GSH content of RBC (2.1-fold) and platelets (1.7-fold) and reduced their ROS (1.5-fold), membrane lipid peroxidation and externalization of PS. Intraperitoneal injection of EPO to heterozygotes (Hbbth3/+) beta-thalassemic mice (3,000U/kg) significantly reduced ROS and increased GSH in their RBC within 3 hours. The in vitro effects of EPO on oxidative stress resulted in reduced sensitivity of thalassemic RBC to undergo hemolysis and phagocytosis by macrophages, and reduced tendency of platelets to undergo activation, as reflected by fewer platelets carrying external PS. Our results suggest that in addition to its effect on erythropoiesis and fetal hemoglobin production, EPO might alleviate symptoms in thalassemia and other hemolytic anemias as a potent antioxidant.
Outcomes of Second Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) in Children.
Authors: Schechter Tal , Avila Laura , Dupuis L.Lee , Pollock-BarZiv Stacey , Gassas Adam , Finkelstein Yaron , Armstrong Christine , Doyle John J.
Published: October 2019

Introduction: Relapse of the malignancy, secondary malignancy and rejection of the graft (GF) remain important risk factors for mortality after HSCT. The optimal treatment strategy for GF is second HSCT, but for recurrent malignancy it has yet to be determined; however, second HSCT provides a chance for long term survival. This study reports outcomes and clinical experience of HSCT retransplantation in pediatric patients with GF, relapse or secondary malignancy after primary HSCT.

Methods: Outcomes of all pediatric recipients of second HSCT for recurrent malignancy or GF between June 1, 1996 and June 30, 2006 at SickKids Hospital were reviewed. Demographic data, the conditioning regimen chosen, morbidity and mortality after second HSCT were described. EFS was defined as evidence of relapse of malignancy or failure to engraft. Univariate analysis was utilized to assess factors that may influence survival.

Results: Forty-one children (25 male; median age 7.8yrs (range 0.6–17.5yrs)) received a second HSCT during the study period. Initial diagnosis was malignancy in 25 (AML/MDS-11, ALL-6, lymphoma-4, CML-3, JMML-1) and nonmalignant disease in 16 (SAA-4, SCID-4, inherited marrow failure symdrome-5, HLH- 1, MPS-1, thalasemia-1). Indications for second HSCT were: malignancy (recurrent/ secondary) in 21 and GF in 20. Mean time to second HSCT in children with GF was 10.5 months (range: 0.8–57.2 months) compared to 22.7 months (range: 10.1–58.1months) for malignancy (p=0.004). All patients transplanted for malignancy received the second HSCT ≥ 6 months from the first HSCT and only 5 (24%) were transplanted within a year from first HSCT. 8 patients (40%) receiving a second HSCT for GF were retransplanted within the first 6 months. Conditioning regimens at second HSCT included myeloablative treatment (23); reduced intensity (RIC) (16) and none (2). The following factors were not statistically associated with survival: underlying disease, type of conditioning regimen (myeloablative vs reduced intensity) and indication for second HSCT (GF vs. malignancy). Overall survival (OS) and EFS rates are summarized below. Non-relapse related mortality was 45%; 9 patients died from infections.

parameters OS EFS Underlying disease Malignant 0.37±0.1 Non-Malignant 0.49±0.13 p=0.64 Reason for second HSCT Recurrent/secondary malignancy 0.43±0.11 Graft failure 0.42±0.12 p=0.69 Conditioning regimen Myeloablative 0.48±0.1 Reduce-intensity 0.33±0.13 p=0.3 All patients 0.425 0.44

Discussion and conclusion: Children who underwent second HSCT had an OS of 43%. We conclude that a second HSCT can result in prolonged EFS in children relapsing more than 6months after first HSCT and provides an acceptable therapeutic option for patients with GF.
Defibrotide in Prevention of Liver Toxicity in Patients at High Risk of VOD after HSC Transplantation.
Authors: Milone G. , Poidomani M. , Coppoletta S. , Mauro E. , Marturano E. , Crispi F. , Spadaro A. , Di Marco A. , Leotta S.
Published: October 2019

Defibrotide has been proposed as preventive treatment of Veno Occlusive Disease (VOD), data on its use are, however, limited and its effectiveness not yet demonstrated. We have administered Defibrotide as prevention of VOD in pts treated with HSC transplantation because of haematological malignancies, all patients were at high VOD risk because of hyper-ferritinemia (>800 ng/ml) or because not in CR of their underlying disease at time of transplant or being overweight (Actual BW>20% of Ideal BW) or because Hepatitis virus B or C sero-positive. 120 pts were treated with Defibrotide, 77 pts received allogeneic HSC tx and 43 autologous HSC tx, 105 received a myeloablative conditioning (55 pts it was based on busulfan) and 15 pts received a RIC. 48% of patients were affected with Acute leukemia, 23% with Lymphomas, 17% with Multiple Myeloma, 12% by other malignancies. Defibrotide was administered i.v. at dosage of 600 mg/d. from the day of conditioning was started to day +25 together with heparin at low dose (100 IU/Kg c.i.).

RESULTS: after prophylaxis with Defibrotide a bilirubin value above 2.5 mg/dl during the first 30 days was observed in 16/120 pts (13%), 7/120 pts (5%) reached Seattle’s VOD criteria but had spontaneous resolution of liver toxicity, only 1 patient (0.8%) had a severe VOD and MOF. Overall survival at 3 years was 60% and it was 54% in allo-transplanted patients. We compared results with those obtained in a group of 78 pts treated by allogeneic or autologous transplants and who received low dose heparin only and not Defibrotide because they were considered at low risk of VOD. Percentage of pts who developed a bilirubin level > 2.5 mg/dl (p=0.12) and percentage of patients that reached criteria for VOD were not different (p= 0.08) in these two groups, numbers of red blood cell transfusions were comparable (p=0.07). When all the 198 studied patients were analyzed using logistic regression for factors important in the development of a bilirubinemia above 2.5 mg/dl we found to be important: use of MTX as prophylaxis of GVHD (P=0.004; Odds ratio 5,153), allogeneic transplant (P=0.007; Odds ratio 7,127) and baseline value of bilirubin (P=0.02; Odds Ratio 4,690).

Conclusions: Use of Defibrotide in prophylaxis of VOD after HSC transplantation is safe and when employed in patients at high risk of VOD, it leads to an incidence of severe VOD below 1% with a frequency of liver toxicity equivalent to that found in patients having low risk features. To conclude on efficacy of Defibrotide in comparison to low dose heparin alone, a large randomised comparison is warranted.
Outcomes for Unrelated Donor Hematopoietic Cell Transplantation (URD-HCT) for Patients with Acute Lymphoblastic Leukemia (ALL) Using Fractionated Total Body Irradiation (FTBI) and Etoposide (VP16).
Authors: O’Donnell Margaret , Parker P , Dagis A , Slovak ML , Snyder D , Stein A , Spielberger R. , Pawlowska A , Rosenthal J , Palmer J , Kogut N. , Forman S. J
Published: October 2019

Although treatment outcomes for adult ALL remain stagnant at 35–40% 5 yr event free survival (EFS), allogeneic transplant particularly from unrelated donors has frequently been reserved for patient beyond first complete remission (CR1) with the exception of patients with a t(9;22) or Philadelphia (Ph+) cytogenetic abnormality. Between 4/1/2001 and 11/14/2007, 67 patients with ALL received URD-HCT; disease status at HCT was (CR1) in 27 pts (40%), CR2 in 20 pts (30%) and advanced disease (AD) which included induction failure, relapse or CR3 in 20 pts (30%). Age ranged from 2–58 yrs with median age of 29.5 yr. One third of the patients were Ph+. An additional 18 pts had high or very high risk cytogenetics abnormalities. The majority (≥75%) of Ph+ patients had received Gleevec prior to HCT. The conditioning regimen was FTBI 1320 Gy given as 11 fractions over four days followed by VP-16 60 mg/kg. Graft vs. host disease (GVHD) prophylaxis was tacrolimus (FK) and methotrexate (MTX) in 20 pts; sirolimus (Siro) was added to this regimen in 15 pts. Other GVHD regimens used were FK, MTX and Cellcept (12 pts) FK and siro (4 pts) and anti thymocyte globulin was added in 11 pts. Graft source was marrow in 12 pts and peripheral blood stem cells in 55 pts. Median time to ANC > 500 was 17 days (range 11–36d) and for platelets was 24 days (range 15–218d). Three patients died prior to day 18 of infectious complications and were considered unevaluable for engraftment. The incidence of acute GVHD Grade II-IV was 57% with Grade III-IV in 18%. Chronic GVHD occurred in 52% of 56 patients at risk. Treatment related mortality was 16% at 100 days and 28% at 1yr. At two years the event free survival (EFS) was 58% for CR1 pts vs 23% for CR2 and 17% for AD. Relapse rates were 18%, 24% and 40% respectively EFS was 62% at 2 yrs for Ph- pts and 50% for Ph+ in CR1 vs 19% for pt > CR1 who were Ph-and 40% for Ph+ (p=0.31). Remission status (CR1 vs > CR1) was a significant predictor of survival (p=0.01) and EFS (p=0.03) in multivariate analysis while age and Ph+ status had no impact on survival end points.

Conclusion: The preparative regimen of FTBI and VP16 offers 2 yr EFS rates of 50–60% in patients receiving URD-HCT in CR1, including patients with Ph+ and other high risk cytogenetic risk groups. These results are comparable to those reported with sibling donor transplants and provide a curative option for patients in CR1 lacking a family donor.

Figure Figure
Reduced-Intensity Conditioning for Allograft (RICT) after Cytoreductive Autograft (ASCT) in Relapsed/Resistant Hodgkin’s Lymphoma (HL)
Authors: Carella A. M. , Todisco E. , Castagna L. , Santoro A. , Catania G. , Nati S , Congiu A , Vimercati R , Rossi E , Spriano M , Varaldo R
Published: October 2019

Reduced-intensity conditioning for transplant (RICT) aims to exploit graft vs lymphoma (GvLy) effects while reducing conditioning-related toxicity. Because GvLy responses might be insufficient when HL are bulky and lymphoma growth is rapid, we pionered that intensive cytoreduction prior to RICT may allow GvLy reaction to be exploited (Carella et al. JCO2000; 18:3918). Thirty-eight patients with relapsed (n=26) or refractory (n=12) HL underwent RICT from an HLA-identical sibling preceeded by ASCT. Previous therapy consisted of 2–6 lines. High-dose therapy with ASCT consisted of BEAM protocol (n=29) or melphalan 200 mg/m2 (n=9). RICT consisted of fludarabine-cyclophosphamide (n=30) or fludarabine-melphalan (n=8). The two groups had similar prognostic factors. The median time to neutrophils and platelets recovery was 10 days and 16 days, respectively. Chimerism studies indicated 100% donor-derived engraftment. Day 100 and cumulative (2 yrs) TRM were 5,3 % (2 pts) and 18% (7 pts), respectively. Seventeen patients (44%) are alive (12 in complete remission and 5 with stable disease) with a median follow-up of 41 months (7–110 months). Twenty-one patients expired (TRM n=7, disease progression n=14). In conclusion, tandem ASCT/RICT is feasible and effective salvage therapy for patients with advanced HL. The long-term results obtained appear encouraging.
Higher Incidence of Relapse with Peripheral Blood Rather Than Marrow as a Source of Stem Cells in Adults with Acute Myelocytic Leukemia, but Not in Adults with Acute Lymphocytic Leukemia Autografted during the First Remission
Authors: Gorin Norbert C , Labopin Myriam , Polge Emmanuelle , Samey Benedicte , Rocha Vanderson
Published: October 2019

The cell source for autologous stem cell transplantation has shifted since 1994 from bone marrow (BM) to peripheral blood (PB). However, no study has compared outcomes. We analyzed 2165 adults with acute myelocytic leukemia (AML) and 1545 adults with acute lymphocytic leukemia (ALL) in first complete remission (CR1) who received autografts (AML: 1607 PB and 558 BM, ALL: 1126 PB and 416 BM) from 1994 to 2006.

AML: Relative to the time of CR1, PB transplants were performed earlier than BM transplants. Since a poorer outcome was associated with a shorter interval from CR1 to PB transplantation, patients were divided into three groups: BM, early PB (≤ 80 days after CR1) and late PB transplants. BM recipients were younger, and more received total body irradiation. In multivariate analysis, transplant-related mortality was not different among groups, but relapse incidence (RI) was higher for early PB (p = .0006) and late PB (p = .01) compared to BM (56 ± 3%,.46 ± 2%, 39 ± 2% respectively). Earlier PB (p= .02) and later PB transplants (p= .06) were associated with a lower three year LFS rate than marrow (36± 3%, 46 ± 2%, 52± 2% respectively).

ALL:: In multi variate analysis the only poor prognostic factors were a high white cell count at diagnosis and cytogenetics (presence of the Phi/bcr-abl). The RI and LFS at 2 years were identical in patients receiving BM and PB ( 45 ± 3% vs 47 ± 2% and 47 ± 3% vs 46 ± 2% respectively)

In conclusion, in AML patients autografted in CR1 but not in ALL, risk of relapse was lower with BM than with PB, independent of the CR1 to transplantation interval.
Double Transplantation with Melphalan (200 mg/m2) Compared with Triple Transplantation with Intermediate Dose Melphalan (100 mg/m2) in Patients with Multiple Myeloma
Authors: Ludwig Heinz , Spicka Ivan , Linkesch Werner , Greil Richard , Kasparu Hedwig , Gunsilius Eberhard , Thaler Josef , Drach Johannes , Kuhn Ingrid , Weiβmann Adalbert , Hinke Axel
Published: October 2019

Previous studies showed a survival advantage of double over single transplantation in proportion of patients. In this study we aimed to explore whether triple transplantation with intermediate melphalan 100mg/m2 (M100) yields at least similar treatment results with less toxicity than double transplantation with melphalan 200mg/m2 (M200). 219 Patients were entered in the study and started on 3 cycles of VAD induction therapy followed by IEV (ifosfamide (2g/m2)-etoposide (150mg/m2), d1-3; epirubicin (50mg/m2) d1) and G-CSF (5mg/kg) from d4 for stem cell priming and collection. Thereafter, 178 patients were randomized to either double or triple transplantation with M200 or M100. Reasons for non-randomization were PD (n=21), toxic side effects during induction or insufficient mobilization (n=9), withdrawal of consent (n=3), too early (n=1), death (n=1), second neoplasia (n=1), protocol violation (n=2), other (n=3). Patients achieving better than PD had subsequently been randomized to prednisolone plus interferon-a2b (Schering Plough) 3MU TIW or to interferon only maintenance therapy. Median age was 58 years (range: 27–71 years, n=172). DS stage I: 7%, stage II: 18%, stage III: 75%, M-component: IgG: 60%, IgA: 24%, IgD: 1%, IgM: 1%, light chain: 11%, non-secretory:1%. QoL was assessed with the EORTC QLQ C30 instrument. Chi2 test for trend was used for comparison of response and survival endpoints were estimated by the Kaplan-Meier product limit method. Median follow up is 34 months. Response rates in 186 patients evaluable for response after VAD (≥ 2 cycles of VAD) were: CR: 10%, PR: 58%, SD: 21% and PD: 11%. Median time from start of VAD to start of transplantation was 141 in the double and 144 days in the triple transplantation arm. Response rates after transplantation did not differ between pts treated with double or triple transplantation (CR: 41% vs. 41%, PR: 50% vs. 49%, SD 8% vs. 8%, PD: 1% vs. 3%, p=0.91. Median PFS for both groups combined was 39 mos, but showed a tendency for shorter PFS in pts on triple transplantation (median 44 mos. vs. 38 mos; HR 1.18, 95% CI: 0.77–1.81, p=0.22). Median OS has not been reached as yet, but did not differ between both groups with 3-year rates of 85% and 81%, respectively, and a HR of 1.01. Except for more cases of grade 1 and 2 anemia in the triple transplantation arm, hematological toxicity was similar in both groups. There was a tendency for more grade 2–3 mucositis and grade 3–4 vomiting, nausea in the double transplantation group. In conclusion, triple transplantation with intermediate dose M resulted in similar response rates, slightly shorter PFS but similar OS and was associated with less mucositis and slightly less vomiting, and nausea.

Figure Figure
Superior Incidence of Progression-Free Survival (PFS) Following High- Dose Melphalan (HDM) and Autologous Stem Cell Transplant (ASCT) for Multiple Myeloma (MM) Patients Receiving Pre-Transplant Induction with IMiDs Versus Conventional VAD Induction Chemotherapy.
Authors: Holland H. Kent , Solomon Scott R. , Bashey Asad , Morris Lawrence E.
Published: October 2019

Previous randomized trials evaluating the benefit of high-dose Melphalan and ASCT for MM patients receiving conventional induction chemotherapy have demonstrated that patients who achieve either a CR or VgPR post transplant have a superior PFS and overall survival (OS) compared to those who do not. We report on our 6-year experience comparing the outcome benefit of newly diagnosed MM patients who received either conventional VAD versus IMiDs based induction pre-transplant therapy and the impact on response on 112 consecutive patients transplanted between 5/2002 through 2/2008. The median age was 58 years (33–74). Disease status was Stage III in 92 (82%), Stage II in 11 (10%) and Stage in 9 (8%) patients. Conventional VAD induction chemotherapy was administered pre-transplant in 36 patients and IMiDs (thalidomide, lenalidomide) based therapy in 76 patients. Single ASCT was administered in 78 patients and Tandem ASCT in 34 patients (8 received conventional therapy and 26 received IMiDs, p=NS). Pre-transplant achievement of CR/VgPR was observed in 1/36 (1%) of patients receiving VAD chemotherapy and in 15/76 (20%) of patients receiving IMiDs (p=.017). Post-transplant, 5/36 (14%) patients that received VAD conventional therapy and 39/76 (51%) that received IMiDs achieved CR/VgPR (p=.002). Patients that entered into a CR/VgPR had both a superior PFS and OS (Median PFS 1,118 days vs. 670 days, p=.02/HR.49; Median OS “Not achieved” vs. 1,387 days, p=.05/HR 0.36). Single vs. Tandem transplant had no impact on outcome. These observations support the use of IMiDs as pre-transplant induction therapy over historical VAD chemotherapy.

Figure Figure
Genes Dysregulated in a Murine Model of Leukemogenesis Comprise a Signature for Identification of Therapeutics in Humans.
Authors: Hassane Duane C , Ashton John M , Guzman Monica L , Jordan Craig T.
Published: October 2019

The identification of genes and pathways that are critical for the development and maintenance of leukemia is important for designing new anti-leukemia therapeutics. However, selection of aberrantly regulated genes and pathways and the translation of these features into targeted anti-cancer drugs represent a significant challenge. Studies have characterized the various oncogenic lesions associated with leukemia, and have shown that specific combinations of mutations are sufficient to induce malignancy in model systems. Thus, we sought to identify the gene signature arising from the introduction of cooperating oncogenic mutations into non-malignant cells. We hypothesized that compounds capable of maximally antagonizing the aberrant expression of this subset of genes would represent potential anti-leukemia therapeutics. To test this hypothesis, we turned to the use of murine genetics and chemical genomics. To this end, we adapted mouse systems created by retroviral transduction of primary marrow cells with the BCR-ABL translocation alone or in combination with the NUP98-HOXA9 translocation. This approach creates authentic models of chronic and blast crisis myeloid leukemia (CML and bcCML, respectively), which has been shown to closely resemble human disease. Next, we employed flow cytometry to separate the transduced marrow into purified primitive lin- populations of either normal, BCR-ABL+, or BCR-ABL+/NUP98-HOXA9+ cells. Importantly, lin- cells were used to focus specifically on those genes most relevant to leukemic stem and progenitor populations. Microarray gene expression profiling was performed on these purified populations in six independent experiments using the Affymetrix Mouse 430 2.0 platform. Gene expression signatures for each transduced population relative to normal lin- cells were obtained. Next, in silico screening for agents that antagonize these signatures was performed comparing the resulting signatures to a local microarray compendium that included the CMap dataset. Gene expression signatures arising from the introduction of BCR-ABL alone identified traditional chemotherapeutics including cytarabine, etoposide, idarubicin – agents considered less effective against leukemic stem cell populations – as well as agents acting along the PI3 kinase pathway. Surprisingly, drugs shown to antagonize the gene signature derived by the introduction the two oncogenic mutations (BCR-ABL and NUP98-HOXA9) included 15-delta-prostaglandin-J2, celastrol, MG- 132, and parthenolide. Each of these drugs eradicate malignant stem and progenitor cells, while sparing normal cells. Thus, the murine-derived signature successfully identified drugs highly relevant to human disease. Our studies therefore demonstrate the utility of combining gene expression-based drug screening and mouse cancer genetics to reveal novel anti-leukemia compounds in humans. DCH and JMA contributed equally.