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1.  Effect of Cord Blood Processing on Transplant Outcomes after Single Myeloablative Umbilical Cord Blood Transplantation 
Variations in cord blood manufacturing and administration are common, and the optimal practice, not known. We compared processing and banking practices at 16 public cord blood banks (CBB) in the United States, and assessed transplant outcomes on 530 single umbilical cord blood (UCB) myeloablative transplantations for hematologic malignancies, facilitated by these banks. UCB banking practices were separated into three mutually exclusive groups based on whether processing was automated or manual; units were plasma and red blood cell reduced or buffy coat production method or plasma reduced. Compared to the automated processing system for units, the day-28 neutrophil recovery was significantly lower after transplantation of units that were manually processed and plasma reduced (red cell replete) (odds ratio [OR] 0.19 p=0.001) or plasma and red cell reduced (OR 0.54, p=0.05). Day-100 survival did not differ by CBB. However, day-100 survival was better with units that were thawed with the dextran-albumin wash method compared to the “no wash” or “dilution only” techniques (OR 1.82, p=0.04). In conclusion, CBB processing has no significant effect on early (day 100) survival despite differences in kinetics of neutrophil recovery.
PMCID: PMC4359657  PMID: 25543094
2.  Impact of conditioning intensity in T-replete haplo-identical stem cell transplantation for acute leukemia: a report from the acute leukemia working party of the EBMT 
Increasing numbers of patients are receiving haplo-identical stem cell transplantation (haplo-SCT) for treatment of acute leukemia with reduced intensity (RIC) or myeloablative (MAC) conditioning regimens. The impact of conditioning intensity in haplo-SCT is unknown.
We performed a retrospective registry-based study comparing outcomes after T-replete haplo-SCT for patients with acute myeloid (AML) or lymphoid leukemia (ALL) after RIC (n = 271) and MAC (n = 425). Regimens were classified as MAC or RIC based on published criteria.
A combination of post-transplant cyclophosphamide (PT-Cy) with one calcineurin inhibitor and mycophenolate mofetil (PT-Cy-based regimen) for graft-versus-host disease (GVHD) prophylaxis was used in 66 (25 %) patients in RIC and 125 (32 %) in MAC groups. Patients of RIC group were older and had been transplanted more recently and more frequently for AML with active disease at transplant. Percentage of engraftment (90 vs. 92 %; p = 0.58) and day 100 grade II to IV acute GVHD (24 vs. 29 %, p = 0.23) were not different between RIC and MAC groups. Multivariable analyses, run separately in AML and ALL, showed a trend toward higher relapse incidence with RIC in comparison to MAC in AML (hazard ratio (HR) 1.34, p = 0.09), and no difference in both AML and ALL in terms of non-relapse mortality (NRM) chronic GVHD and leukemia-free survival. There was no impact of conditioning regimen intensity in overall survival (OS) in AML (HR = 0.97, p = 0.79) but a trend for worse OS with RIC in ALL (HR = 1.44, p = 0.10). The main factor impacting outcomes was disease status at transplantation (HR ≥ 1.4, p ≤ 0.01). GVHD prophylaxis with PT-Cy-based regimen was independently associated with reduced NRM (HR 0.63, p = 0.02) without impact on relapse incidence (HR 0.99, p = 0.94).
These data suggest that T-replete haplo-SCT with both RIC and MAC, in particular associated with PT-Cy, are valid options in first line treatment of high risk AML or ALL.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-016-0248-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4791867  PMID: 26980295
Allogeneic stem cell transplantation; Haplo-identical donor; Conditioning regimen; Acute Leukemia; Toxicity; Anti-leukemic effect
3.  Prediction of Hematopoietic Stem Cell Transplantation Related Mortality- Lessons Learned from the In-Silico Approach: A European Society for Blood and Marrow Transplantation Acute Leukemia Working Party Data Mining Study 
PLoS ONE  2016;11(3):e0150637.
Models for prediction of allogeneic hematopoietic stem transplantation (HSCT) related mortality partially account for transplant risk. Improving predictive accuracy requires understating of prediction limiting factors, such as the statistical methodology used, number and quality of features collected, or simply the population size. Using an in-silico approach (i.e., iterative computerized simulations), based on machine learning (ML) algorithms, we set out to analyze these factors. A cohort of 25,923 adult acute leukemia patients from the European Society for Blood and Marrow Transplantation (EBMT) registry was analyzed. Predictive objective was non-relapse mortality (NRM) 100 days following HSCT. Thousands of prediction models were developed under varying conditions: increasing sample size, specific subpopulations and an increasing number of variables, which were selected and ranked by separate feature selection algorithms. Depending on the algorithm, predictive performance plateaued on a population size of 6,611–8,814 patients, reaching a maximal area under the receiver operator characteristic curve (AUC) of 0.67. AUCs’ of models developed on specific subpopulation ranged from 0.59 to 0.67 for patients in second complete remission and receiving reduced intensity conditioning, respectively. Only 3–5 variables were necessary to achieve near maximal AUCs. The top 3 ranking variables, shared by all algorithms were disease stage, donor type, and conditioning regimen. Our findings empirically demonstrate that with regards to NRM prediction, few variables “carry the weight” and that traditional HSCT data has been “worn out”. “Breaking through” the predictive boundaries will likely require additional types of inputs.
PMCID: PMC4778768  PMID: 26942424
4.  Chemotherapy Dose Adjustment for Obese Patients Undergoing Hematopoietic Stem Cell Transplantation: A Survey on Behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation 
The Oncologist  2014;20(1):50-55.
An electronic survey was used to assess current practice of dose adjustment of chemotherapy in obese and overweight patients undergoing hematopoietic stem cell transplantation. It revealed large diversity among transplant centers regarding dose-adjustment practice. This novel survey is an important step toward defining the right dose adjustment for pretransplantation conditioning to improve efficacy, to reduce toxicity, and thus to improve transplantation outcome.
Appropriate chemotherapy dosing for obese patients with malignant diseases is a significant challenge because limiting chemotherapy doses in these patients may negatively influence outcome. There is a paucity of information addressing high-dose chemotherapy in obese patients undergoing hematopoietic stem cell transplantation (HSCT).
The Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT) designed an electronic survey to assess current practice of dose adjustment of chemotherapy in obese patients undergoing HSCT.
A total of 56 EBMT centers from 27 countries responded to the online survey. Overall, 45 centers declared that they routinely adjust chemotherapy doses for obese patients (80.5%), and only 11 (19.5%) declared they do not adjust dose. Among the former group, most used body mass index as the parameter for defining obesity (28 centers, 62%). The method for determining the weight for chemotherapy calculation was actual body weight (ABW) in 16 centers, ideal body weight (IBW) in 10 centers, IBW plus 25% of the difference between IBW and ABW in 16 centers, and other methods for the rest. Among centers that used dose adjustment, 44% also capped the dose at 2 m2 for a chemotherapy dose based on body surface area (BSA), whereas 56% did not cap. Interestingly, most of the centers (9 of 11) that did not adjust dose for weight also did not cap the BSA at 2 m2.
This EBMT survey revealed large diversity among transplant centers regarding dose-adjustment practice for high-dose conditioning chemotherapy. Our next step is to analyze outcomes of transplantation according to dose-adjustment practice and, subsequently, to formulate a methodology for future prospective studies.
PMCID: PMC4294603  PMID: 25480827
Obesity; Dose adjustment; Hematopoietic stem cell transplantation; Conditioning
5.  Economics and Outcome After Hematopoietic Stem Cell Transplantation: A Retrospective Cohort Study 
EBioMedicine  2015;2(12):2101-2109.
Hematopoietic stem cell transplantation (HSCT) is a lifesaving expensive medical procedure. Hence, more transplants are performed in more affluent countries. The impact of economic factors on patient outcome is less defined. We analyzed retrospectively a defined cohort of 102,549 patients treated with an allogeneic (N = 37,542; 37%) or autologous (N = 65,007; 63%) HSCT. They were transplanted by one of 404 HSCT centers in 25 European countries between 1999 and 2006. We searched for associations between center-specific microeconomic or country-specific macroeconomic factors and outcome. Center patient-volume and center program-duration were significantly and systematically associated with improved survival after allogeneic HSCT (HR 0·87; 0·84–0·91 per 10 patients; p < 0·0001; HR 0·90;0·85–0·90 per 10 years; p < 0·001) and autologous HSCT (HR 0·91;0·87–0·96 per 10 patients; p < 0·001; HR 0·93;0·87–0·99 per 10 years; p = 0·02). The product of Health Care Expenditures by Gross National Income/capita was significantly associated in multivariate analysis with all endpoints (R2 = 18%; for relapse free survival) after allogeneic HSCT. Data indicate that country- and center-specific economic factors are associated with distinct, significant, systematic, and clinically relevant effects on survival after HSCT. They impact on center expertise in long-term disease and complication management. It is likely that these findings apply to other forms of complex treatments.
•Hematopoietic stem cell transplants (HSCTs) are expensive; economics plays a role on use and outcome.•102,549 patients treated with HSCT in 404 European centers between 1999 and 2006 were investigated.•Center program duration, patient volume, accreditation and country economics were associated with survival.•Effects were significant, clinically relevant but distinct for allogeneic and autologous HSCT.
Hematopoietic stem cell transplantation (HSCT) is lifesaving but expensive; it's more frequently used in richer countries. We asked whether economics impact on outcome. For 102,549 patients treated with an allogeneic or autologous HSCT in 404 European centers between 1999 and 2006 survival was significantly better in centers with longer program duration and a higher patient volume. Survival was better in economically advantaged countries. Data indicate distinct, significant, systematic, and clinically relevant effects on long-term disease and complication management by country- and center-specific economic factors. It is likely that these effects apply to other forms of complex treatments.
PMCID: PMC4703735  PMID: 26844291
Hematopoietic stem cell transplantation; Macroeconomics; Microeconomics; Center effect; Patient volume; Program duration; Outcome; Survival; GNI/cap; HCE/cap; HDI; Risk assessment
6.  Heparanase enhances myeloma progression via CXCL10 down regulation 
Leukemia  2014;28(11):2178-2187.
In order to explore the mechanism(s) underlying the pro-tumorigenic capacity of heparanase we established an inducible Tet-on system. Heparanase expression was markedly increased following addition of doxycycline (Dox) to the culture medium of CAG human myeloma cells infected with the inducible heparanase gene construct, resulting in increased colony number and size in soft agar. Moreover, tumor xenografts produced by CAG-heparanase cells were markedly increased in mice supplemented with Dox in their drinking water compared with control mice maintained without Dox. Consistently, we found that heparanase induction is associated with decreased levels of CXCL10, suggesting that this chemokine exerts tumor suppressor properties in myeloma. Indeed, recombinant CXCL10 attenuated the proliferation of CAG, U266 and RPMI-8266 myeloma cells. Similarly, CXCL10 attenuated the proliferation of human umbilical vein endothelial cells (HUVEC), implying that CXCL10 exhibits anti-angiogenic capacity. Strikingly, development of tumor xenografts produced by CAG-heparanase cells over expressing CXCL10 was markedly reduced compared with control cells. Moreover, tumor growth was significantly attenuated in mice inoculated with human or mouse myeloma cells and treated with CXCL10-Ig fusion protein, indicating that CXCL10 functions as a potent anti-myeloma cytokine.
PMCID: PMC4185261  PMID: 24699306
Heparanase; myeloma; CXCL10; tumor suppressor
7.  Unrelated cord blood transplantation for adult patients with acute myeloid leukemia: higher incidence of acute graft-versus-host disease and lower survival in male patients transplanted with female unrelated cord blood—a report from Eurocord, the Acute Leukemia Working Party, and the Cord Blood Committee of the Cellular Therapy and Immunobiology Working Party of the European Group for Blood and Marrow Transplantation 
In the setting of allogeneic human leukocyte antigen (HLA)-matched bone marrow transplantation, transplanting male patients with grafts from female donors has been associated with a higher incidence of graft-versus-host disease (GVHD) and of nonrelapse mortality (NRM). The aim of the current analysis was to compare transplantation outcomes in male patients given female unrelated cord blood (UCB) versus other gender combinations.
Patients and methods
Data from 552 consecutive patients with acute myeloid leukemia (AML) given a single UCB transplantation between 2000 and 2014 were included.
In comparison with other gender combination, male patients given female UCB (n = 131) had a trend for a higher incidence of grades II–IV acute GVHD (33 versus 25 %, P = 0.08), a trend for a higher incidence of NRM (41 versus 33 %, P = 0.06), and a lower leukemia-free (LFS, 30 versus 41 %, P = 0.01) and overall survival (OS, 33 versus 45 %, P = 0.008). In multivariate analyses, taking into consideration all patients for which data on HLA-matching and cell dose transplanted were fully available (n = 363), male patients transplanted with a female UCB had a trend for a higher incidence of grade III–IV acute GVHD (hazard ratio (HR) = 2.0, P = 0.06), a trend for a higher NRM (HR = 1.5, P = 0.06), and a worse LFS (HR = 1.4, P = 0.04) and OS (HR = 1.3, P = 0.06).
Our data suggest that male patients transplanted with female UCB might have higher risk of acute GVHD and of NRM leading to worse LFS and OS. These results should be confirmed in other large cohorts of patients before used for determining the choice of an UCB unit.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-015-0207-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4594748  PMID: 26445106
Unrelated cord blood; Female; Male; AML; GVHD; Transplantation
8.  Neurotherapeutic Effect of Cord Blood Derived CD45+ Hematopoietic Cells in Mice after Traumatic Brain Injury 
Journal of Neurotrauma  2014;31(16):1405-1416.
Treatment of traumatic brain injury (TBI) is still an unmet need. Cell therapy by human umbilical cord blood (HUCB) has shown promising results in animal models of TBI and is under evaluation in clinical trials. HUCB contains different cell populations but to date, only mesenchymal stem cells have been evaluated for therapy of TBI. Here we present the neurotherapeutic effect, as evaluated by neurological score, using a single dose of HUCB-derived mononuclear cells (MNCs) upon intravenous (IV) administration one day post-trauma in a mouse model of closed head injury (CHI). Delayed (eight days post-trauma) intracerebroventricular administration of MNCs showed improved neurobehavioral deficits thereby extending the therapeutic window for treating TBI. Further, we demonstrated for the first time that HUCB-derived pan-hematopoietic CD45 positive (CD45+) cells, isolated by magnetic sorting and characterized by expression of CD45 and CD11b markers (96–99%), improved the neurobehavioral deficits upon IV administration, which persisted for 35 days. The therapeutic effect was in a direct correlation to a reduction in the lesion volume and decreased by pre-treatment of the cells with anti-human-CD45 antibody. At the site of brain injury, 1.5-2 h after transplantation, HUCB-derived cells were identified by near infrared scanning and immunohistochemistry using anti-human-CD45 and anti-human-nuclei antibodies. Nerve growth factor and vascular endothelial growth factor levels were differentially expressed in both ipsilateral and contralateral brain hemispheres, thirty-five days after CHI, measured by enzyme-linked immunosorbent assay. These findings indicate the neurotherapeutic potential of HUCB-derived CD45+ cell population in a mouse model of TBI and propose their use in the clinical setting of human TBI.
PMCID: PMC4132581  PMID: 24640955
brain trauma; CD45+ hematopoietic cells; cell transplantation; cord blood; neurotherapy
9.  Halofuginone inhibits phosphorylation of SMAD-2 reducing angiogenesis and leukemia burden in an acute promyelocytic leukemia mouse model 
Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-β (TGF-β) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model.
NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 μg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells.
HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-β-signaling.
Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-015-0181-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4486128  PMID: 26099922
Acute Promyelocytic Leukemia; Halofuginone; SMAD; TGF-β; VEGF; Angiogenesis
10.  Bioactive magnetic near Infra-Red fluorescent core-shell iron oxide/human serum albumin nanoparticles for controlled release of growth factors for augmentation of human mesenchymal stem cell growth and differentiation 
Iron oxide (IO) nanoparticles (NPs) of sizes less than 50 nm are considered to be non-toxic, biodegradable and superparamagnetic. We have previously described the generation of IO NPs coated with Human Serum Albumin (HSA). HSA coating onto the IO NPs enables conjugation of the IO/HSA NPs to various biomolecules including proteins. Here we describe the preparation and characterization of narrow size distribution core-shell NIR fluorescent IO/HSA magnetic NPs conjugated covalently to Fibroblast Growth Factor 2 (FGF2) for biomedical applications. We examined the biological activity of the conjugated FGF2 on human bone marrow mesenchymal stem cells (hBM-MSCs). These multipotent cells can differentiate into bone, cartilage, hepatic, endothelial and neuronal cells and are being studied in clinical trials for treatment of various diseases. FGF2 enhances the proliferation of hBM-MSCs and promotes their differentiation toward neuronal, adipogenic and osteogenic lineages in vitro.
The NPs were characterized by transmission electron microscopy, dynamic light scattering, ultraviolet–visible spectroscopy and fluorescence spectroscopy. Covalent conjugation of the FGF2 to the IO/HSA NPs significantly stabilized this growth factor against various enzymes and inhibitors existing in serum and in tissue cultures. IO/HSA NPs conjugated to FGF2 were internalized into hBM-MSCs via endocytosis as confirmed by flow cytometry analysis and Prussian Blue staining. Conjugated FGF2 enhanced the proliferation and clonal expansion capacity of hBM-MSCs, as well as their adipogenic and osteogenic differentiation to a higher extent compared with the free growth factor. Free and conjugated FGF2 promoted the expression of neuronal marker Microtubule-Associated Protein 2 (MAP2) to a similar extent, but conjugated FGF2 was more effective than free FGF2 in promoting the expression of astrocyte marker Glial Fibrillary Acidic Protein (GFAP) in these cells.
These results indicate that stabilization of FGF2 by conjugating the IO/HSA NPs can enhance the biological efficacy of FGF2 and its ability to promote hBM-MSC cell proliferation and trilineage differentiation. This new system may benefit future therapeutic use of hBM-MSCs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12951-015-0090-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4432958  PMID: 25947109
11.  Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents 
British journal of haematology  2012;157(6):718-731.
Multiple Myeloma (MM), a malignancy of plasma cells, remains incurable despite the use of conventional and novel therapies. Halofuginone (HF), a synthetic derivative of quinazolinone alkaloid, has recently been shown to have anti-cancer activity in various preclinical settings. This study demonstrated the anti-tumour activity of HF against a panel of human MM cell lines and primary patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. HF showed anti-MM activity in vivo using a myeloma xenograft mouse model. HF suppressed proliferation of myeloma cells alone and when co-cultured with bone marrow stromal cells. Similarly, HF induced apoptosis in MM cells even in the presence of insulin-like growth factor 1or interleukin 6. Importantly, HF, even at high doses, did not induce cytotoxicity against CD40 activated peripheral blood mononuclear cells from normal donors. HF treatment induced accumulation of cells in the G0/G1 cell cycle and induction of apoptotic cell death associated with depletion of mitochondrial membrane potential; cleavage of poly (ADP-ribose) polymerase and caspases-3, 8 and 9 as well as down-regulation of anti-apoptotic proteins including Mcl-1and X-IAP. Multiplex analysis of phosphorylation of diverse components of signalling cascades revealed that HF induced changes in P38MAPK activation; increased phosphorylation of c-jun, c-jun NH(2)-terminal kinase (JNK), p53 and Hsp-27. Importantly, HF triggered synergistic cytotoxicity in combination with lenalidomide, melphalan, dexamethasone, and doxorubicin. Taken together, these preclinical studies provide the preclinical framework for future clinical studies of HF in MM.
PMCID: PMC4414398  PMID: 22533681
Halofuginone; TGF-β; JNK; c-jun; p-53
12.  NK Cell Receptor NKp46 Regulates Graft-versus-Host Disease 
Cell reports  2014;7(6):1809-1814.
Hematopoietic stem cell transplantation (HSCT) is often the only curative treatment for a wide variety of hematologic malignancies. Donor selection in these diseases is crucial, given that transplanted cells can mediate not only the desired graft-versus-leukemia effect but also graft-versus-host disease (GVHD). Here, we demonstrate that in the absence of NKp46, a major killer receptor expressed by human and mouse natural killer (NK) cells, GVHD is greatly exacerbated, resulting in rapid mortality of the transplanted animals because of infection with commensal bacteria. Furthermore, we demonstrate that the exacerbated GVHD is the result of an altered ability of immune cells to respond to stimulation by immature dendritic cells. Because high and low expression of NKp46 on NK cells is observed in different individuals, our data indicate that choosing NKp46-high donors for the treatment of different hematologic malignancies might lead to better tumor eradication while minimizing GVHD.
PMCID: PMC4074424  PMID: 24882008
13.  P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway 
Biochemical pharmacology  2013;86(5):584-596.
The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors.
PMCID: PMC4000015  PMID: 23774624
Apoptosis; P-glycoprotein; TRAIL; Resistance; Modulators
14.  SH2B3 (LNK) mutations from Myeloproliferative Neoplasms patients have mild loss of function against wild type JAK2 and JAK2 V617F 
British journal of haematology  2013;161(6):811-820.
Somatic point mutations in the PH domain of SH2B3 (LNK), an adaptor protein that is highly expressed in haematopoietic cells, were recently described in patients with myeloproliferative neoplasms. We studied the effect of these mutations on the JAK2 signalling pathway in cells expressing either wild type JAK2 or the JAK2 V617F mutation. Compared to wild type SH2B3, PH domain mutants have mild loss of function, with no evidence for a dominant-negative effect. Mutants retain binding capacity for JAK2, an established SH2B3 target, as well as for the adaptor proteins 14-3-3 and CBL. Our data suggest that the loss of SH2B3 inhibitory function conferred by the PH domain mutations is mild and may collaborate with JAK2 V617F and CBL mutations in order to promote either the development or the progression of myeloproliferative neoplasms.
PMCID: PMC3672250  PMID: 23590807
MPN; JAK2 mutation; SH2B3 (LNK) mutation; 14-3-3; CBL
15.  Biosimilar G-CSF Based Mobilization of Peripheral Blood Hematopoietic Stem Cells for Autologous and Allogeneic Stem Cell Transplantation 
Theranostics  2014;4(3):280-289.
The use of granulocyte colony stimulating factor (G-CSF) biosimilars for peripheral blood hematopoietic stem cell (PBSC) mobilization has stimulated an ongoing debate regarding their efficacy and safety. However, the use of biosimilar G-CSF was approved by the European Medicines Agency (EMA) for all the registered indications of the originator G-CSF (Neupogen®) including mobilization of stem cells. Here, we performed a comprehensive review of published reports on the use of biosimilar G-CSF covering patients with hematological malignancies as well as healthy donors that underwent stem cell mobilization at multiple centers using site-specific non-randomized regimens with a biosimilar G-CSF in the autologous and allogeneic setting.
A total of 904 patients mostly with hematological malignancies as well as healthy donors underwent successful autologous or allogeneic stem cell mobilization, respectively, using a biosimilar G-CSF (520 with Ratiograstim®/Tevagrastim, 384 with Zarzio®). The indication for stem cell mobilization in hematology patients included 326 patients with multiple myeloma, 273 with Non-Hodgkin's lymphoma (NHL), 79 with Hodgkin's lymphoma (HL), and other disease. 156 sibling or volunteer unrelated donors were mobilized using biosimilar G-CSF. Mobilization resulted in good mobilization of CD34+ stem cells with side effects similar to originator G-CSF. Post transplantation engraftment did not significantly differ from results previously documented with the originator G-CSF. The side effects experienced by the patients or donors mobilized by biosimilar G-CSF were minimal and were comparable to those of originator G-CSF.
In summary, the efficacy of biosimilar G-CSFs in terms of PBSC yield as well as their toxicity profile are equivalent to historical data with the reference G-CSF.
PMCID: PMC3915091  PMID: 24505236
Biosimilar G-CSF; hematopoietic stem cells; mobilization; autologous & allogeneic transplantation; healthy donors
16.  Effect of HLA-Matching Recipients to Donor Noninherited Maternal Antigens on Outcomes after Mismatched Umbilical Cord Blood Transplantation for Hematologic Malignancy 
Transplantation-related mortality (TRM) is high after HLA-mismatched umbilical cord blood (UCB) transplantation (UCBT). In utero, exposure to noninherited maternal antigen (NIMA) is recognized by the fetus, which induces Tregulator cells to that haplotype. It is plausible that UCBTs in which recipients are matched to donor NIMAs may alleviate some of the excess mortality associated with this treatment. To explore this concept, we used marginal matched-pair Cox regression analysis to compare outcomes in 48 NIMA-matched UCBTs (ie, the NIMA of the donor UCB unit matched to the patient) and in 116 non–NIMA-matched UCBTs. All patients had a hematologic malignancy and received a single UCB unit. Cases and controls were matched on age, disease, disease status, transplantation-conditioning regimen, HLA match, and infused cell dose. TRM was lower after NIMA-matched UCBTs compared with NIMA-mismatched UCBTs (relative risk, 0.48; P=.05; 18% versus 32% at 5 years posttransplantation). Consequently, overall survival was higher after NIMA-matched UCBT. The 5-year probability of overall survival was 55% after NIMA-matched UCBTs versus 38% after NIMA-mismatched UCBTs (P=.04). When faced with the choice of multiple HLA-mismatched UCB units containing adequate cell doses, selecting an NIMA-matched UCB unit may improve survival after mismatched UCBT.
PMCID: PMC3826155  PMID: 22814031
Permissive match; Regulatory T cells; Fetal immune response
17.  Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC) 
Background: Several studies revealed that MSC from human bone marrow can downregulate graft-versus-host disease (GVHD) after allogeneic HSCT. Methods: Herein we present 50 patients with acute GVHD who got 74 (1-4) MSC infusions for 54 separate episodes of aGVHD. Results: aGVHD was defined as steroid resistant grade IV aGVHD in 42 cases. The major presentation was gastrointestinal GVHD; two (n=18) or more (n=21) systems were involved in the majority of cases. The 1st infusion with MSC was given on day +27 (range, 1 to 136); d+45 (range, +11 to +150) post diagnosis of aGVHD and HSCT, respectively. In 2/3 of the cases treatment was performed with frozen stocked MSCs; in 62 cases early passages (1-3) were used. The median number of infused cells was 1.14±0.47 million per kg in the first injection and up to 4.27 (1.70±1.10) millions in total. The two patients with aggressive liver GVHD received MSCs injections intra hepatic arteries without changes of blood flow or evidence cytolysis, but also without a visible effect. Disease free survival at 3.6 years was 56%. We observed better overall survival in patients with GVHD grade < 4, in responders to the 1st treatment with MSC, and in pediatric group. The multivariate analysis demonstrated independent influence on survival of initial response and younger age. There were no immediate or late toxicity or side effects. Conclusion: Injection of MSCs seems to be a promising and safe treatment of GVHD. The encouraging results obviously should be confirmed in a randomized prospective study.
PMCID: PMC3755522  PMID: 23997985
Mesenchymal stromal cells (MSC); mesenchymal stem cells; hematopoietic stem cell transplantation; graft versus host disease; steroid resistance
19.  Regulatory T Cells in Allogeneic Stem Cell Transplantation 
Growing evidence suggests that cellular adoptive immunotherapy is becoming an attractive though challenging approach in regulating tumor immunity and alloresponses in clinical transplantation. Naturally arising CD4+CD25+Foxp3+ regulatory T cells (Treg) have emerged as a key component in this regard. Over the last decade, a large body of evidence from preclinical models has demonstrated their crucial role in auto- and tumor immunity and has opened the door to their “first-in-man” clinical application. Initial studies in clinical allogeneic stem cell transplantation are very encouraging and may pave the way for other applications. Further improvements in Treg ex vivo or in vivo expansion technologies will simplify their global clinical application. In this review, we discuss the current knowledge of Treg biology and their potential for cell-based immunotherapy in allogeneic stem cell transplantation.
PMCID: PMC3662184  PMID: 23737813
20.  Older patients with normal karyotype acute myeloid leukemia have a higher rate of genomic changes compared to young patients as determined by SNP array analysis 
Leukemia Research  2011;36(4):467-473.
Older patients with AML have a worse outcome compared to young patients. To study for potential contributors to their poor prognosis, we compared two NK-AML cohorts, young (< 60 years old) and old (> 60 years old), via high density SNP array analysis. Older patients had more genomic changes (1.83±0.23 vs. 1.16±0.2, p=0.037) and a trend for a higher number of copy number neutral loss of heterozygosity (0.5±0.2 vs. 0.24±0.08, p=0.088) compared to young patients. We speculate that complex genomic changes in NK-AML may be a sign of an increase in genomic instability and an indicator of a worse prognosis.
PMCID: PMC3288295  PMID: 22071139
AML; Normal karyotype; SNP array; Old age
21.  Development of Allogeneic NK Cell Adoptive Transfer Therapy in Metastatic Melanoma Patients: In Vitro Preclinical Optimization Studies 
PLoS ONE  2013;8(3):e57922.
Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients. Until today most studies utilized autologous NK cells and yielded disappointing results. Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC). We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching. Combination of the various strategies yielded improved effects. In addition, we developed various clinically-compatible ex vivo expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors. The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes. This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.
PMCID: PMC3587427  PMID: 23483943
22.  Immunotherapy for Multiple Myeloma 
PMCID: PMC3546469  PMID: 23346187
23.  RNA Inhibition Highlights Cyclin D1 as a Potential Therapeutic Target for Mantle Cell Lymphoma 
PLoS ONE  2012;7(8):e43343.
Mantle cell lymphoma is characterized by a genetic translocation results in aberrant overexpression of the CCND1 gene, which encodes cyclin D1. This protein functions as a regulator of the cell cycle progression, hence is considered to play an important role in the pathogenesis of the disease. In this study, we used RNA interference strategies to examine whether cyclin D1 might serve as a therapeutic target for mantle cell lymphoma. Knocking down cyclin D1 resulted in significant growth retardation, cell cycle arrest, and most importantly, induction of apoptosis. These results mark cyclin D1 as a target for mantle cell lymphoma and emphasize the therapeutic potential hidden in its silencing.
PMCID: PMC3419170  PMID: 22905260
24.  Involvement of Host Stroma Cells and Tissue Fibrosis in Pancreatic Tumor Development in Transgenic Mice 
PLoS ONE  2012;7(7):e41833.
Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.
Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.
Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.
The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.
PMCID: PMC3404977  PMID: 22848627
25.  Factors Affecting Mortality following Myeloablative Cord Blood Transplantation in Adults: a Pooled Analysis of Three International Registries 
Bone Marrow Transplantation  2010;46(1):70-76.
A retrospective analysis was conducted to examine factors affecting early mortality following myeloablative, single-unit cord blood transplantation (CBT) for hematological malignancies in adolescents and adults. Data were collected from the three main CBT registries pooling 514 records of unrelated, single, unmanipulated, first myeloablative allogeneic CBTs conducted in North America or Europe from 1995 to 2005, with an HLA match ≥4/6 loci, in patients aged 12 to 55. Overall 100-, 180- day and 1-year survival (Kaplan-Meier method) were 56%, 46% and 37%, respectively, with no significant heterogeneity across registries. Multivariate analysis showed cell dose < 2.5×107/Kg (Odds Ratio [OR] 2.76, p<0.0001), older age (p=0.002), advanced disease (p=0.02), positive CMV sero-status (OR 1.37 p=0.11), female gender (OR 1.43, p=0.07) and limited CBT center experience (<10 records contributed, OR 2.08, p=0.0003) to be associated with higher 100-day mortality. A multivariate model predictive of 1-year mortality included similar prognostic factors except female gender. Transplant year did not appear as a significant independent predictor. This is the first analysis to pool records from three major CBT registries in the US and Europe. Despite some differences in practice patterns, survival was remarkably homogeneous. The resulting model may contribute to better understanding factors affecting CBT outcomes.
PMCID: PMC3366187  PMID: 20436518
Cord blood transplantation; registry; leukemia; mortality; leukemia

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