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1.  Integrating a prospective pilot trial and patient-derived xenografts to trace metabolic changes associated with acute myeloid leukemia 
Despite the considerable progress in understanding the molecular bases of acute myeloid leukemia (AML), new tools to link disease biology to the unpredictable patient clinical course are still needed. Herein, high-throughput metabolomics, combined with the other “-omics” disciplines, holds promise in identifying disease-specific and clinically relevant features.
In this study, we took advantage of nuclear magnetic resonance (NMR) to trace AML-associated metabolic trajectory employing two complementary strategies. On the one hand, we performed a prospective observational clinical trial to identify metabolic changes associated with blast clearance during the first two cycles of intensive chemotherapy in nine adult patients. On the other hand, to reduce the intrinsic variability associated with human samples and AML genetic heterogeneity, we analyzed the metabolic changes in the plasma of immunocompromised mice upon engraftment of primary human AML blasts.
Combining the two longitudinal approaches, we narrowed our screen to seven common metabolites, for which we observed a mirror-like trajectory in mice and humans, tracing AML progression and remission, respectively. We interpreted this set of metabolites as a dynamic fingerprint of AML evolution.
Overall, these NMR-based metabolomic data, to be consolidated in larger cohorts and integrated in more comprehensive system biology approaches, hold promise for providing valuable and non-redundant information on the systemic effects of leukemia.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-016-0346-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13045-016-0346-2
PMCID: PMC5086061  PMID: 27793157
Acute myeloid leukemia; Metabolomics; Patient-derived xenografts; Nuclear magnetic resonance
2.  Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer 
Nature medicine  2012;18(5):807-815.
The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile.
doi:10.1038/nm.2700
PMCID: PMC5019824  PMID: 22466705
3.  Design and Validation of an Augmented Hematopoietic Cell Transplantation-Comorbidity Index Comprising Pre-transplant Ferritin, Albumin and Platelet Count for Prediction of Outcomes after Allogeneic Transplantation 
Purpose
Pre-transplant values of serum ferritin, albumin and peripheral blood counts were previously suggested to provide prognostic information about hematopoietic cell transplantation (HCT) outcomes. Whether these “biomarkers” have prognostic value independent of each other and the HCT-comorbidity index (HCT-CI) is unknown.
Patients and Methods
We analyzed data from 3917 allogeneic HCT recipients at multiple sites in the US and Italy using multivariate models including each biomarker and the HCT-CI. Data from all sites were then randomly divided into a training set (n=2352) to develop weights for the relevant biomarkers to be added to the HCT-CI scores and a validation set (n=1407) to validate an augmented HCT-CI compared to the original index.
Results
Multivariate analysis with data from one site showed that ferritin, albumin and platelets-- not neutrophils or hemoglobin--were independently associated with increased non-relapse mortality (NRM) and decreased overall survival. Findings were validated in data from the other sites. Subsequently, in a training set from all sites, ferritin >2500 mg/dL (HR:1.69); albumin 3–3.5 g/dL (HR:1.61) and <3.0 g/dL (HR:2.27); and platelets 50–<100,000 (HR:1.28), 20–<50,000 (HR:1.29) and <20,000 (HR:1.55) were statistically significantly associated with NRM. Weights were assigned to these laboratory values following the same equation used to design the original index. In the validation set, The addition of the biomarkers to the original index to develop an augmented HCT-CI resulted in a statistically significant increase in higher c-statistic estimate for prediction of NRM. (p=0.0007).
Conclusion
Ferritin, albumin, and platelet counts are important prognostic markers that further refine the discriminative power of the HCT-CI for transplant outcomes.
doi:10.1016/j.bbmt.2015.04.002
PMCID: PMC4506728  PMID: 25862589
5.  RIC versus MAC UCBT in adults with AML: A report from Eurocord, the ALWP and the CTIWP of the EBMT 
Oncotarget  2016;7(28):43027-43038.
Nonrelapse mortality (NRM) is the first cause of treatment failure after unrelated cord blood transplantation (UCBT) following myeloablative conditioning (MAC). In the last decade, reduced-intensity conditioning (RIC) regimens have been developed with the aim of reducing NRM and allowing older patients and those with medical comorbidities to benefit from UCBT. The aim of the current retrospective study was to compare transplantation outcomes of acute myeloid leukemia (AML) patients given UCBT after either RIC or MAC. Data from 894 adults with AML receiving a single or double UCBT as first allograft from 2004 to 2013 at EBMT centers were included in this study. 415 patients were given UCBT after RIC while 479 patients following a MAC. In comparison to MAC recipients, RIC recipients had a similar incidence of neutrophil engraftment and of acute and chronic graft-versus-host disease (GVHD). However, RIC recipients had a higher incidence of disease relapse and a lower NRM, translating to comparable leukemia-free (LFS), GVHD-free, relapse-free survival (GRFS) and overall survival (OS). These observations remained qualitatively similar after adjusting for differences between groups in multivariate analyses. In conclusion, these data suggest that LFS and OS are similar with RIC or with MAC in adults AML patients transplanted with UCBT. These observations could serve as basis for a future prospective randomized study.
doi:10.18632/oncotarget.9599
PMCID: PMC5190005  PMID: 27250025
unrelated cord blood; AML; reduced-intensity; myeloablative; transplantation
6.  In Vivo Tracking of Human Hematopoiesis Reveals Patterns of Clonal Dynamics during Early and Steady-State Reconstitution Phases 
Cell Stem Cell  2016;19(1):107-119.
Summary
Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.
Graphical Abstract
Highlights
•Hematopoietic reconstitution occurs in two distinct clonal waves•A few thousand HSPC clones stably sustain multilineage blood cell production•Steady-state hematopoiesis after transplant is maintained by both HSCs and MPPs•Natural killer clones have closer relationships to myeloid cells than to lymphoid cells
Biasco et al. report a clonal tracking study on the dynamics and nature of hematopoietic reconstitution in humans after transplant. Using integration sites as molecular tags, they measured, in gene therapy patients, repopulating waves, population size and dynamics, activity of progenitor subtypes during the early and late post-transplant phases, and hierarchical relationships among lineages.
doi:10.1016/j.stem.2016.04.016
PMCID: PMC4942697  PMID: 27237736
7.  Longitudinal MRI quantification of muscle degeneration in Duchenne muscular dystrophy 
Abstract
Objective
The aim of this study was to evaluate the usefulness of magnetic resonance imaging (MRI) in detecting the progression of Duchenne muscular dystrophy (DMD) by quantification of fat infiltration (FI) and muscle volume index (MVI, a residual‐to‐total muscle volume ratio).
Methods
Twenty‐six patients (baseline age: 5–12 years) with genetically proven DMD were longitudinally analyzed with lower limb 3T MRI, force measurements, and functional tests (Gowers, 10‐m time, North Star Ambulatory Assessment, 6‐min walking test). Five age‐matched controls were also examined, with a total of 85 MRI studies. Semiquantitative (scores) and quantitative MRI (qMRI) analyses (signal intensity ratio – SIR, lower limb MVI, and individual muscle MVI) were carried out. Permutation and regression analyses according to both age and functional test‐outcomes were calculated. Age‐related quantitative reference curves of SIRs and MVIs were generated.
Results
FI was present on glutei and adductor magnus in all patients since the age of 5, with a proximal‐to‐distal progression and selective sparing of sartorius and gracilis. Patients' qMRI measures were significantly different from controls' and among age classes. qMRI were more sensitive than force measurements and functional tests in assessing disease progression, allowing quantification also after loss of ambulation. Age‐related curves with percentile values were calculated for SIRs and MVIs, to provide a reference background for future experimental therapy trials. SIRs and MVIs significantly correlated with all clinical measures, and could reliably predict functional outcomes and loss of ambulation.
Interpretations
qMRI‐based indexes are sensitive measures that can track the progression of DMD and represent a valuable tool for follow‐up and clinical studies.
doi:10.1002/acn3.319
PMCID: PMC4999593  PMID: 27606343
8.  A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells 
Autophagy  2015;11(7):1161-1178.
Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.
doi:10.1080/15548627.2015.1052928
PMCID: PMC4590585  PMID: 26043024
aggregate; autophagy; bortezomib; endoplasmic reticulum; multiple myeloma; p62; plasma cells; proteasome; proteasome inhibitors; proteostasis; SQSTM1; ubiquitin
10.  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 
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1186/s13045-016-0248-3
PMCID: PMC4791867  PMID: 26980295
Allogeneic stem cell transplantation; Haplo-identical donor; Conditioning regimen; Acute Leukemia; Toxicity; Anti-leukemic effect
11.  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.
doi:10.1371/journal.pone.0150637
PMCID: PMC4778768  PMID: 26942424
12.  miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells 
Cancer Cell  2016;29(2):214-228.
Summary
To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.
Graphical Abstract
Highlights
•Clinical outcome in AML correlates with LSC-associated miRNA expression•miR-126 targets multiple components of the PI3K/AKT/MTOR signaling pathway•miR-126 promotes chemotherapy resistance by preserving LSC in a quiescent state•miR-126 governs opposing self-renewal outcomes in normal and malignant stem cells
Lechman et al. show that miR-126 targets the PI3K/AKT/MTOR signaling pathway to preserve quiescence, increase self-renewal, and promote chemotherapy resistance of acute myeloid leukemia stem cells (LSC). Reducing the miR-126 level impairs LSC maintenance in contrast to expanding normal hematopoietic stem cells.
doi:10.1016/j.ccell.2015.12.011
PMCID: PMC4749543  PMID: 26832662
13.  Th22 cells increase in poor prognosis multiple myeloma and promote tumor cell growth and survival 
Oncoimmunology  2015;4(5):e1005460.
There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17−IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10− and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17−IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.
doi:10.1080/2162402X.2015.1005460
PMCID: PMC4485827  PMID: 26155400
bone marrow microenvironment; bone marrow mesenchymal stromal cells; CD4+ T helper lymphocytes; IL-22RA1; interleukin-13; interleukin-22; multiple myeloma; Th22 cells; Ab, antibody; BM, bone marrow; BMMCs, bone marrow mononuclear cells; DCs, dendritic cells; Dx, dexamethasone; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; ISS, International Staging System; LCL, Epstein–Barr virus-transformed B lymphoblastoid cell line; Ln, lenalidomide; MGUS, monoclonal gammopathy of undetermined clinical significance; MM, multiple myeloma; MSC, mesenchymal stromal cell; PB, peripheral blood; PBMCs, peripheral blood mononuclear cells; pDCs, plasmacytoid dendritic cells; SMM, smoldering multiple myeloma; Th, T helper; TNF, tumor necrosis factor; Treg, regulatory T cells; WB, Western blot
14.  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.
Background.
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).
Methods.
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.
Results.
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.
Conclusion.
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.
doi:10.1634/theoncologist.2014-0187
PMCID: PMC4294603  PMID: 25480827
Obesity; Dose adjustment; Hematopoietic stem cell transplantation; Conditioning
15.  Intra‐arterial transplantation of HLA‐matched donor mesoangioblasts in Duchenne muscular dystrophy 
EMBO Molecular Medicine  2015;7(12):1513-1528.
Abstract
Intra‐arterial transplantation of mesoangioblasts proved safe and partially efficacious in preclinical models of muscular dystrophy. We now report the first‐in‐human, exploratory, non‐randomized open‐label phase I–IIa clinical trial of intra‐arterial HLA‐matched donor cell transplantation in 5 Duchenne patients. We administered escalating doses of donor‐derived mesoangioblasts in limb arteries under immunosuppressive therapy (tacrolimus). Four consecutive infusions were performed at 2‐month intervals, preceded and followed by clinical, laboratory, and muscular MRI analyses. Two months after the last infusion, a muscle biopsy was performed. Safety was the primary endpoint. The study was relatively safe: One patient developed a thalamic stroke with no clinical consequences and whose correlation with mesoangioblast infusion remained unclear. MRI documented the progression of the disease in 4/5 patients. Functional measures were transiently stabilized in 2/3 ambulant patients, but no functional improvements were observed. Low level of donor DNA was detected in muscle biopsies of 4/5 patients and donor‐derived dystrophin in 1. Intra‐arterial transplantation of donor mesoangioblasts in human proved to be feasible and relatively safe. Future implementation of the protocol, together with a younger age of patients, will be needed to approach efficacy.
doi:10.15252/emmm.201505636
PMCID: PMC4693504  PMID: 26543057
cell therapy; Duchenne; dystrophin; mesoangioblast; MRI; Genetics, Gene Therapy & Genetic Disease; Musculoskeletal System
16.  Improving the safety of cell therapy with the TK-suicide gene 
While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy.
doi:10.3389/fphar.2015.00095
PMCID: PMC4419602  PMID: 25999859
cellular adoptive immunotherapy; gene therapy; allogeneic hematopoietic stem cell transplantation; suicide gene therapy; TK cells
17.  BAT2 and BAT3 polymorphisms as novel genetic risk factors for rejection after HLA-related stem cell transplantation 
Bone marrow transplantation  2014;49(11):1400-1404.
The genetic background of donor and recipient is an important factor determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We applied a whole genome analysis to investigate genetic variants - other than HLA class I and II - associated with negative outcome after HLA-identical sibling allo-HSCT in a cohort of 110 β-Thalassemic patients. We identified two single nucleotide polymorphisms in BAT2 (A/G) and BAT3 (T/C) genes, SNP rs11538264 and SNP rs10484558, both located in the HLA class III region, in strong Linkage Disequilibrium between each other (R2=0.92). When considered as single SNP, none of them reached a significant association with graft rejection (nominal P < 0.00001 for BAT2 SNP rs11538264, and P < 0.0001 for BAT3 SNP rs10484558). Whereas, the BAT2/BAT3 A/C haplotype was present at significantly higher frequency in patients who rejected as compared to those with functional graft (30.0% vs. 2.6%, nominal P = 1.15×10−8; and adjusted P = 0.0071).
The BAT2/BAT3 polymorphisms and specifically the A/C haplotype may represent novel immunogenetic factor associated with graft rejection in patients undergoing allo-HSCT.
doi:10.1038/bmt.2014.177
PMCID: PMC4222814  PMID: 25111513
Beta-thalassemia; Allogeneic hematopoietic stem transplantation; whole genome analysis; BAT2; BAT3
18.  Long term follow up of patients after allogeneic stem cell transplantation and transfusion of HSV-TK transduced T-cells 
Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated ΔLNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005–2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.
doi:10.3389/fphar.2015.00076
PMCID: PMC4407574  PMID: 25954199
gene transfer; horizontal; gene therapy; proteomics data; allogeneic stem cell transplantation; graft vs. host disease
19.  Lentivirus-based Gene Therapy of Hematopoietic Stem Cells in Wiskott-Aldrich Syndrome 
Science (New York, N.Y.)  2013;341(6148):1233151.
Wiskott-Aldrich Syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and re-infused the cells after reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical score. Vector integration analyses revealed highly polyclonal and multi-lineage haematopoiesis resulting from the gene corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes and no aberrant clonal expansion was observed after 20–32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.
doi:10.1126/science.1233151
PMCID: PMC4375961  PMID: 23845947
20.  Risk-associations between HLA-DPB1 T cell epitope matching and outcome of unrelated hematopoietic cell transplantation are independent from HLA-DPA1 
Bone marrow transplantation  2014;49(9):1176-1183.
HLA-DP antigens are beta-alpha heterodimers encoded by polymorphic HLA-DPB1 and -DPA1 alleles, respectively, in strong linkage disequilibrium (LD) with each other. Non-permissive unrelated donor (UD)-recipient HLA-DPB1 mismatches across three different T cell epitope (TCE) groups are associated with increased mortality after hematopoietic cell transplantation (HCT), but the role of HLA-DPA1 is unclear. We studied 1281 onco-hematologic patients after 10/10 HLA-matched UD-HCT facilitated by the National Marrow Donor Program. Non-permissive mismatches defined solely by HLA-DPB1 TCE groups were associated with significantly higher risks of treatment-related mortality compared to permissive mismatches (HR 1.30, CI 1.06–1.53; p=0.009) or allele matches. Moreover, non-permissive HLA-DPB1 TCE group mismatches in the graft versus host (GvH) direction significantly decreased the risk of relapse compared to permissive mismatches (HR 0.55, CI 0.37–0.80; p=0.002) or allele matches. Splitting each group into HLA-DPA1*02:01 positive or negative, in frequent LD with HLA-DPB1 alleles from two of the three TCE groups, or into HLA-DPA1 matched or mismatched, did not significantly alter the observed risk associations. Our findings suggest that the effects of clinically non-permissive HLA-DPB1 TCE group mismatches are independent of HLA-DPA1, and that selection of donors with non-permissive DPB1 TCE mismatches in GvH direction might provide some protection from disease recurrence.
doi:10.1038/bmt.2014.122
PMCID: PMC4154997  PMID: 24955785
Non-permissive mismatch; HLA-DPB1 T cell epitope matching; Unrelated HCT; relapse; transplant related mortality; HLA-DPA1
21.  A novel self-lipid antigen targets human T cells against CD1c+ leukemias 
The Journal of Experimental Medicine  2014;211(7):1363-1377.
CD1c self-reactive T cells recognize a novel class of self-lipids that are accumulated on leukemia cells.
T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c+ acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c+ human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy.
doi:10.1084/jem.20140410
PMCID: PMC4076585  PMID: 24935257
22.  Autologous Pancreatic Islet Transplantation in Human Bone Marrow 
Diabetes  2013;62(10):3523-3531.
The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans.
doi:10.2337/db13-0465
PMCID: PMC3781459  PMID: 23733196
23.  HLA-G Expression on Blasts and Tolerogenic Cells in Patients Affected by Acute Myeloid Leukemia 
Journal of Immunology Research  2014;2014:636292.
Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4+ T cells in the peripheral blood of AML patients at diagnosis. Moreover, we explored the possible influence of the 3′ untranslated region (3′UTR) of HLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4+ T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4+ T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the host's immune system. Further studies on larger populations are required to verify our findings.
doi:10.1155/2014/636292
PMCID: PMC3987970  PMID: 24741612
24.  Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells 
T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.
doi:10.3389/fimmu.2014.00016
PMCID: PMC3907718  PMID: 24550909
hematopoietic stem cell transplantation; haploidentical; IL-10; T regulatory type 1 cells; cell therapy; tolerance
25.  Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity 
Clinical Immunology (Orlando, Fla.)  2013;146(3):248-261.
Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8+ T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8+STAT5+ T cells with lytic potential infiltrated the skin, even though FOXP3+ Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.
Highlights
► CD25 deficiency leads to profound immune dysregulation. ► Preferential CD8+ T cell expansion and high cytokine serum levels were present. ► Proliferating CD8+ T cells infiltrated the skin but failed to respond to pathogens. ► CD4+FOXP3+CD127lowCD25null Tregs could be detected. ► Altered IL2 signaling events and failure of IL2 consumption contribute to autoimmunity.
doi:10.1016/j.clim.2013.01.004
PMCID: PMC3594590  PMID: 23416241
CD25;; IPEX-like;; Immunodeficiency;; Autoimmunity;; Tregs;; IL-2

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