Secondary solid cancer following hematopoietic cell transplantation in patients with thalassemia major.

Hematopoietic cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers (SSCs). The aim of this retrospective study was to compare the incidence of SSC in a monocentric cohort of thalassemia major (TM) patients (n=122) who received HCT versus an hematopoietic cell donor monocentric cohort (n=122) and versus a large multicenter cohort of age- and sex-matched TM patients (n=244) who received conventional therapy. With a median follow-up of 24 years, 8 transplanted patients were diagnosed with SSC at a median of 18 years after HCT and at a median age of 33 years. Three patients died of cancer progression and 5 are living after a follow-up ranging from 10 months to 16 years after SSC diagnosis. The 30-year cumulative incidence of developing SSC was 13.24%. The occurrence of solid cancers in the hematopoietic cell donor cohort was limited to only one case for a significantly lower cumulative incidence (3.23%, P=0.02) and to 3 cases in the cohort of nontransplant patients for a significantly lower cumulative incidence (1.32%, P=0.005). This study shows that the magnitude of increased risk of SST is fourfold to sixfold for patients treated with HCT as compared with hematopoietic cell donors and nontransplant patients.

Tregs: hype or hope for allogeneic hematopoietic stem cell transplantation?

The discovery of T regulatory cells has been one of the most important advances in basic immunology and has opened the door to the development of innovative therapeutic strategies for improving the outcome of solid organ and hematopoietic stem cell transplantation. Basic immunology is rapidly elucidating the complex biology of these cells even though the difficulties in purifying or even expanding them in vitro represent a major limitation to the development of clinical studies. The clinical benefit potentially associated with this therapeutic approach remains to be demonstrated. Meanwhile, several drugs used for the treatment of hematologic malignancies or for other purposes have been shown to upregulate the number and function of Tregs in vivo. In the near future, both ex vivo or in vivo expanded T cells are likely to enter the therapeutic armamentarium of clinical transplantation.

To breathe or not to breathe: the haematopoietic stem/progenitor cells dilemma.

UNLABELLED: Adult haematopoietic stem/progenitor cells (HSPCs) constitute the lifespan reserve for the generation of all the cellular lineages in the blood. Although massive progress in identifying the cluster of master genes controlling self-renewal and multipotency has been achieved in the past decade, some aspects of the physiology of HSPCs still need to be clarified. In particular, there is growing interest in the metabolic profile of HSPCs in view of their emerging role as determinants of cell fate. Indeed, stem cells and progenitors have distinct metabolic profiles, and the transition from stem to progenitor cell corresponds to a critical metabolic change, from glycolysis to oxidative phosphorylation. In this review, we summarize evidence, reported in the literature and provided by our group, highlighting the peculiar ability of HSPCs to adapt their mitochondrial oxidative/bioenergetic metabolism to survive in the hypoxic microenvironment of the endoblastic niche and to exploit redox signalling in controlling the balance between quiescence versus active cycling and differentiation. Especial prominence is given to the interplay between hypoxia inducible factor-1, globins and NADPH oxidases in managing the mitochondrial dioxygen-related metabolism and biogenesis in HSPCs under different ambient conditions. A mechanistic model is proposed whereby 'mitochondrial differentiation' is a prerequisite in uncommitted stem cells, paving the way for growth/differentiation factor-dependent processes. Advancing the understanding of stem cell metabolism will, hopefully, help to (i) improve efforts to maintain, expand and manipulate HSPCs ex vivo and realize their potential therapeutic benefits in regenerative medicine; (ii) reprogramme somatic cells to generate stem cells; and (iii) eliminate, selectively, malignant stem cells. LINKED ARTICLES: This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

Mesenchymal stem cells (MSCs) from scleroderma patients (SSc) preserve their immunomodulatory properties although senescent and normally induce T regulatory cells (Tregs) with a functional phenotype: implications for cellular-based therapy.

Systemic sclerosis (SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc-mesenchymal stem cells (MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls (HC). Senescence was evaluated by assessing cell cycle, ß-galactosidase (ß-Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co-culturing MSCs with peripheral blood mononuclear cells (PBMCs) and CD4(+) cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells (Tregs) induction. SSc-MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0-G1 phase, without significant differences between SSc and HC. SSc-MSCs showed an increased positive ß-Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, ß-Gal staining increased significantly in SSc-MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc- and HC-MSCs. Interleukin (IL)-6 and transforming growth factor (TGF)-ß-related transcripts and their protein levels were significantly higher in SSc-MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL-6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression.

The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Prognostic significance of genetic variants in the IL-23/Th17 pathway for the outcome of T cell-depleted allogeneic stem cell transplantation.

T helper (Th) 17 cells have emerged as important mediators in infectious and inflammatory diseases and, recently, in transplant rejection. We analyzed the associations between five common genetic variants in the IL-23/Th17 signaling pathway, namely in IL17A, IL17F and IL23R genes, and clinical outcome in T cell-depleted allogeneic SCT (allo-SCT). In the multivariate analysis, variants in IL23R and IL17A genes were the most important prognostic factors. Thus, patient GA genotype at rs11209026 in IL23R was associated with improved overall survival (hazard ratio (HR)=0.48; P=0.028) and, in donor, with decreased risk of fungal infections (P=0.05). In contrast, patient TC and CC genotypes at rs8193036 in IL17A gene were associated with increased risk of CMV infection (HR=3.68; P=0.011) and patient acute GVHD (HR=7.08; P=0.008), respectively. These results suggest that genetic variants in the IL-23/Th17 inflammatory pathway are important prognostic factors for the clinical outcome of allo-SCT. Although validation studies are ultimately required, our results would suggest the potential usefulness of IL-23/Th17 genotyping in donor selection and patient evaluation.

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

Recurrent primary plasmacytoma of the eyelid with rapid regional metastasis.

In this case, originally reported as primary eyelid plasmacytoma, the tumor recurred on the same eyelid within 2 years of surgery. No plasma cell infiltration was observed at bone marrow biopsy. No serum or urinary monoclonal component was detected at immunofixation. Histology and immunohistochemistry confirmed plasma cell infiltration. Tumor cell clonality was determined by immunohistological staining; cells were positive for kappa light chain like the first eyelid tumor. Surgery was followed by radiotherapy. Twenty months later, biopsy of one enlarged right cervical lymph node showed massive diffuse infiltration of atypical plasma cells (CD20(-), CD79a(+), CD138(+), MUM1/IRF4(+)). Given the rapid diffusion to lymph nodes and the appearance of the monoclonal component, the lymph node was removed surgically. No adjuvant chemotherapy was given. Unexpectedly, the serum monoclonal component normalized. No plasma cell infiltration was observed at bone marrow biopsy. As this case might be a particularly slow-progressing extra-medullary plasmacytoma, this study recommends closely monitored follow-ups so that the aggressive form can be treated in time.

Mesenchymal cells: a vehicle for gene therapy.

T cell-depleted allogeneic stem cell transplantation is associated with delayed immunological reconstitution. Bone marrow stroma and interleukin 7 (IL-7) regulate homeostasis of T lymphocytes. We engineered human stromal cells with a retroviral vector containing the IL-7 gene and studied in vitro effects on T cells. Human stromal cells were successfully transduced and generated a layer that was morphologically and phenotypically normal. IL-7-engineered stromal cells conserve the biological properties of unmanipulated stromal cells. Through their production of IL-7, they enhance survival and homeostatic proliferation of naive T cells. Because of this cytokine production, they might be an ideal vehicle for gene therapy aimed at supporting lymphopoiesis in the T cell-deficient host.

In vivo demethylation of a MoMuLV retroviral vector expressing the herpes simplex thymidine kinase suicide gene by 5' azacytidine.

We constructed a functional MoMuLV-based bicistronic retroviral vector encoding the herpes simplex virus type I thymidine kinase gene, which induces sensitivity to the prodrug ganciclovir (gcv), and the reporter beta-galactosidase gene (MFG-tk-IRES-lacZ). The U937 histiocytic cell line was transduced with this vector, and a clone (VB71) with high-level transgene expression was selected. Severe combined immunodeficient (SCID) mice were injected with VB71 cells to evaluate the role of long terminal repeat methylation in transgene silencing in vivo and to see whether 5-azacytidine (5' aza-C) demethylating agent prevented it. We found 5' aza-C maintained gene expression at high level in vitro. In vivo, time to tumor onset was significantly longer in SCID mice receiving the VB71 cells, 5' aza-C, and gcv compared with animals treated with either 5' aza-C or gcv alone. The number of injected tumor cells influences tumor onset time and the efficacy of 5' aza-C and gcv treatment. The standard gcv treatment schedule (10 mg/kg from d + 1 until the onset of tumor) controlled tumor onset better than short-term treatment with high doses. In conclusion, the results extend our previous findings that transgene methylation in vivo may be prevented with an appropriate schedule of 5' aza-C and gcv.

T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection.

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect.

Primary eyelid plasmacytoma.

We report a case of eyelid plasmacytoma. The patient presented with a superficial lesion on the upper left eyelid, 10x7 mm in size. Histological and immunohistochemical findings were indicative of plasmacytoma with slightly abnormal sub-epidermic plasma cell infiltrates. Immunoperoxidase staining was strongly positive for intracytoplasmic kappa light chains. No infiltration by atypical plasma cells could be documented in the bone marrow biopsy and aspirate. Skeletal survey showed no osteolytic lesions or osteoporosis. The tumor on the left eyelid was surgically removed. No radiotherapy was administered.

5-Azacytidine prevents transgene methylation in vivo.

Retroviral sequence can silence transgene expression in vitro and in vivo. We report that this effect can be efficiently prevented by in vivo administration of the demethylating agent 5-azacytidine (aza-C). We engineered the U937 human cell line with a retroviral vector consisting of the thymidine kinase suicide gene (tk), which induces sensitivity to ganciclovir (gcv) and through an IRES sequence, the bacterial beta-galactosidase gene (lacZ) as a marker gene. About 90% of the U937 cells expressed the transgene. By injecting the transduced U937 cells in severe combined immunodeficient disease (SCID) mice, we generated a tumor which, during in vivo treatment with aza-C, maintained the high expression of lacZ and tk genes at the baseline values. LacZ-positive cells in the tumour masses after death was weak (1-2%) in the control group, while in mice treated with aza-C it was maintained at 90%. The delay in tumour onset was significantly longer when animals were treated with both aza-C and gcv (P < 0.0001) compared with animals treated with gcv or with aza-C alone. The prevention of silencing phenomena has important implications for gene therapy, because an efficient transduction associated with appropriate drug therapy, might be a powerful strategy for successful application of gene therapy protocols.