Gene Therapy for Pediatric Cancer: State of the Art and Future Perspectives

While modern treatments have led to a dramatic improvement in survival for pediatric malignancy, toxicities are high and a significant proportion of patients remain resistant. Gene transfer offers the prospect of highly specific therapies for childhood cancer. “Corrective” genes may be transferred to overcome the genetic abnormalities present in the precancerous cell. Alternatively, genes can be introduced to render the malignant cell sensitive to therapeutic drugs. The tumor can also be attacked by decreasing its blood supply with genes that inhibit vascular growth. Another possible approach is to modify normal tissues with genes that make them more resistant to conventional drugs and/or radiation, thereby increasing the therapeutic index. Finally, it may be possible to attack the tumor indirectly by using genes that modify the behavior of the immune system, either by making the tumor more immunogenic, or by rendering host effector cells more efficient. Several gene therapy applications have already been reported for pediatric cancer patients in preliminary Phase 1 studies. Although no major clinical success has yet been achieved, improvements in gene delivery technologies and a better understanding of mechanisms of tumor progression and immune escape have opened new perspectives for the cure of pediatric cancer by combining gene therapy with standard therapeutic available treatments

Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies

Chimeric T-cell receptors (ChTCR), are a fascinating technological step in the field of immunotherapy for orienting the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumors, including hematologic malignancies. The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. ChTCR are, in fact, artificial T-cell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the bright expression of specific antigens on the surface of tumor cells. Thus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. Even though technical and safety progresses are still needed to improve the profile of gene transfer and protein expression of ChTCR, phase 1 trials will be carried out in the near future to demonstrate the feasibility of their clinical translation and, it is be hoped, give preliminary indications about their anti-tumor efficacy

Prolonged extracorporeal membrane oxygenation therapy for severe acute respiratory distress syndrome in a child affected by rituximab-resistant autoimmune hemolytic anemia: a case report

<p>Abstract</p> <p>Introduction</p> <p>Autoimmune hemolytic anemia in children younger than 2 years of age is usually characterized by a severe course, with a mortality rate of approximately 10%. The prolonged immunosuppression following specific treatment may be associated with a high risk of developing severe infections. Recently, the use of monoclonal antibodies (rituximab) has allowed sustained remissions to be obtained in the majority of pediatric patients with refractory autoimmune hemolytic anemia.</p> <p>Case presentation</p> <p>We describe the case of an 8-month-old Caucasian girl affected by a severe form of autoimmune hemolytic anemia, which required continuous steroid treatment for 16 months. Thereafter, she received 4 weekly doses of rituximab (375 mg/m²/dose) associated with steroid therapy, which was then tapered over the subsequent 2 weeks. One month after the last dose of rrituximab, she presented with recurrence of severe hemolysis and received two more doses of rrituximab. The patient remained in clinical remission for 7 months, before presenting with a further relapse. An alternative heavy immunosuppressive therapy was administered combining cyclophosphamide 10 mg/kg/day for 10 days with methylprednisolone 40 mg/kg/day for 5 days, which was then tapered down over 3 weeks. While still on steroid therapy, the patient developed an interstitial pneumonia with Acute Respiratory Distress Syndrome, which required immediate admission to the intensive care unit where extracorporeal membrane oxygenation therapy was administered continuously for 37 days. At 16-month follow-up, the patient is alive and in good clinical condition, with no organ dysfunction, free from any immunosuppressive treatment and with a normal Hb level.</p> <p>Conclusions</p> <p>This case shows that aggressive combined immunosuppressive therapy may lead to a sustained complete remission in children with refractory autoimmune hemolytic anemia. However, the severe life-threatening complication presented by our patient indicates that strict clinical monitoring must be vigilantly performed, that antimicrobial prophylaxis should always be considered and that experienced medical and nursing staff must be available, to deliver highly specialized supportive salvage therapies, if necessary, during intensive care monitoring.</p

New advances in leukaemia immunotherapy by the use of Chimeric Artificial Antigen Receptors (CARs): state of the art and perspectives for the near future

Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs), which are artificial molecules containing antibody-derived fragments (to bind the specific target), joined with potent signalling T-Cell Receptor (TCR)-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future

CD40 activation of BCP-ALL cells generates IL-10-producing, IL-12-defective APCs that induce allogeneic T-cell anergy

The use of leukemia cells as antigenpresenting cells (APCs) in immunotherapy is critically dependent on their capacity to initiate and sustain an antitumor-specific immune response. Previous studies suggested that pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells could be manipulated in vitro through the CD40-CD40L pathway to increase their immunostimulatory capacity. We extended the APC characterization of CD40L-activated BCP-ALL for their potential use in immunotherapy in a series of 19 patients. Engaging CD40 induced the up-regulation of CCR7 in 7 of 11 patients and then the migration to CCL19 in 2 of 5 patients. As accessory cells, CD40Lactivated BCP-ALL induced a strong proliferation response of naive T lymphocytes. Leukemia cells, however, were unable to sustain proliferation over time, and T cells eventually became anergic. After CD40-activation, BCP-ALL cells released substantial amounts of interleukin-10 (IL-10) but were unable to produce bioactive IL-12 or to polarize TH1 effectors. Interestingly, adding exogenous IL-12 induced the generation of interferon- (IFN- )–secreting TH1 effectors and reverted the anergic profile in a secondary response. Therefore, engaging CD40 on BCP-ALL cells is insufficient for the acquisition of full functional properties of immunostimulatory APCs. These results suggest caution against the potential use of CD40L-activated BCP-ALL cells as agents for immunotherapy unless additional stimuli, such as IL-12, are provided.Fil: D'Amico, Giovanna. Università Milano Bicocca; ItaliaFil: Vulcano, Marisa. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Bugarin, Cristina. Università Milano Bicocca; ItaliaFil: Bianchi, Giancarlo. Università Milano Bicocca; ItaliaFil: Pirovano, Gisella. Università Milano Bicocca; ItaliaFil: Bonamino, Martin. Università Milano Bicocca; ItaliaFil: Marin, Virna. Università Milano Bicocca; ItaliaFil: Allavena, Paola. Università Milano Bicocca; ItaliaFil: Biagi, Ettore. Università Milano Bicocca; ItaliaFil: Biondi, Andrea. Università Milano Bicocca; Itali

Exploration of the Lysis Mechanisms of Leukaemic Blasts by Chimaeric T-Cells

Adoptive transfer of specific cytotoxic T lymphocytes (CTL) and Cytokine Induced Killer Cells (CIK) following genetic engineering of T-cell receptor zeta hold promising perspective in immunotherapy. In the present work we focused on the mechanisms of anti-tumor action of effectors transduced with an anti-CD19 chimaeric receptor in the context of B-lineage acute lymphoblastic leukemia (B-ALL). Primary B-ALL blasts were efficiently killed by both z-CD19 CTL and z-CD19 CIK effectors. The use of death receptor mediated apoptosis of target cells was excluded since agonists molecules of Fas and TRAIL-receptors failed to induce cell death. Perforin/granzyme pathway was found to be the mechanism of chimaeric effectors mediated killing. Indeed, cytolytic effector molecules perforin as well as granzymes were highly expressed by CTL and CIK. CD19 specific stimulation of transduced effectors was associated with degranulation as attested by CD107 membrane expression and high IFN-γ and TNF-α release. Moreover inhibitors of the perforin-based cytotoxic pathway, Ca2+-chelating agent EGTA and Concanamycin A, almost completely abrogated B-ALL blast killing. In conclusion we show that the cytolysis response of z-CD19 chimaeric effectors is predominantly mediated via perforin/granzyme pathway and is independent of death receptors signaling in primary B-ALL

Platelet-Lysate-Expanded Mesenchymal Stromal Cells for the Treatment of Resistant GVHD

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Mesenchymal Stromal Cells Do Not Increase the Risk of Viral Reactivation Nor the Severity of Viral Events in Recipients of Allogeneic Stem Cell Transplantation

Mesenchymal stromal cells (MSC) are tested in clinical trials to treat graft versus host disease (GvHD) after stem cell transplantation (SCT). In vitro studies demonstrated MSC's broad immunosuppressive activity. As infections represent a major risk after SCT, it is important to understand the role of MSC in this context. We analyzed 24 patients (pts) receiving MSC for GvHD in our Unit between 2009 and 2011. We recorded viral reactivations as measured in whole blood with polymerase chain reaction for 100 days following MSC administration. In patients with a documented viral reactivation in the first 3 days following MSCs infusion the frequency of virus-specific IFNgamma-producing cells was determined through enzyme-linked immunospot assay. In our cohort of patients viral reactivation after MSC infusion occurred in 45% of the cases, which did not significantly differ from the incidence in a historical cohort of patients affected by steroid resistant GvHD and treated with conventional immunosuppression. No patient presented severe form of infection. Two cases could be checked for immunological response to viral stimulus and demonstrated virus specific T-cytotoxic lymphocyte activity. In our experience MSC infusion did not prove to trigger more frequent or severer viral reactivations in the post transplantation setting

Platelet-lysate-Expanded Mesenchymal Stromal Cells as a Salvage Therapy for Severe Resistant Graft-versus-Host Disease in a Pediatric Population

Despite advances in graft-versus-host-disease (GVHD) treatment, it is estimated that overall survival (OS) at 2 years for hematopoietic cell transplantation (HCT) recipients who experience steroid-resistant GVHD is 10%. Among recent therapeutic approaches for GVHD treatment, mesenchymal stromal cells (MSCs) hold a key position. We describe a multicenter experience of 11 pediatric patients diagnosed with acute or chronic GVHD (aGVHD, cGVHD) treated for compassionate use with GMP-grade unrelated HLA-disparate donors' bone marrow-derived MSCs, expanded in platelet-lysate (PL)-containing medium. Eleven patients (aged 4-15 years) received intravenous (i.v.) MSCs for aGVHD or cGVHD, which was resistant to multiple lines of immunosuppression. The median dose was 1.2 × 10 6 /kg (range: 0.7-3.7 × 10 6 /kg). No acute side effects were observed, and no late side effects were reported at a median follow-up of 8 months (range: 4-18 months). Overall response was obtained in 71.4% of patients, with complete response in 23.8% of cases. None of our patients presented GVHD progression upon MSC administration, but 4 patients presented GVHD recurrence 2 to 5 months after infusion. Two patients developed chronic limited GVHD. This study underlines the safety of PL-expanded MSC use in children. MSC efficacy seems to be greater in aGVHD than in cGVHD, even after failure of multiple lines of immunosuppression