Search

The transcription factor Wilms’ tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. WT1 is highly expressed in leukemia cells and its overexpression is associated with a poor response to therapy. Recently the Krüppel-like zinc-finger protein, ZNF224 was identified as a novel WT1-interacting factor involved in WT1 transcriptional regulation. ZNF224 itself could be modulated by cytosine arabinoside (ara-C), a drug widely used in the treatment of myeloid leukemia and that ZNF224 overexpression increases susceptibility to apoptosis of Ph+ K562 cell lines. In our retrospective analysis we evaluated the relative expression of ZNF224 mRNA in 30 adult patients with BCR-ABL–positive chronic phase chronic myeloid leukaemia (CP-CML) as a determinant of imatinib sensitivity. Methods: Response to tyrosine kinase inhibitor (TKI) imatinib is assessed with standardized real quantitative polymerase chain reaction and/or cytogenetics at 3, 6, and 12 months. Response to the therapy was classified as optimal, warning, and failure, according to the recent ELN criteria. We compared the ZNF224 expression at diagnosis with molecular response over the first 12 month of imatinib therapy. Sample have been selected, for retrospective analysis, for them interim molecular results a 12 month, showing 15 patients in optimal response (OR), 10 patients in a warning response (WR) and 5 patients in failure response (FR). 5 healthy donors (HDs) were included to the study. All patients signed informed consent in accordance with the Declaration of Helsinki. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN).Results:ZNF224 mRNA were significantly up-regulated in PB samples at diagnosis of patients with OR compared to patients with WR/FR, (1.13±0.76 vs 0.62±0.25 NCN,respectively; p=0.05). Interesting the ZNF224 mRNA expression in HDs was significantly higher (2.11±0.98 NCN vs OR patients, p=0.05 and WR/FR patients; p=0.0005). The treatment for 12 month with imatinib increase the ZNF224 expression in both CML categories (2.91±1.72 NCN in OR and1.77±1.52 NCN in WR/FR; p=0.05). Conclusions:We observed that the OR patients express a significantly higher number of copies of the ZNF224 transcript than WR/FR. Furthermore, in both groups of patients at diagnosis, ZNF224 protein levels are lower than those after therapy with TKI at 12 months

Southern anal. of the MLL Locus in DNA from bone marrow cells samples from a 5-mo old child with acute lymphoblastic leukemiasampled at diagnosis revealed a rearrangement at locus MLL of chromosome 11q23. Panhandle PCR amplification of the fragment comprising the der(11) translocation breakpoints yielded a 4.6 kb fragment corresponding to the bamHI-rearranged fragment detected by Southern anal. The new fusion gene lacks both the AF4-derived and the MLL-derived (nucleotide 2772-3579) transactivating domain. The resulting fusion protein is able to bind DNA through the MLL-derived AT hook domain, and through the AF4-derived nuclear targeting sequence domain but could lose its capacity to activate the expression of these genes.

Summary Several observations have suggested that lipoprotein (a) (Lp(a)) is a risk factor for coronary artery disease because of potential interference with fibrinolysis secondary to its activation of plasminogen. However, there are few data on the possible role of Lp(a) in liver cirrhosis. The present study was carried out, to better elucidate its relationship to the fibrinolytic system in liver cirrhosis. We studied the plasma levels of Lp(a) and the fibrinolytic parameters of 95 patients with liver cirrhosis (57 men, 38 women, aged 26–81). Patients in Child-Pugh class C (n = 32) had significantly lower levels of Lp(a) than those in class B (n = 45), and the class B had lower Lp(a) values than class A (n = 18) (1.4 (0.0–3.7) vs 2.9 (0.0–6.1) vs 3.4 (1.8–5.5); the data are log-transformed). Alpha-2-antiplasmin and plasminogen, had patterns similar to those of Lp(a), tissue plasminogen activator (t-PA) was significantly increased only in class C (class A: 7.5 ± 5.8 ng/ml; class B: 10.8 ± 7.7 ng/ml; class C: 19.1 ± 11.3 ng/ml). Patients with systemic hyperfibrinolysis (cross-linked fibrin degradation products, XDP > 200 ng/ml) also had lower levels of Lp(a) than those without 1.6 (0.0–4.4) vs (0.0–6.1); p = 0.0002. There was a significant correlation between Lp(a) and plasminogen (r = 0.43; p = 0.001). Lipoprotein (a) progressively decreases as liver cirrhosis worsens but it appears unlikely to be involved in causing the hyperfibrinolytic state often observed in advanced liver cirrhosis, in which there are marked abnormalities of several other fibrinolytic parameters, also including increased t-PA and decreased inhibitors.

Programmed cell death‐1 (PD‐1) signaling downregulates the T‐cell response, promoting an exhausted state in tumor‐infiltrating T cells, through mostly unveiled molecular mechanisms. Dynamin‐related protein‐1 (Drp1)‐dependent mitochondrial fission plays a crucial role in sustaining T‐cell motility, proliferation, survival, and glycolytic engagement. Interestingly, such processes are exactly those inhibited by PD‐1 in tumor‐infiltrating T cells. Here, we show that PD‐1pos CD8+ T cells infiltrating an MC38 (murine adenocarcinoma)‐derived murine tumor mass have a downregulated Drp1 activity and more elongated mitochondria compared with PD‐1neg counterparts. Also, PD‐1pos lymphocytic elements infiltrating a human colon cancer rarely express active Drp1. Mechanistically, PD‐1 signaling directly prevents mitochondrial fragmentation following T‐cell stimulation by downregulating Drp1 phosphorylation on Ser616, via regulation of the ERK1/2 and mTOR pathways. In addition, downregulation of Drp1 activity in tumor‐infiltrating PD‐1pos CD8+ T cells seems to be a mechanism exploited by PD‐1 signaling to reduce motility and proliferation of these cells. Overall, our data indicate that the modulation of Drp1 activity in tumor‐infiltrating T cells may become a valuable target to ameliorate the anticancer immune response in future immunotherapy approaches., PD‐1 signaling prevents TCR‐dependent Drp1 activation and subsequent mitochondrial fragmentation in T cells. In turn, this reduces both proliferation and migration of activated T cells. Cancer cells exploit this mechanism to downregulate T‐cell functionality within the tumor microenvironment, favoring tumor progression. These findings shed light on a new possible therapeutical approach for the treatment of solid cancers. (image made in BioRender).

[Abstract]: Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. [Methods]: We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. [Results]: The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. [Conclusions]: Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts., The experimental work was supported by grants awarded by Ricerca Finalizzata GR-2013 02359212 (CQ), GR-2016-02364546 (BDA), RF-2016- 02364388 (FL), Accelerator Award-Cancer Research UK/AIRC/AECC-INCAR project (FL and AO), Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5×1000 no. 9962 (FL), AIRC IG 2018 id. 21724 (FL), Ricerca Corrente (FL, CQ, BDA), Ministero dell’Università e della Ricerca (Grant PRIN 2017 to FL); Italian Healthy Ministry project on CAR T RCR-2019-23669115 (coordinator FL), Independent Research grant AIFA (FL PI: 2016 call).

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4+CD25+ Treg cells and other CD4+CD25− regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4+ T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.

Pathophysiology of graft failure (GF) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing GF, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing GF had serum levels of interferon (IFN)-γ and CXCL9 (a chemokine specifically induced by IFNγ) significantly higher than those of controls (8859±7502 vs. 0 pg/mL, P=0.03, and 1514.0±773 vs. 233.6±50.1 pg/mlL, P=0.0006, respectively). The role played by IFNγ in HSCT-related GF was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1-/- mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing GF documented a predominance of effector memory CD8+ cells, which showed markers of activation (overexpression of CD95 and downregulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing GF, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the US Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that the IFNγ pathway plays a major role in GF occurring after HSCT. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of GF and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.

CAR-NK cells may represent a valuable tool, complementary to CAR-T cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer.

GD2-redirected chimeric antigen receptor (CAR) T lymphocytes represent a promising therapeutic option for immunotherapy of neuroblastoma (NB). However, despite the encouraging therapeutic effects observed in some hematological malignancies, clinical results of CAR T cell immunotherapy in solid tumors are still modest. Tumor driven neo-angiogenesis supports an immunosuppressive microenvironment that influences treatment responses and is amenable to targeting with antiangiogenic drugs. The latter agents promote lymphocyte tumor infiltration by transiently reprogramming tumor vasculature, and may represent a valid combinatorial approach with CAR T cell immunotherapy. In light of these considerations, we investigated the anti-NB activity of GD2-CAR T cells combined with bevacizumab (BEV) in an orthotopic xenograft model of human NB. Two weeks after tumor implantation, mice received BEV or GD2-CAR T cells or both by single intravenous administration. GD2-CAR T cells exerted a significant anti-NB activity only in combination with BEV, even at the lowest concentration tested, which per se did not inhibit tumor growth. When combined with BEV, GD2-CAR T cells massively infiltrated tumor mass where they produced interferon-γ (IFN-γ), which, in turn, induced expression of CXCL10 by NB cells. IFN-γ, and possibly other cytokines, upregulated NB cell expression of PD-L1, while tumor infiltrating GD2-CAR T cells expressed PD-1. Thus, the PD-1/PD-L1 axis can limit the anti-tumor efficacy of the GD2-CAR T cell/BEV association. This study provides a strong rationale for testing the combination of GD2-CAR T cells with BEV in a clinical trial enrolling NB patients. PD-L1 silencing or blocking strategies may further enhance the efficacy of such combination.

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell–related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A*02+ medulloblastoma. Significance: These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells. Cancer Res; 78(12); 3337–49. ©2018 AACR.

Background: Neuroblastoma is the most common extracranial solid tumor in infancy. The majority of children have a disseminated disease at diagnosis with bone marrow as the most common site of metastasis. Although several prognostic factors have been defined (i.e. age, stage, histology, recurrent genetic anomalies), the identification of non-invasive biomarkers for disease follow-up and therapy monitoring is indeed still a clinical need. Aberrant regulation of microRNAs (miRNAs) expression has been implicated in several malignancies. Objectives: In this mini-review, we describe the recent findings about miRNAs in neuroblastoma, both in the tumor and circulation, with particular focus on those involved in tumor progression and drug resistance. Furthermore, we will discuss the use of specific miRNAs as potential therapeutic tools in this tumor. Results: Several miRNAs have been identified to be down- or up-regulated in primary tumors and have been associated with MYCN amplification, differentiation, dissemination and chemoresistance. Little evidence is available in the literature about circulating miRNAs which are of particular interest as potential biomarkers for liquid biopsy. Conclusion: Identification of body-fluid markers for non-invasive diagnosis, risk stratification, treatment monitoring and tumor follow-up, is gaining growing interest, especially in the pediatric field. miRNAs are suitable candidates as biomarkers in neuroblastoma but further investigations are needed to expand knowledge regarding their role in this malignancy to design specific approaches of miRNAs-mediated therapies.