Barbara Carlini

CXCL12 Does Not Attract CXCR4 + Human Metastatic Neuroblastoma Cells: Clinical Implications

Purpose: The role of CXCR4 in bone marrow localization of neuroblastoma cells has been recently proposed. The aim of this study was to investigate the expression and chemotactic functionality of CXCR4 in human metastatic neuroblastoma cells isolated from the bone marrow and, for comparison, in a panel of neuroblastoma cell lines. Experimental Design: CXCR4 expression and chemotactic functionality were investigated in metastatic neuroblastoma cells isolated from patient bone marrow and in neuroblastoma cell lines. The former cells were isolated as CD45− or GD2+ cells by immunomagnetic bead manipulation. Chemotactic assays were done in a transwell system. Regulator of G protein signaling expression was investigated by reverse transcription-PCR. Results: Metastatic neuroblastoma cells consistently expressed CXCR4, which was also detected in 5 of 10 neuroblastoma cell lines. CXCL12 did not stimulate the chemotaxis of primary tumor cells or cell lines in either normoxia or hypoxia, irrespective of CXCR4 up-regulation detected under the latter condition. Accordingly, neuroblastoma cells failed to modulate filamentous actin and to activate mitogen-activated protein kinase upon treatment with CXCL12. RGS16 mRNA was consistently expressed in primary tumor cells and cell lines, but its down-regulation by RNA interference did not restore CXCR4 chemotactic functionality. Conclusions: These results show unambiguously that CXCR4 expressed in human metastatic neuroblastoma cells is not functional and do not support the clinical use of CXCR4 antagonists to prevent neuroblastoma metastasis.

Immunotherapy of neuroblastoma by an Interleukin-21-secreting cell vaccine involves survivin as antigen

AimIL-21 is the most recently identified member of the IL-2 cytokine family. Here we studied the therapeutic efficacy of IL-21-gene-modified cells (Neuro2a/IL-21) in a syngeneic metastatic neuroblastoma (NB) model.Materials and methodsNeuro2a/IL-21 cells were tested as subcutaneous (sc) vaccine both in prophylactic and therapeutic settings. Depletion studies, cytotoxicity assay and immunohistochemical analyses were carried out to evaluate the mechanisms involved in tumor rejection.ResultsWhen injected sc in syngeneic A/J mice viable Neuro2a/IL-21 cells were rejected and induced resistance to a subsequent iv challenge with Neuro2a parental cells (Neuro2a/pc), suggesting the involvement of an immune response. More importantly, in mice bearing Neuro2a/pc micrometastases, a single sc injection of Neuro2a/IL-21 cells significantly increased the mean tumor-free survival of treated animals (43 vs. 22xa0days) and cured 14% of them. The administration of two or three doses of Neuro2a/IL-21 cell vaccine further increased the mean survival time to 54 and 75xa0days, and the cure rate to 27 and 33%, respectively, whereas the use of unmodified Neuro2a or mock-transfected cells had no effect. In vivo cell subset depletion and a Winn-assay indicated the involvement of CD8xa0+xa0CTLs. Immunohistochemical analysis indicated a reduction of CD31+ and VEGFR2+ microvessels in late metastases from therapeutically vaccinated mice. A role of survivin as antigen was suggested by in vitro assays using survivin-synthetic CTL-epitopes.ConclusionsOur present data indicate that IL-21-secreting NB cells are effective as therapeutic vaccine in mice bearing metastatic NB, through a specific CTL response involving survivin as antigen, and suggest a potential interest for IL-21 in NB immuno-gene therapy.

Peripheral Blood Stem Cell Tumor Cell Contamination and Survival of Neuroblastoma Patients

Purpose: Contribution of peripheral blood stem cell (PBSC) contaminating tumor cells to subsequent relapse and overall survival of neuroblastoma patients remains controversial. Experimental Design: Neuroblastoma cell contamination of 27 PBSC harvests from stage IV neuroblastoma patients was assessed by quantitative RT-PCR for both tyrosine hydroxylase (TH) and GD2 synthase (GD2-s). The effect of PBSC contamination on survival was then analyzed. Results: Seven PBSC tested negative for both markers; 19 were positive for GD2-s, 6 for TH, with 5 positive for both. Survival of the 20 patients with positive PBSC did not differ from that of the patients with negative PBSC (log-rank test, P = 0.134 and 0.218 for event-free survival and overall survival, respectively). By considering the TH and GD2-s results independently, a borderline (P = 0.053) negative effect on event-free survival was observed in patients reinfused with GD2-s-positive PBSC. When the status at transplant was taken into account, only the event-free survival of the patients rescued when in complete remission with GD2-s-negative PBSC was better, although not significantly, than that of patients infused with GD2-s-positive PBSC. Conclusions: Our results obtained in a small cohort of homogeneously treated stage IV patients suggest that patient survival is not affected by PBSC contamination with the exception of a borderline negative effect on event-free survival in patients rescued when in complete remission.

Angiogenesis in a human neuroblastoma xenograft model: mechanisms and inhibition by tumour-derived interferon- γ

Tumour progression in neuroblastoma (NB) patients correlates with high vascular index. We have previously shown that the ACN NB cell line is tumorigenic and angiogenic in immunodeficient mice, and that interferon-γ (IFN-γ) gene transfer dampens ACN tumorigenicity. As IFN-γ represses lymphocyte-induced tumour angiogenesis in various murine models and inhibits proliferation and migration of human endothelial cells, we have investigated the antiangiogenic activity of tumour-derived IFN-γ and the underlying mechanism(s). In addition, we characterised the tumour vasculature of the ACN xenografts, using the chick embryo chorioallantoic membrane assay. We show that the ACN/IFN-γ xenografts had a lower microvessel density and less in vivo angiogenic potential than the vector-transfected ACN/neo. The vascular channels of both xenografts were formed by a mixed endothelial cell population of murine and human origin, as assessed by the FICTION (fluorescence immunophenotyping and interphase cytogenetics) technique. With respect to ACN/neo, the ACN/IFN-γ xenografts showed more terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive human and murine endothelial cells, suggesting that inhibition of angiogenesis by IFN-γ was dependent on the induction of apoptosis, likely mediated by nitric oxide. Once the dual origin of tumour vasculature is confirmed in NB patients, the xenograft model described here will prove useful in testing the efficacy of different antiangiogenic compounds.

Multiple Target Molecular Monitoring of Bone Marrow and Peripheral Blood Samples From Patients With Localized Neuroblastoma and Healthy Donors

Multiple target molecular monitoring of minimal residual disease in neuroblastoma (NB) patients may increase sensitivity and overcome tumor heterogeneity. However, multiple target analysis is costly and time consuming, thus improvement with respect to single target monitoring needs to be achieved.

Transient depletion of CD4+ T cells augments IL-21-based immunotherapy of disseminated neuroblastoma in syngeneic mice

IL‐21 is a member of the IL‐2 cytokine family, produced by CD4+ T cells. We previously showed that immunotherapy (IT) with IL‐21‐transduced neuroblastoma cells (Neuro2a/IL‐21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL‐21 vaccine. The administration of anti‐CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti‐CD4 mAb had a more potent effect leading to 80% cure rate. Anti‐CD25 mAb, indeed, only partially depleted CD4+CD25+FoxP3+ Treg cells, whereas anti‐CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine+anti‐CD4 mAb, which developed systemic immunity to NB, CD4+ T cells counts completely recovered in 90 days. Depletion of CD8+ T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD8+ T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD‐SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL‐21 vaccination showed increased expression of IFN‐alpha2, ‐beta1 and ‐gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine+anti‐CD4 mAb showed increased IFN‐gamma serum levels and IFN‐gamma‐producing CD8+ T cells were found in spleen cells. In conclusion, anti‐CD4 mAb potentiated IL‐21‐based IT by removing Treg cells and/or their precursors and other potentially immune‐suppressive CD4+ cell subsets, thus allowing the development of an IL‐21‐driven CD8+ T cell response, which mediates NB rejection.

Neuroblastoma and bone metastases: Clinical significance and prognostic value of Dickkopf 1 plasma levels

The critical role of the Wnt pathway inhibition in sustaining the onset of bone lesions has been demonstrated in a variety of bone diseases and tumors, and it has been associated with cancer aggressiveness. We have previously demonstrated that neuroblastoma cells express Dickkopf 1 (Dkk1), an inhibitor of the canonical Wnt pathway which prevents the differentiation of bone-forming cells. Since Dkk1 is a secreted factor, it could have potential clinical application as tumor marker for detecting bone metastasis and monitoring of disease. In this study, we investigated the diagnostic and prognostic value of Dkk1 plasma levels in 92 children affected by neuroblastoma, including 32 with bone metastases. Fifty-seven children hospitalized for minor surgical problems served as control group. Circulating levels of Dkk1 were higher in healthy children than in normal adults and were comparable to those found in adult patients with aggressive tumors. No significant differences were found between neuroblastoma patients and controls and between patients with and without bone metastases. However, when only patients with metastatic neuroblastoma were considered, the highest Dkk1 levels were detected in patients that poorly responded to induction chemotherapy and in subjects with unamplified MYCN and three or more different metastatic sites. The Receiver Operating Characteristic curve enabled us to identify a threshold value to distinguish patients who were unresponsive to induction treatment. The relationship between Dkk1 and drug resistance was supported by in vitro experiments, since an increased sensitivity to doxorubicin was found in neuroblastoma cells releasing low Dkk1 levels, either constitutively or experimentally following the treatment with specific siRNA. In conclusion, Dkk1 is released by neuroblastoma cells and is able to affect the balance between osteoblastogenesis and osteoclastogenesis, thus favoring the onset of osteolytic metastases. Nevertheless, Dkk1 plasma levels do not allow the detection of bone lesions in neuroblastoma but seem to have a predictive value with regard to the severity and the prognosis of the disease in a subset of patients with metastatic tumor. New knowledge on the biological role of Dkk1 in driving the natural history of neuroblastoma has to be further investigated and could help to establish specific therapeutic strategies able to target key factors of tumor progression.

Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma.

The impact of bone marrow (BM) GD2-positive cells on survival has been evaluated in 145 Italian children with localised neuroblastoma (NB) evaluated at diagnosis by anti-GD2 immunocytochemistry. Nineteen of these (13.1%) were found to be BM GD2-positive, with the number of positive cells ranging between 1 and 155 out of 1 × 106 total cells analysed. Seven/19 (38.8%) GD2-positive vs 12/126 (9.5%) GD2-negative patients relapsed. The 5-year event-free survival (EFS) and overall survival of the GD2-positive patients was significantly worse than that of the GD2-negative ones (62.2 vs 89.9%, P<0.001; and 74.9 vs 95.9%, P=0.005, respectively). GD2 positivity was not associated to other known risk factors, and in particular to Myc-N amplification and 1p deletion. Among Myc-N-negative patients, the EFS of those negative for both GD2 and 1p deletion was significantly better than in children positive for either one of these two markers (EFS=96.9 vs 66.0%, P<0.001). In conclusion, GD2 positivity may represent a prognostic marker for patients with non-metastatic NB without Myc-N amplification, and its combination with genetic alterations might help identifying patients that require a more careful follow-up.

IL-10 and ARG-1 Concentrations in Bone Marrow and Peripheral Blood of Metastatic Neuroblastoma Patients Do Not Associate with Clinical Outcome

The expression of the immunosuppressive molecules IL-10 and arginase 1 (ARG-1), and of FOXP3 and CD163, as markers of regulatory T cells (Treg) and macrophages, respectively, was evaluated in bone marrow (BM) and peripheral blood (PB) samples collected at diagnosis from patients with metastatic neuroblastoma (NB). IL-10 and ARG-1 plasma concentrations were measured and the association of each parameter with patients outcome was tested. The percentages of immunosuppressive Treg and type-1 regulatory (Tr1) cells were also determined. In both BM and PB samples, IL-10 mRNA expression was higher in metastatic NB patients than in controls. IL-10 plasma concentration was higher in patients with NB regardless of stage. Neither IL-10 expression nor IL-10 plasma concentration significantly associated with patient survival. In PB samples from metastatic NB patients, ARG-1 and CD163 expression was higher than in controls but their expression did not associate with survival. Moreover, ARG-1 plasma concentration was lower than in controls, and no association with patient outcome was found. Finally, in metastatic NB patients, the percentage of circulating Treg was higher than in controls, whereas that of Tr1 cells was lower. In conclusion, although IL-10 concentration and Treg percentage were increased, their contribution to the natural history of metastatic NB appears uncertain.

CD4+CD25hiCD127− Treg and CD4+CD45R0+CD49b+LAG3+ Tr1 cells in bone marrow and peripheral blood samples from children with neuroblastoma

ABSTRACT Metastatic spread in the bone marrow (BM) at diagnosis is the worst prognostic factor for neuroblastoma (NB) patients. Here, we analyzed the presence of two immunosuppressive cell subsets, CD4+CD25hiCD127− regulatory T (Treg) cells and CD4+CD45R0+CD49b+LAG3+ type 1 regulatory (Tr1) cells, in BM and peripheral blood (PB) samples from NB patients and controls. Frequency of both regulatory cell subsets was lower in BM and PB samples from NB patients than in respective healthy controls. No correlation was found between the frequency of Treg and Tr1 cells and prognostic factors at diagnosis, such as age and stage. Only MYCN amplification correlated to a higher number of Treg in BM and of Tr1 in PB. These findings suggested an altered trafficking of regulatory T cells in NB, but delineated a limited role of these subsets in BM microenvironment and/or periphery in NB. These observations should be considered designing immunotherapeutic approaches for metastatic NB.