Molecular Determinants of Neuroblastoma.

The aim of this Special Issue was to discuss novel findings regarding the different mechanisms involved in the progression of neuroblastoma (NB), which represents the most common pediatric extra-cranial solid tumor [...].

HLA-G and Other Immune Checkpoint Molecules as Targets for Novel Combined Immunotherapies.

HLA-G is an HLA-class Ib molecule that is involved in the establishment of tolerance at the maternal/fetal interface during pregnancy. The expression of HLA-G is highly restricted in adults, but the de novo expression of this molecule may be observed in different hematological and solid tumors and is related to cancer progression. Indeed, tumor cells expressing high levels of HLA-G are able to suppress anti-tumor responses, thus escaping from the control of the immune system. HLA-G has been proposed as an immune checkpoint (IC) molecule due to its crucial role in tumor progression, immune escape, and metastatic spread. We here review data available in the literature in which the interaction between HLA-G and other IC molecules is reported, in particular PD-1, CTLA-4, and TIM-3, but also IDO and TIGIT. Clinical trials using monoclonal antibodies against HLA-G and other IC are currently ongoing with cancer patients where antibodies and inhibitors of PD-1 and CTLA-4 showed encouraging results. With this background, we may envisage that combined therapies using antibodies targeting HLA-G and another IC may be successful for clinical purposes. Indeed, such immunotherapeutic protocols may achieve a better rescue of effective anti-tumor immune response, thus improving the clinical outcome of patients.

Molecular dynamics of targeting CD38 in multiple myeloma.

Multiple functions of CD38 need exploring to expand clinical application of anti-CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co-cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody-exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti-CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism-related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody-exposed cells showed increased CD73 and CD39 expression, presence of programmed death-ligand 1 and significant up-/down-modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor-positive (FcR+ ) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR+ cells with significant functional modifications. These observations have relevance for improving anti-CD38 therapeutic antibodies through targeting this mechanism and its sequelae.

Ectonucleotidase Expression on Human Amnion Epithelial Cells: Adenosinergic Pathways and Dichotomic Effects on Immune Effector Cell Populations.

This study investigates the mechanism(s) underlying the immunoregulatory activities of placenta-derived human amnion epithelial cells (hAEC). The working hypothesis is that NAD+ and ATP, along with ectoenzymes involved in their metabolism, play a significant role in hAEC-mediated immune regulation. Proof of principle of the hypothesis was obtained by analyzing the interactions between hAEC and the main human leukocyte populations. The results obtained indicate that hAEC constitutively express a unique combination of functional ectoenzymes, driving the production of adenosine (ADO) via canonical (CD39, CD73) and alternative (CD38, CD203a/PC-1, CD73) pathways. Further, the picture is completed by the observation that hAEC express A1, A2a, and A2b ADO receptors as well as ADO deaminase, the enzyme involved in ADO catabolism. The contribution of the purinergic mediator to immunomodulation was confirmed by exposing in vitro different immune effector cells to the action of primary hAECs. B cells showed an enhanced proliferation and diminished spontaneous apoptosis when in contact with hAEC. T cell proliferation was partially inhibited by hAEC through ADO production, as confirmed by using specific ectoenzyme inhibitors. Further, hAEC induced an expansion of both T and B regulatory cells. Last, hAEC inhibited NK cell proliferation. However, the involvement of ADO-producing ectoenzymes is less apparent in this context. In conclusion, hAEC exert different in vitro immunoregulatory effects, per se, as a result of interactions with different populations of immune effector cells. These results support the view that hAEC are instrumental for regenerative medicine as well as in therapeutic applications for immune-related diseases.

The Role of Extracellular Vesicles in the Progression of Human Neuroblastoma.

The long-underestimated role of extracellular vesicles in cancer is now reconsidered worldwide by basic and clinical scientists, who recently highlighted novel and crucial activities of these moieties. Extracellular vesicles are now considered as king transporters of specific cargoes, including molecular components of parent cells, thus mediating a wide variety of cellular activities both in normal and neoplastic tissues. Here, we discuss the multifunctional activities and underlying mechanisms of extracellular vesicles in neuroblastoma, the most frequent common extra-cranial tumor in childhood. The ability of extracellular vesicles to cross-talk with different cells in the tumor microenvironment and to modulate an anti-tumor immune response, tumorigenesis, tumor growth, metastasis and drug resistance will be pinpointed in detail. The results obtained on the role of extracellular vesicles may represent a panel of suggestions potentially useful in practice, due to their involvement in the response to chemotherapy, and, moreover, their ability to predict resistance to standard therapies-all issues of clinical relevance.

The Circular Life of Human CD38: From Basic Science to Clinics and Back.

Monoclonal antibodies (mAbs) were initially considered as a possible "magic bullet" for in vivo elimination of tumor cells. mAbs represented the first step: however, as they were murine in nature (the earliest experience on the field), they were considered unfit for human applications. This prompted the development of techniques for cloning the variable regions of conventional murine antibodies, genetically mounted on human IgG. The last step in this years-long process was the design for the preparation of fully human reagents. The choice of the target molecule was also problematic, since cancer-specific targets are quite limited in number. To overcome this obstacle in the planning phases of antibody-mediated therapy, attention was focused on a set of normal molecules, whose quantitative distribution may balance a tissue-dependent generalized expression. The results and clinical success obtained with anti-CD20 mAbs revived interest in this type of strategy. Using multiple myeloma (MM) as a tumor model was challenging first of all because the plasma cells and their neoplastic counterpart eluded the efforts of the Workshop on Differentiation Antigens to find a target molecule exclusively expressed by these cells. For this reason, attention was turned to surface molecules which fulfill the requisites of being reasonably good targets, even if not specifically restricted to tumor cells. In 2009, we proposed CD38 as a MM target in virtue of its expression: it is absent on early hematological progenitors, has variable but generalized limited expression by normal cells, but is extremely high in plasma cells and in myeloma. Further, regulation of its expression appeared to be dependent on a variety of factors, including exposure to all-trans retinoic acid (ATRA), a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells that are now approved for in vivo use. This review discusses the history of human CD38, from its initial characterization to its targeting in antibody-mediated therapy of human myeloma.

CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.

IL-27 in human secondary lymphoid organs attracts myeloid dendritic cells and impairs HLA class I-restricted antigen presentation.

Different cytokines play crucial roles in inflammation and in polarizing immune responses, including IL-27 that exerts pro- and anti-inflammatory functions. Although the activity of IL-27 is well characterized in murine immune cells, only limited information is available regarding the natural cellular sources of IL-27 in humans and its effects on human immune cells. Dendritic cells (DCs) are the most potent professional APCs that in the immature state are positioned throughout peripheral tissues by acting as sentinels, sensing the presence of Ags. Activated DCs migrate into the lymph nodes and direct Ag-specific T cell responses, thus acting as key players in both adaptive and innate immunity. In this study we asked whether IL-27 is produced by human secondary lymphoid organs and what is its functional role on human DCs. To our knowledge, we provide the first evidence that 1) in lymph nodes, macrophages are the major source for IL-27; 2) immature and mature human DCs express functional IL-27R; 3) IL-27 exerts immunosuppressive activity by crippling the Ag processing machinery in immature DCs under steady-state conditions and after pulsing with a viral Ag; and 4) IL-27 is chemotactic for human DCs. Our findings highlight novel mechanisms underlying the immunosuppressive activity of IL-27, suggesting that this cytokine may function as a homeostatic cytokine in secondary lymphoid organs by limiting duration and/or intensity of ongoing adaptive immune responses. The results presented in this study pave the way to future studies aimed at investigating whether dysregulation of IL-27 expression and function may be involved in pathogenesis of autoimmune disease and cancer.

Binding of HLA-G to ITIM-bearing Ig-like transcript 2 receptor suppresses B cell responses.

Inhibition of B cells constitutes a rational approach for treating B cell-mediated disorders. We demonstrate in this article that the engagement of the surface Ig-like transcript 2 (ILT2) inhibitory receptor with its preferential ligand HLA-G is critical to inhibit B cell functions. Indeed, ILT2-HLA-G interaction impedes both naive and memory B cell functions in vitro and in vivo. Particularly, HLA-G inhibits B cell proliferation, differentiation, and Ig secretion in both T cell-dependent and -independent models of B cell activation. HLA-G mediates phenotypic and functional downregulation of CXCR4 and CXCR5 chemokine receptors on germinal center B cells. In-depth analysis of the molecular mechanisms mediated by ILT2-HLA-G interaction showed a G0/G1 cell cycle arrest through dephosphorylation of AKT, GSK-3ß, c-Raf, and Foxo proteins. Crucially, we provide in vivo evidence that HLA-G acts as a negative B cell regulator in modulating B cell Ab secretion in a xenograft mouse model. This B cell regulatory mechanism involving ILT2-HLA-G interaction brings important insight to design future B cell-targeted therapies aimed at reducing inappropriate immune reaction in allotransplantation and autoimmune diseases.

Soluble HLA-G modulates miRNA-210 and miRNA-451 expression in activated CD4+ T lymphocytes.

In this study, we have investigated the expression of 87 micro (mi)RNAs in activated CD4(+) T cells cultured in the presence or absence of the immunoregulatory molecule soluble HLA-G (sHLA-G). We observed (i) a decreased miR-451 expression and (ii) an increased miR-210 expression in sHLA-G-treated CD4(+) T cells. By transfecting CD4(+) T cells with miR-210 and miR-451 mimics or inhibitors, we found that sHLA-G-mediated modulation of these miRNAs was not related to sHLA-G-mediated inhibition of (i) proliferation and (ii) CXCR3 expression in CD4(+) T cells. Finally, we investigated the expression of 14 genes targeted by miR-210 or miR-451 in activated CD4(+) T cells, treated or not with sHLA-G. We observed an increased expression of OSR-1 (odd-skipped related 1) and HBP-1 (HMG-box transcription factor 1) and a decreased expression of CXCL16 (chemokine C-X-C motif ligand 16) and C11orf30 (chromosome 11 open reading frame 30) in sHLA-G-treated CD4(+) T cells. In conclusion, sHLA-G triggered a modulation of miRNA expression that may in turn modulate downstream gene expression, thus affecting CD4(+) T-cell function.

Mechanisms of the antitumor activity of human Vγ9Vδ2 T cells in combination with zoledronic acid in a preclinical model of neuroblastoma.

Low expression of surface major histocompatibility complex (MHC) class I molecules and defects in antigen processing machinery make human neuroblastoma (NB) cells appropriate targets for MHC unrestricted immunotherapeutic approaches. Human T-cell receptor (TCR) Vγ9Vδ2 lymphocytes exert MHC-unrestricted antitumor activity and are activated by phosphoantigens, whose expression in cancer cells is increased by aminobisphosphonates. With this background, we have investigated the in vivo anti-NB activity of human Vγ9Vδ2 lymphocytes and zoledronic acid (ZOL). SH-SY-5Y human NB cells were injected in the adrenal gland of immunodeficient mice. After 3 days, mice received ZOL or human Vγ9Vδ2 T cells or both agents by intravenous administration once a week for 4 weeks. A significantly improved overall survival was observed in mice receiving Vγ9Vδ2 T cells in combination with ZOL. Inhibition of tumor cell proliferation, angiogenesis and lymphangiogenesis, and increased tumor cell apoptosis were detected. Vγ9Vδ2 T lymphocytes were attracted to NB-tumor masses of mice receiving ZOL where they actively modified tumor microenvironment by producing interferon-γ (IFN-γ), that in turn induced CXCL10 expression in NB cells. This study shows that human Vγ9Vδ2 T cells and ZOL in combination inhibit NB growth in vivo and may provide the rationale for a phase I clinical trial in patients with high-risk NB.

Validation of analytical methods for the production of expanded γδ T lymphocytes useful for therapeutic purposes.

The use of γδ T lymphocytes as advanced therapeutic medicinal product has attracted much interest in the last years. Indeed, such cells are an ideal tool for the reconstitution of the immune system in patients receiving hematopoietic stem cell transplantation, due to their MHC-independent anti-tumor and anti-viral activities. We have here setup a protocol for the production of pure and functional γδ T lymphocytes, expanded from healthy donors' mononuclear cells, and validated the analytical methods to identify them and to analyze their potency. Next, we performed stability studies to ensure that the cell product (γδ T cells) can be used after freezing and thawing. Notably, such protocol can be promptly translated to GMP-facility, since it has been designed using only clinical grade reagents.

Microbes identified from monitoring cell manipulations in 5-year life of the Cell Factory G. Gaslini.

Introduction: Quality and safety of a cell product, essential to guarantee the health of patients, depends on many factors including an appropriate environmental monitoring of the manufacturing rooms. Nonetheless, the maintenance of a controlled environment is requested to minimize the risk of contamination. Thus, a timely detection of changes in microbiological trends is important to adopt promptly effective measures against resistant strains that, in turn, may invalidate not only the sanitization procedures but also the safety of the cell product. Methods: We analyzed microbes found in our cell processing clean room over the last 5 years. We used 10.147 plates for air sampler, passive air monitoring and for checking instruments and operators of the production unit. Results: From these plates, 747 colonies were subjected to identification by the MALDI-TOF Vitek® MS system and the large majority of them was gram positive (97.8%) as witnessed by the finding that the most represented genera harvested from the classified areas were Staphylococcus (65%), Micrococcus (13%), Kocuria (8%) and Bacillus (5%). We never detected fungi. Most microbes found in the operators (both from class A and B) were collected from forearms and resulted of the Staphylococcus genus. Conclusions: The observed microbial contamination is to be attributed to the personnel and no substantial microbial pitfalls in our Cell Factory has been detected.

Human TCRγδ+ T cells represent a novel target for IL-27 activity.

IL-27 and TCRγδ(+) T lymphocytes play critical roles in both innate and adaptive immune responses in health and disease, including infection and tumors. Although the activity of IL-27 is well characterized in different human immune cells, no information is available on the role of IL-27 in human TCRγδ(+) T lymphocytes. Here, we provide the first evidence that TCRγδ(+) T lymphocytes express both gp130 and WSX-1 chains of IL-27R, and that IL-27 may function in TCRγδ(+) T cells by (i) inducing STAT1 and STAT3 phosphorylation, (ii) stimulating cytotoxicity against tumor cells through upregulation of cytotoxic granules production, (iii) reducing the release of Th2-related cytokines, such as IL-5 and IL-13, and inducing IFN-γ production, and (iv) upregulating the expression of CD62L. These results highlighted a novel immunoregulatory property of human IL-27 that may be relevant in the immune response against tumors. Our results may offer new perspectives for the development of future clinical trials using IL-27 and TCRγδ(+) cells for cancer immunotherapy.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells.

Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G-mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56brigh) and CD56dim NK cells. Such sHLA-G-mediated down-modulations were reverted by adding anti-HLA-G or anti-ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G-treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G-mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G-mediated inhibition of NK-cell functions.

Blood Lymphocyte Subsets and Proinflammatory Cytokine Profile in ROHHAD(NET) and non-ROHHAD(NET) Obese Individuals.

Context: Rapid-onset obesity with central hypoventilation, hypothalamic dysfunction, and autonomic dysregulation with neural crest tumors (ROHHAD-NET) syndrome pathophysiology remains elusive. Acquired neuroimmunological dysfunction has been proposed as a possible pathogenetic pathway. Objective: The aim of our study was to characterize lymphocyte subpopulations subsets in peripheral blood (PB) and to evaluate a panel of proinflammatory cytokines/chemokines in ROHHAD(NET) patients vs controls. Methods: We included 11 ROHHAD(NET) patients, 7 ROHHAD and 4 ROHHAD-NET, selected by clinical criteria. Controls were 11 simple obese children, matched for age and sex. Flow cytometric analysis and enzyme-linked immunosorbent assay were performed on PB and serum samples of the 2 groups. Results: Analysis revealed that T lymphocytes are significantly increased in ROHHAD(NET) patients (P = .04) with a prevalence of CD4-T cells (P = .03) and a lower number of activated CD8-T cells (P = .02). With regard to regulatory subset, patients displayed increased regulatory B cells (P = .05) and type-1 regulatory T cells (P = .03). With regard to CD8-T cells, a lower number of T effector memory was observed (P = .02). In contrast, among CD4-T cells, we found a higher number of T naive (P = .04) and T effector (P = .0008). Interleukin-8 (IL-8) levels and monocyte chemotactic protein-1 were increased in patients vs controls (P = .008 and P = .01, respectively). Furthermore, IL-8 levels were higher in the subgroup with neural tumor (P = .0058) (ROHHAD-NET) than in patients without neural tumor (ROHHAD). Soluble HLA-G was significantly lower in patients vs controls (P = .03). Conclusion: Our findings contribute to support the hypothesis of immune dysregulation, which may underlie this complex, often fatal disease. Because ROHHAD(NET) syndrome is an ultra-rare disease, multicentric studies are needed to improve the effect of our data in the management of this condition.

Bone marrow of neuroblastoma patients shows downregulation of CXCL12 expression and presence of IFN signature.

BACKGROUND: At diagnosis, children with neuroblastoma (NB) present with either localized or metastatic disease. Since the mechanisms responsible for BM invasion are not well known, we investigated the transcriptome of resident BM cells from NB patients as compared to healthy children. PROCEDURE: Ninety-two and 88 children with localized and metastatic NB, respectively, and 15 healthy children were included in the study. BM resident cells recovered from BM aspirates by immunomagnetic bead manipulation were subjected to genome-wide microarray analysis. After validation in an independent set of samples, the genes significantly modulated in resident BM cells from NB patients were tested for their diagnostic/prognostic values. RESULTS: BM resident cells, irrespective of neoplastic cell invasion, significantly overexpressed genes involved in innate immune responses, and interferon (IFN) and IFN-DRS signatures were enriched. Genes coding for metallothioneins and zinc finger proteins, and involved in histone and nucleosome/chromatin organization were also overexpressed. Resident BM cells from NB patients significantly downregulated genes involved in cell adhesion, and in erythrocyte, myeloid, and platelet differentiation pathways. Among downregulated genes, CXCL12 expression reached near complete silencing in patients with metastatic disease. The downregulation of CXCL12 expression was independent of contact between NB cell and resident BM cell. CONCLUSIONS: We demonstrated that NB tumor growth at the primary site can alter the BM microenvironment, and the presence of BM-infiltrating NB cells makes the alterations more pronounced. Therefore, the restoration of a BM physiological state by means of IFN-α monoclonal antibody, Sifalimumab, and selective noradrenaline receptor blockers should be further studied to ameliorate patients' clinical management.

HLA-G in organ transplantation: towards clinical applications.

HLA-G plays a particular role during pregnancy in which its expression at the feto-maternal barrier participates into the tolerance of the allogenic foetus. HLA-G has also been demonstrated to be expressed in some transplanted patients, suggesting that it regulates the allogenic response. In vitro data indicate that HLA-G modulates NK cells, T cells, and DC maturation through its interactions with various inhibitory receptors. In this paper, we will review the data reporting the HLA-G involvement of HLA-G in human organ transplantation, then factors that can modulate HLA-G, and finally the use of HLA-G as a therapeutic tool in organ transplantation.

Emerging topics and new perspectives on HLA-G.

Following the Fifth International Conference on non-classical HLA-G antigens (HLA-G), held in Paris in July 2009, we selected some topics which focus on emerging aspects in the setting of HLA-G functions. In particular, HLA-G molecules could play a role in: (1) various inflammatory disorders, such as multiple sclerosis, intracerebral hemorrhage, gastrointestinal, skin and rheumatic diseases, and asthma, where they may act as immunoregulatory factors; (2) the mechanisms to escape immune surveillance utilized by several viruses, such as human cytomegalovirus, herpes simplex virus type 1, rabies virus, hepatitis C virus, influenza virus type A and human immunodeficiency virus 1 (HIV-1); and (3) cytokine/chemokine network and stem cell transplantation, since they seem to modulate cell migration by the downregulation of chemokine receptor expression and mesenchymal stem cell activity blocking of effector cell functions and the generation of regulatory T cells. However, the immunomodulatory circuits mediated by HLA-G proteins still remain to be clarified.

Serum levels of cytoplasmic melanoma-associated antigen at diagnosis may predict clinical relapse in neuroblastoma patients.

The high molecular weight melanoma-associated antigen (HMW-MAA) and the cytoplasmic melanoma-associated antigen (cyt-MAA/LGALS3BP) are expressed in melanoma. Their serum levels are increased in melanoma patients and correlate with clinical outcome. We investigated whether these molecules can serve as prognostic markers for neuroblastoma (NB) patients. Expression of cyt-MAA and HMW-MAA was evaluated by flow cytometry in NB cell lines, patients' neuroblasts ((FI)-NB), and short-term cultures of these latter cells (cNB). LGALS3BP gene expression was evaluated by RT-qPCR on (FI)-NB, cNB, and primary tumor specimens. Soluble HMW-MAA and cyt-MAA were tested by ELISA. Cyt-MAA and HMW-MAA were expressed in NB cell lines, cNB, and (FI)-NB samples. LGALS3BP gene expression was higher in primary tumors and cNB than in (FI)-NB samples. Soluble cyt-MAA, but not HMW-MAA, was detected in NB cell lines and cNBs supernatants. NB patients' serum levels of both antigens were higher than those of the healthy children. High cyt-MAA serum levels at diagnosis associated with higher incidence of relapse, independently from other known risk factors. In conclusion, both HMW-MAA and cyt-MAA antigens, and LGALS3BP gene, were expressed by NB cell lines and patients' neuroblasts, and both antigens' serum levels were increased in NB patients. Elevated serum levels of cyt-MAA at diagnosis correlated with relapse, supporting that cyt-MAA may serve as early serological biomarker to individuate patients at higher risk of relapse that may require a more careful follow-up, after being validated in a larger cohort of patients at different time-points during follow-up. Given its immunogenicity, cyt-MAA may also be a potential target for NB immunotherapy.