Neuroblastoma (NB) is the most common pediatric extracranial solid tumor of childhood, and 45% of patients have high-risk tumors, nearly all of which are metastatic (stage 4) when diagnosed . Patients with neuroblastoma are risk stratified based on presenting factors including age, stage and location of disease, and specific biologic molecular markers of the tumor, including NMYC status and ploidy [1-3]. Treatment given is tailored to whether a patient has low, intermediate or high-risk disease. The overall prognosis for those with high risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy followed by stem cell rescue. Additionally, many patients who survive suffer from complications related to their treatment. In this chapter, we review the literature that provides a rationale for the use of novel targeted agents to improve the treatment and survival while lessening toxicity of patients with neuroblastoma who have failed standard therapies.
In particular, we focus our discussion on a few specific signaling pathways. The central role of the phosphatidylinositol 3-kinase-Akt-phosphatase and tensin homolog (PI3K-Akt-PTEN) axis and RAF-MEK-ERK as potential molecular targets to control downstream effectors of coordinated cell division, tumor growth, angiogenesis, apoptosis, invasion and cellular metabolism in the tumor and surrounding stromal compartments. The PI3K and RAF-MEK-ERK pathways have also been implicated in modulating p53, the hypoxia-inducible factor 1 (HIF1α), mycN and others.
NMYC is known to play a role in the tumorigenesis of certain high-risk neuroblastoma tumors and its control has many implications in targeting therapy. Additional pathways and targets explored in this chapter are the RAS/Raf/MEK/ERK pathway, specific angiogenesis inhibitors including VEGF, ALK 1 mutations and inhibitors, and control of apoptosis through caspase 8. We also discuss the idea of synthetic lethality and the concepts of sequential versus simultaneous inhibition. We will discuss the emerging importance of genomic and metabolomic profiling in tumor interrogation with therapeutic considerations.
We will review the literature supporting a role for cancer stem cells (CSCs) in the pathogenesis of neuroblastoma and the signaling pathways that define the CSC phenotype. We discuss the role targeted therapies in CSC related therapeutics and the adaptive responses that such cells have when exposed to targeted therapeutic agents.
Lastly, the emerging role of immunotherapeutics into both standard and targeted therapies for neuroblastoma is explored. This includes areas of T cell and macrophage infiltration of tumors, interleukin and cytokine involvement, and anti-GD2 human and mouse monoclonal antibodies.
2. PTEN and PI-3 kinase and mycN signaling as targets for NB therapeutics — The intercept node hypothesis
The idea that some signaling pathways are more central to tumorigenesis than others was suggested by our laboratory and others . From connectivity map analysis, some signaling proteins appear connected to a large number of upstream and downstream effector pathways. These are considered central “intercept nodes” [4, 5] which provide coordinate control over the output of a large number of cell surface receptor input. The specificity of signaling downstream of such intercept nodes is generally fine tuned by more specialized signaling effector proteins e.g. Rac2, HIF1α, NFκB or mycN which encode more specific signaling content. Two such central pathways, PTEN-PI-3-AKT and Raf-MEK-ERK are critical for NB survival, proliferation, invasion and angiogenesis
3. Role of angiogenesis in tumorigenicity of neuroblastoma / PI3 kinase and VEGF inhibitors in treatment of neuroblastoma
Work from a number of laboratories indicates that the angiogenic response is coordinately and highly regulated physiologic response to hypoxia and inflammation. Hence it is not surprising to learn that central node in mammalian cells control output from many cell surface receptors to regulate this response . We and others have shown that PTEN a major tumor suppressor protein regulates angiogenesis and loss of PTEN results in deregulation of PTEN and multiple downstream signaling pathways shown in Fig. 1 and 2 which have all been implicated in the literature to exert coordinate control of angiogenesis
In general, angiogenesis plays an important role in the progression and metastasis of malignant tumors . In neuroblastoma, tumor vascularity is correlated with an aggressive phenotype [15, 16]. Pro-angiogenic factors are differentially expressed in high-risk neuroblastoma [17, 18]. Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen that stimulates angiogenesis and plays a crucial role in tumor growth . Over expression of VEGF has been demonstrated in neuroblastoma, nephroblastoma, as well as in various other cancers [20-22]. Recent studies have validated inhibition of VEGF as an effective antiangiogenic therapy in some of these cancers [23-25]. Although several preliminary studies have demonstrated that expression of angiogenic growth factors, including VEGF, correlate with a high-risk phenotype in neuroblastoma, clinical data are still insufficient to draw conclusions [17, 21, 26, 27]. Therefore, further clinical studies, are needed to evaluate the possible significance of these factors for use in a routine clinical practice. Preclinical studies also suggest that antiangiogenic strategies may be effective in the treatment of neuroblastoma . In addition, phase I clinical trials (COG study) using the human anti-VEGF antibody, bevacizumab, in pediatric patients with refractory solid tumors reported promising results . Recently, Jakovljevic
4. Cancer stem cells in neuroblastoma tumorigenicity
The Cancer Stem Cell Theory postulates that tumors contain a subset of cells that are capable of increased self-renewal and differentiation, can propagate tumor growth and are resistant to apoptosis [31, 32]. These stem-like cancer cells are analogous to normal stem cells  but differentiate into diverse cancer cells that form the major portion of the tumor. Recent evidence suggests the presence of stem cells in various cancers including those of the blood , breast , prostrate  and brain .
Evidence for the presence of cancer stem cells in brain tumors first came from the observation that human medulloblastoma, astrocytomas, and ependymomas contain cells that express the neural stem cell marker CD133  . Singh et al.  have shown that human brain tumors contain CD133+ stem-like cells that are capable of growing tumors in immune-deficient mice. Cournoyer et al. have shown that CD133 high neuroblastoma (NB) cells have high tumor initiating cell properties, and Coulon et al. suggest that CD133, ABC transporter, Wnt and NOTCH genes are sphere markers in NB cells. Overall, 19–29 % of cells in glioblastomas and 6–21 % of cells of medulloblastomas are reported to be CD133+ and tumorigenic . Recently, several groups have suggested that CD15 (stage specific embryonic antigen 1 or SSEA-1), which is expressed on neural progenitor and stem cells, may be a better marker than CD133 of tumor-initiating cells in MB, glioma, and ependymoma [42-44]. Hansford et al has recently identified tumor initiating cells from NB bone marrow metastases that have several properties of cancer stem cells including the expression of stem cell markers, the ability to self renew and the capability to form metastatic NB in immunodeficient animals with as few as 10 cells . Kaplan’s laboratory has further defined the NB tumor initiating cell (TIC) with stem cell like properties to express, CD133 and CD44. These cells isolated from NB bone marrow have tumor initiating activity and upon profiling display sensitivity to a number of targeted therapeutic agents.
A key aspect of the tumor stem cell (TSC) niche is the balance of signals received, and over recent years considerable attention has been directed towards understanding the role of signaling pathways, which are critical mediators of normal stem cell biology, in cancers. The embryonic signaling pathways most commonly implicated in tumorigenesis include Hedgehog, Notch, and Wnt pathways. Sonic Hedgehog (SHH) signaling is important in embryonic cell development and proliferation and aberrant pathway activation can lead to tumor formation, tumor cell self-renewal and the development of metastatic disease .Similarly, Notch plays a crucial role in biological functions of development and cell fate including cell differentiation and proliferation . Constitutive activation of Notch can lead to tumorigenesis and cell survival, and Notch activity is involved in tumor angiogenesis . The Wnt family proteins help direct a wide range of developmental processes including cell fate, proliferation, motility, and polarity . Dysregulation of the Wnt pathways has been implicated in tumor formation, proliferation, and maintenance . All of the current pediatric studies demonstrating that progenitor and stem cells can respond to embryonic signaling have been in MB or primitive neuroectodermal tumors (PNET). Aberrant SHH signaling has been implicated in MB, and recently was used to define one of four distinct molecular variants of MB .
In order to identify pathways required for proliferation and cell survival characteristics of TIC in neuroblastoma, Grinshtein et al. has performed drug screen on bone marrow derived tumor initiating cells (TICs) with a unique collection of pharmacological inhibitors. They identified that PI3K (phosphoinositide 3 kinase)/AKT, PKC (protein kinase C), Aurora, ErbB2, Trk and Polo-like kinase 1 (PLK1) are the potential kinase targets for survival of TIC . Their studies demonstrated that PLK1 inhibitors are an attractive candidate therapy for metastatic NB. Another group suggested that both PI-3 kinase as well as Ras-RAF-MEK-Erk signaling pathways promote the tumorigenicity of the glioblastoma cancer stem like cells, and combined treatment with MEK and PI-3 kinase inhibitors can block the differentiation of glioblastoma cancer stem like cell into non tumor initiating status .
The therapeutic resistance of cancer stem cell to current treatment modalities such as chemotherapy and radiation make these cells clinically relevant irrespective of their origin. Resistance to chemotherapeutic agents has been demonstrated in neuroblastoma stem cells and sarcoma stem cells including Ewing’s sarcoma and osteosarcoma. Recent work by Hambardzumyan suggests that the PI-3 kinase pathway activity promotes post-radiation survival in cancer stem cells in medulloblastoma . Although lots of literatures are available on the cancer stem cell in neuroblastoma but yet the novel signaling pathways controlling the proliferation and survival of cancer stem cell and the mechanism behind resistance developed due to chemotherapy needs to be investigated.
5. Neuroblastoma and cancer metabolism
It has been known from a long time that cancer cells take up and metabolize glucose and glutamine to a degree that far exceeds their needs for these molecules in anabolic macromolecular synthesis . Commonly occurring oncogenic signal transduction pathways initiated by receptor tyrosine kinases or Ras engage PI3K-Akt signaling to directly stimulate glycolytic metabolism under aerobic conditions a condition termed the Warburg and Pasteur effects [56-58]. Myc-activation/amplification is one of the most common oncogenic events observed in cancer and is known to drive the progression of a certain subgroup of neuroblastoma . The activation of mycN could occur through amplification of the mycN gene or through upstream activation of signaling pathways that would stabilize mycN e.g. trkB, IGF-1 or the activation of Raf and/or PI-3K-AKT stimulation. Oncogenic levels of Myc have recently been linked to increased glutaminolysis through a coordinated transcriptional program program [60-62]. Quantitative RTPCR and ChIP experiments support Myc’s binding and transcriptional activation of two high affinity glutamine transporters: SLC38A5 (also called SN2) and SLC1A5 (ASCT2), the transporter required for glutamine-dependent mTORC1 activation [60, 63]. In addition to facilitating glutamine uptake, Myc promotes the metabolism of imported glutamine into glutamic acid and ultimately into lactic acid . Whether the tendency of Myc to complement Ras and PI3K-Akt [64, 65] is related to the interdependence of glutamine and glucose metabolism in support of cell growth remains an open question. The work of C. Dang and other points to a potential important metabolic requirement for glutamine in c-myc and mycN driven tumors where glutamine can serve a role in promoting tumor growth [58, 66]. This might suggest a role of agents which deplete glutamine (glutaminases) as a therapeutic target for mycN driven malignancies like neuroblastoma and the SHH subtype of medulloblastoma.
6. Role of tumor infiltrating immune cells in tumorigenicity of neuroblastoma
Solid tumors are composed of tumor stromal cells, blood vessels, infiltrating immune cells and tumor cells themselves. Over the last decade, a growing body of literature has highlighted the importance of the tumor microenvironment for the prognosis of different types of cancer . The tumor microenvironment contains many resident cell types, such as adipocytes and fibroblasts, but it is also populated by migratory hematopoietic cells, including lymphoid cells, granulocytes, mast cells, dendritic cells, natural killer cells, neutrophils and macrophages. These haematopoietic cells have pivotal roles in the progression and metastasis of tumors [69, 70]. The significance of tumor stroma for the overall prognosis may be in part due to the fact that several components of the tumor-microenvironment have been shown to compromise immune effect functions against tumor cells . The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers .
The first evidence suggesting immune responses to neuroblastoma was provided in 1968 when blood leukocytes, which were 50–70% lymphocytes, were reported to inhibit colony formation by neuroblastoma cells . These lymphocytes inhibited colony formation by both autologous and allogeneic neuroblastoma cells but did not affect growth of fibroblasts from the same donors. Plasma from these patients also was reported to inhibit tumor cell colony formation in the presence of complement. In this same time, primary tumors were reported to contain leukocytes [74, 75], and some localized and metastatic neuroblastomas were reported to regress spontaneously [76, 77]. Together, these studies suggest that the immune system could develop an anti-neuroblastoma response. In this section, we will highlight the role of tumor infiltrating immune cells in progression of this disease and how blocking the function of these infiltrating cells may prove beneficial in its treatment of NB.
Tumor infiltrating Lymphocytes
Tumor-associated lymphocyte population includes CD8+ cytotoxic T cells, CD4+ T helper cells, regulatory T cells (Tregs), NKT or γδT cells. Tregs are immunosuppressive regulatory T cells. Tregs are able to suppress the activity of CTLs by direct cell-cell contact and also secrete immunoregulatory cytokines such as transforming growth factor β (TGF-β) and interleukin-10 (IL-10). However, the role of Tregs is much less clear and to our knowledge there are no published data on the presence (or otherwise) of Tregs in pediatric tumors.
CD8+ cytotoxic T lymphocytes (CTL) are a primary source of anti-tumor activity in the immune system [1, 3]. In many adult cancers the presence of significant numbers of tumor-infiltrating lymphocytes, potentially represents the host immune response against the tumor and is associated with improved prognosis [78-80]. In neuroblastoma, Martin
However, despite these cellular responses to NB, the presence of tumor-infiltrating CTL is rare, suggesting a block in T cell trafficking that may protect the tumor from CTL-mediated cytotoxicity. Therefore, strategies aiming to generate CTLs must take into account mechanisms by which neuroblastoma cells may avoid immune elimination. These include decreased expression of peptide presenting HLA class I molecules by tumor cells, which can impair target peptide recognition by CTLs [82, 86, 87]. Also, neuroblastoma cells express low levels of antigen processing genes, including LMP-2, LMP-7, and TAP-1, which are necessary for preparation of peptides from proteins for presentation by HLA class I molecules to CTLs [88, 89]. Neuroblastoma cells also induce monocytes to release HLA-G, which suppresses both CTL and NK mediated cytotoxicity by interacting with inhibitory receptors or inducing apoptosis via CD8 ligation or the Fas-FasL pathway . Thus, effective CTL anti-tumor responses require that these escape mechanisms be evaluated and, if present, be overcome.
Natural Killer Cells
Natural Killer (NK) cells represent a particular subset of T lymphocytes, which express both T cell markers, such as the αβ T-cell receptor (TCR) and associated CD3 complex, and NK cell markers, such as NK1.1. These cells recognize glycolipids presented by the MHC class I-like molecule CD1d and are believed to play an important role at the interface between the innate and adaptive immune responses to infection and malignancy . Two main subtypes of NKT cell are recognised, with Type I NKT cells expressing an invariant α-TCR chain and being implicated in antitumor immunity, whilst Type II NKT cells express a variety of TCR molecules (in addition to CD1d) and appear to have a more immune inhibitory role . The presence of these immune effector cells within tumors has been examined in a number of different malignancies, including, neuroblastoma. Type I NKT cells were found in 53% of 98 untreated primary stage 4 neuroblastoma samples  and their infiltration correlated with favorable outcome, with expression of the chemokine CCL2 and with absence of MYCN amplification (indicating less aggressive disease). Subsequent investigations have confirmed that expression of CCL2 is repressed in MYCN amplified tumors, leading to a failure of NKT cell infiltration and potentially contributing to tumor immune escape .
Recent studies have suggested anti-tumor role of NK cells in high risk neuroblastoma NK cells are activated to be cytotoxic and secrete IFNγ by IL-2. IL-2 alone has been tested in phase I and II trials for patients with neuroblastoma, and, although immune effects were documented, no objective tumor responses were observed [95, 96]. Lenalidomide is an immune modulating drug that activates T cells to secrete IL-2, which in turn activates NK cell cytotoxicity and ADCC [97, 98]. Clinical trials in children and adults demonstrated increased numbers of NK cells and cytotoxicity, decreased T regulatory cells, and increased secretion of IL-2, IL-15, and GM-CSF after 21 days of lenalidomide treatment [99, 100]. Thus, lenalidomide may be useful for activating NK cells to enhance mAb immunotherapy of neuroblastoma.
Role of tumor associated macrophages
Macrophages represent a further important cellular component of the tumor stroma. Far from being mere bystanders to tumor development, there is increasing evidence that tumor-associated macrophages (TAMs) promote and facilitate tumor growth [101, 102]. Of key importance is the concept of distinct macrophage phenotypes, mirroring the dichotomy between TH1 and TH2 T helper cells and type I and type II immune responses. Alternatively activated M2-macrophages are involved in polarized Th2 inflammatory reactions and characterized by expression of arginase-1 and mannose and scavenger receptors [103, 104]. On the other extreme, classically activated M1 macrophages are IL-12 high, IL-23 high, IL-10 low; produce high levels of inducible nitric oxide synthetase (iNOS); secrete inflammatory cytokines such as IL-1β, IL-6, and TNF; and are inducer and effector cells in Th1 type inflammatory responses . It has been suggested that tumor-associated macrophages (TAMs) display an M2-like phenotype .
TAMs are recruited to tumors when stimulated by growth factors and chemokines, produced by the tumor cells [107, 108]. The conventional wisdom about TAM function is that they are recruited to reject the tumor, which has been recognized as foreign because tumors express unique antigens. However, there is a growing body of evidence that the tumor microenvironment is immunosuppressive , perhaps as a result of selection for such an environment a process recently termed ‘immunoediting. Recent data indicate that TGF-β1 has an important role in suppressing these local responses and that inhibiting this molecule can result in tumor rejection [110, 111]. It is noteworthy that TAMs can both produce TGF-β1 and process latent TGF-βs to produce their active forms. In addition, the local cytokine milieu in the tumor tends to block the immunological functions of these newly recruited mononuclear phagocytes such as antigen presentation and cytotoxicity towards tumors, and diverts them towards specialized TAMs that are immunosuppressed and trophic . A principal component of this cytokine mixture is CSF-1, which locally blocks the maturation of dendritic cells so that they are unable to present antigens and promotes the development of immunosuppressed trophic TAMs. TAMs promote tumor growth by affecting angiogenesis, immune suppression, invasion and metastasis [101, 102]. Existing literature suggests that tumor associated macrophages secrete several genes including matrix metalloproteinases-9 (MMP-9) , urokinase- type plasminogen activator (uPA) , vascular endothelial growth factor (VEGF) , and cyclooxygenase-2 (Cox-2]  which promotes tumor growth by breaking down extracellular matrix. The role of TAMs in tumor growth and progression is highlighted in Figure 3.
Clinical studies have, on balance, shown a correlation between an abundance of TAMs and poor prognosis . These data are particularly strong for breast, prostate, pancreatic, ovarian and cervical cancers; the data for stomach and lung cancers are contradictory [108, 117, 118], and in a small study in colorectal cancer, their presence was associated with good prognosis . However, taking all reports into account regardless of method and sample number more than 80% show a significant correlation between TAM density and poor prognosis, whereas less than 10% associate TAM density with a good prognosis . So, increased TAM density is usually associated with advanced tumor progression and metastasis in most of the cancers. However the prognostic significance of tumor associated inflammatory cells in metastatic disease and in childhood cancers is mostly unknown.
Recent reports suggest that interaction between tumor and inflammatory cells contribute to the clinical metastatic neuroblastoma phenotype . It has been reported that metastatic neuroblastomas had higher infiltration of TAMs than loco regional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation related genes than those in patients diagnosed at age < 18 months. They identified 14 genes, out of which nine were tumor cell related and five were inflammation related that comprises a prognostic signature for neuroblastoma. Expression of inflammation related genes representing TAMs (
7. Multiple ‘Omics’ analysis an emerging concept in treatment of neuroblastoma
The “Omics” is a neologism widely adopted by scientists to refer to large scale analysis of genes (genomic), proteins (proteomics) and lately small metabolites (metabolomics). Modern molecular achievements over the last decade have seen the increase and implementation of multiple 'omics technologies in oncology that promises to provide for a deeper comprehension of complex tumor pathways. It is believed that an integration of multiple “omics” technologies is likely to provide even further insight into the holistic view of the biology networks . The studies of global expression profiles of both mRNA and protein are necessary to reveal the important pathways for an enigmatic disease such as neuroblastoma. During the past several years many studies utilized microarray-based high throughput technologies to investigate gene expression profiles and DNA copy number alterations in neuroblastoma [123, 124]. Guo
Metabolomics falls behind its predecessor genomics and proteomics, but represent a burgeoning field with potential to fill up the gap between genotype and phenotype  The high throughput nature of metabolomics makes it an attractive tool for scientists involved in the process of drug development. The reason for that lies in the principle that a patients response to drugs and toxicities do not depend only on a person genetic make-up, but it is rather a factorial outcome of interactions between intrinsic factors and environment . Therefore, metabolomics technology is a powerful tool that can accurately measure the entire spectrum of biochemical changes and mapping these changes to metabolic pathways [128, 129]. In 1995, Florian et al.  determined the metabolic characteristics of three types of human brain and nervous system tumors by high-resolution in vitro MRS and chromatographic analysis. Signals from leucine, isoleucine, glycine, valine, threonine, lactate, acetate, glutamate, and choline-containing compounds were similarly detected in meningiomas, glioblastomas, and NB. In 2007, Peet et al.  reported the results of in vitro 1H high-resolution magic angle spinning NMR spectroscopy (HRMAS) investigations performed on cell suspension of 13 lines of NB possessing multiple genetic alterations. In their study, a specific metabolite profile associated with
Overall, the emerging concept of analyzing NB-specific 'omics profiles to better understand and define the behavior of advanced-stage tumors along with providing direct and targeted therapy may ultimately translate into improved outcomes for high-risk NB.
8. Antibody dependent cellular toxicity (ADCC) / Role of ITAM and ITIM signaling in neuroblastoma
The Fcγ receptors (FcγRs) expressed on hematopoietic cells play a key role in immune defenses by linking humoral and cellular immunity . FcγRs display coordinate and opposing roles in immune responses depending on their cytoplasmic region and/or their associated chains. Indeed, the activating receptors contain an immunoreceptor tyrosine-based activation motif (ITAM) and initiate inflammatory, cytolytic, and phagocytic activities of immune effector cells. In contrast, the inhibitory receptors that downmodulate the immune responses contain an immunoreceptor tyrosine-based inhibitory motif (ITIM) [134, 135]. There are numerous Fc receptors for IgG (FcγR) that are widely expressed on immune cells. The FcγR family consists of four classes of receptors, FcγRI, FcγRII, FcγRIII, and FcγRIV, that have been identified in both mice and humans. There are significant similarities in the functions of the FcγR receptors between mice and humans, but there is limited homology in receptors themselves . To date, only one inhibitory FcγR, FcγRIIb, has been identified and is the only receptor to have complete homology between mice and humans . FcγRs can be found on virtually all hematopoietic cells except T cells; in most cases, cells coexpress activating and inhibitory FcγR, allowing for the balance between activating and inhibitory receptors to dictate their response . NK cells are an exception to this rule and express only the activating FcγRIIIa. NK cells do not express the inhibitory FcγRIIb.
Antibodies directed against neoplastic cells provide new therapeutic approaches against various malignancies, including lymphoma, leukemia, melanoma, and breast and colorectal carcinoma [137, 138] There is increasing evidence that the Fc portion of the anti-tumor IgG is a major component of their therapeutic activity, along with other mechanisms such as activation of apoptosis, blockade of signaling pathways, or masking of tumor antigens. Thus, by binding to activating FcγRs expressed by immune effector cells, such as macrophages, monocytes, neutrophils, or NK cells, tumor-specific antibodies trigger the destruction of malignant cells via antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis [139, 140].
Because of their rapid and unopposed responses to mAb, NK cells play a major role in the anti-tumor response elicited by tumor-specific mAbs. Multiple clinically successful mAbs utilize NK-mediated ADCC as a mechanism of action. Rituximab (anti-CD20), Herceptin (anti-Her-2/neu), Cetuximab (anti-EGFR), and the anti-GD2-mAbs 3F8 and ch14.18 are examples of tumor-specific mAbs whose clinical activity can be attributed, at least in part, to NK cells. Natural killer (NK) cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs). Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs) designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.
9. Tumor associated gangliosides / GD2 monoclonal antibodies
Gangliosides (GD) are membrane-associated glycosphingolipids which have important regulatory roles during embryogenesis and have also been implicated in tumor development. Particular gangliosides, which show restricted patterns of expression in normal tissue, may be expressed at high levels by tumor cells (e.g. GD3 by melanoma) and are implicated both in tumorigenesis and as mediators of metastatic spread . There is also evidence that gangliosides secreted by tumor cells can modulate the immune response and, in particular, act to inhibit dendritic cell differentiation and function. Neuroblastoma (and other neuroendocrine) tumor cells ubiquitously express the ganglioside GD2, whilst expression in normal tissues is restricted to neurons. Thus, GD2 is an attractive antigen for neuroblastoma immunotherapy strategies  including humanized anti-GD2 monoclonal antibodies such as ch14.18 , or GD2-directed cytotoxic lymphocytes. A chimeric human–murine anti-GD2 monoclonal antibody  called ch14.18 has shown activity against neuroblastoma in preclinical studies  and early-phase clinicaltrials[146, 147], this activity could be enhanced when ch14.18 is used in combination with granulocyte–macrophage colony-stimulating factor (GM-CSF)  or interleukin-2 [149, 150] to augment antibody-dependent cell-mediated cytotoxicity. The feasibility of administering ch14.18 in combination with GM-CSF, interleukin-2, and isotretinoin during the early post-transplantation period has been shown in two sequential pilot phase 1 studies [143, 151]. This progression of clinical trials culminated in the recently completed phase III randomized study of isotretinoin together with ch14.18, IL-2, and GMCSF vs. isotretinoin only for children with high-risk neuroblastoma who had a clinical response to induction therapy and myeloablative consolidation therapy/AHSCT. Immunotherapy after consolidation significantly improve event free survival (EFS) (66 ±5% vs. 46 ±5% at 2 years, P = 0.01) and overall survival (86 ± 4% vs. 75 ± 5% at 2 years, P = 0.02). This was the first demonstration that antibody based therapy improves EFS and overall survival. Although EFS was improved by adding immunotherapy to isotretinoin, approximately 40% of patients still relapsed during or after this therapy . Additionally, the combination of ch14.18 with IL-2 and GM-CSF has significant toxicities, including neuropathic pain, fever without neutropenia, infection, hypokalemia, hypotension, and capillary leak syndrome. Thus, a search for new agents to combine with ch14.18 to improve efficacy and decrease toxicity is justified.
Immunocytokines commonly known as antibody-cytokine fusion proteins combine the targeting ability of antibodies with the functional activity of cytokines, and are known to improve antibody-based therapy by delivering cytokines to the microenvironment to both activate effector cells and modulate the microenvironment. To date, immunocytokine research has focused on ADCC mediated by NK cells and on induction of CTL. An anti-GD2/IL-2 immunocytokine eradicated hepatic metastases of neuroblastomas in SCID mice that had been reconstituted with human lymphokine (IL-2) activated killer cells [153, 154]. In contrast, the combination of monoclonal anti-GD2 antibody and IL-2 at doses equivalent to the immunocytokine only reduced tumor load. In a syngeneic murine model of GD2 expressing melanoma, targeting with an anti-GD2 antibody/IL-2 immunocytokine resulted in generation of CD8+ T lymphocytes that could eradicate tumor as well as prevent tumor growth . Based upon these data, phase I and II studies have tested a humanized anti-GD2/IL-2 immunocytokine (hu14.18/IL-2) in patients with refractory or relapsed neuroblastoma. In the phase I study of 27 patients, treatment with hu14.18/IL2 caused elevated serum levels of soluble IL-2 receptor alpha (sIL2R_) and lymphocytosis. There were no measurable complete or partial responses to hu14.18/IL2; however, three patients showed evidence of antitumor activity . In the phase II study, 39 patients with recurrent or refractory neuroblastoma were enrolled (36 evaluable). No responses were seen for patients with disease measurable by standard radiographic criteria (stratum 1) (n = 13). Of 23 patients with disease evaluable only by 123I-metaiodobenzylguanidine (MIBG) scintigraphy and/or bone marrow histology (stratum 2), five patients (21.7%) responded; all had a complete response of 9, 13, 20, 30, and 35+ months duration. Grade 3 and 4 non-hematologic toxicities included capillary leak, hypoxia, pain, rash, allergic reaction, elevated transaminases, and hyperbilirubinemia, which were reversible within a few days of completing a treatment course. These results support further testing of hu14.18/IL2 in children with non-bulky high-risk neuroblastoma .
Herein, we have reviewed a number of important areas of basic and translational research related to emerging novel therapies for the pediatric solid tumor, neuroblastoma. These include: 1) Signaling pathways within the tumor cell itself e.g. oncogenes and tumor suppressor proteins 2) Signaling pathways that regulate the tumor stromal compartment to control angiogenesis and the immune system and 3) Elements of cancer metabolism related to the oncogene addiction hypothesis.
Future studies will tap into these areas of basic science investigation to illuminate new avenues for therapeutics. We hereby advocate the need to genotype and perform molecular profiling by multi “omic” analysis on the tumor and stromal cells within the tumor and metastatic sites. Moreover, we suggest that we should examine the adaptive responses to targeted therapeutic agents in mouse models and patients treated with these agents in search of most potent combinations and mechanisms for resistance. This will be required to affect a cure of this difficult to treat disease.
Shimada H, Stram DO, Chatten J, Joshi VV, Hachitanda Y, Brodeur GM, et al. Identification of subsets of neuroblastomas by combined histopathologic and N-myc analysis. J Natl Cancer Inst. 1995 Oct 4;87(19):1470-6.
Seeger RC, Brodeur GM, Sather H, Dalton A, Siegel SE, Wong KY, et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med. 1985 Oct 31;313(18):1111-6.
Schmidt ML, Lal A, Seeger RC, Maris JM, Shimada H, O'Leary M, et al. Favorable prognosis for patients 12 to 18 months of age with stage 4 nonamplified MYCN neuroblastoma: a Children's Cancer Group Study. J Clin Oncol. 2005 Sep 20;23(27):6474-80.
Castellino RC, Durden DL. Mechanisms of disease: the PI3K-Akt-PTEN signaling node--an intercept point for the control of angiogenesis in brain tumors. Nat Clin Pract Neurol. 2007 Dec;3(12):682-93.
Castellino RC, Muh CR, Durden DL. PI-3 kinase-PTEN signaling node: an intercept point for the control of angiogenesis. Curr Pharm Des. 2009;15(4):380-8.
Chesler L, Schlieve C, Goldenberg DD, Kenney A, Kim G, McMillan A, et al. Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma. Cancer Res. 2006 Aug 15;66(16):8139-46.
Kang J, Rychahou PG, Ishola TA, Mourot JM, Evers BM, Chung DH. N-myc is a novel regulator of PI3K-mediated VEGF expression in neuroblastoma. Oncogene. 2008 Jun 26;27(28):3999-4007.
Chanthery YH, Gustafson WC, Itsara M, Persson A, Hackett CS, Grimmer M, et al. Paracrine signaling through MYCN enhances tumor-vascular interactions in neuroblastoma. Sci Transl Med. 2012 Jan 4;4(115):115ra3.
Peirce SK, Findley HW, Prince C, Dasgupta A, Cooper T, Durden DL. The PI-3 kinase-Akt-MDM2-survivin signaling axis in high-risk neuroblastoma: a target for PI-3 kinase inhibitor intervention. Cancer Chemother Pharmacol. 2011 Aug;68(2):325-35.
Garlich JR, De P, Dey N, Su JD, Peng X, Miller A, et al. A vascular targeted pan phosphoinositide 3-kinase inhibitor prodrug, SF1126, with antitumor and antiangiogenic activity. Cancer Res. 2008 Jan 1;68(1):206-15.
Wen S, Stolarov J, Myers MP, Su JD, Wigler MH, Tonks NK, et al. PTEN controls tumor-induced angiogenesis. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4622-7.
Fang J, Ding M, Yang L, Liu LZ, Jiang BH. PI3K/PTEN/AKT signaling regulates prostate tumor angiogenesis. Cell Signal. 2007 Dec;19(12):2487-97.
Tian T, Nan KJ, Wang SH, Liang X, Lu CX, Guo H, et al. PTEN regulates angiogenesis and VEGF expression through phosphatase-dependent and -independent mechanisms in HepG2 cells. Carcinogenesis. 2010 Jul;31(7):1211-9.
Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996 Aug 9;86(3):353-64.
Meitar D, Crawford SE, Rademaker AW, Cohn SL. Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma. J Clin Oncol. 1996 Feb;14(2):405-14.
Ribatti D, Vacca A, Nico B, De Falco G, Giuseppe Montaldo P, Ponzoni M. Angiogenesis and anti-angiogenesis in neuroblastoma. Eur J Cancer. 2002 Apr;38(6):750-7.
Eggert A, Ikegaki N, Kwiatkowski J, Zhao H, Brodeur GM, Himelstein BP. High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas. Clin Cancer Res. 2000 May;6(5):1900-8.
Chlenski A, Liu S, Crawford SE, Volpert OV, DeVries GH, Evangelista A, et al. SPARC is a key Schwannian-derived inhibitor controlling neuroblastoma tumor angiogenesis. Cancer Res. 2002 Dec 15;62(24):7357-63.
Goldberg MA, Schneider TJ. Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem. 1994 Feb 11;269(6):4355-9.
Rossler J, Taylor M, Geoerger B, Farace F, Lagodny J, Peschka-Suss R, et al. Angiogenesis as a target in neuroblastoma. Eur J Cancer. 2008 Aug;44(12):1645-56.
Drozynska E, Izycka-Swieszewska E, Balcerska A, Bodalski J, Bohosiewicz J, Brozyna A, et al. [Analysis of microvascular density and the expression of vascular-endothelial growth factor (VEGF) and its membrane receptor Flk-1 in neuroblastoma]. Med Wieku Rozwoj. 2006 Jul-Sep;10(3 Pt 1):745-55.
Ghanem MA, van Steenbrugge GJ, Sudaryo MK, Mathoera RB, Nijman JM, van der Kwast TH. Expression and prognostic relevance of vascular endothelial growth factor (VEGF) and its receptor (FLT-1) in nephroblastoma. J Clin Pathol. 2003 Feb;56(2):107-13.
Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, et al. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med. 2003 Jul 31;349(5):427-34.
Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004 Jun 3;350(23):2335-42.
Herbst RS, Johnson DH, Mininberg E, Carbone DP, Henderson T, Kim ES, et al. Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol. 2005 Apr 10;23(11):2544-55.
Fukuzawa M, Sugiura H, Koshinaga T, Ikeda T, Hagiwara N, Sawada T. Expression of vascular endothelial growth factor and its receptor Flk-1 in human neuroblastoma using in situ hybridization. J Pediatr Surg. 2002 Dec;37(12):1747-50.
Rossler J, Breit S, Havers W, Schweigerer L. Vascular endothelial growth factor expression in human neuroblastoma: up-regulation by hypoxia. Int J Cancer. 1999 Mar 31;81(1):113-7.
Shusterman S, Maris JM. Prospects for therapeutic inhibition of neuroblastoma angiogenesis. Cancer Lett. 2005 Oct 18;228(1-2):171-9.
Glade Bender JL, Adamson PC, Reid JM, Xu L, Baruchel S, Shaked Y, et al. Phase I trial and pharmacokinetic study of bevacizumab in pediatric patients with refractory solid tumors: a Children's Oncology Group Study. J Clin Oncol. 2008 Jan 20;26(3):399-405.
Jakovljevic G, Culic S, Stepan J, Bonevski A, Seiwerth S. Vascular endothelial growth factor in children with neuroblastoma: a retrospective analysis. J Exp Clin Cancer Res. 2009;28:143.
Huntly BJ, Gilliland DG. Cancer biology: summing up cancer stem cells. Nature. 2005 Jun 30;435(7046):1169-70.
Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001 Nov 1;414(6859):105-11.
Cho RW, Clarke MF. Recent advances in cancer stem cells. Curr Opin Genet Dev. 2008 Feb;18(1):48-53.
Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997 Jul;3(7):730-7.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3983-8.
O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007 Jan 4;445(7123):106-10.
Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature. 2004 Nov 18;432(7015):396-401.
Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, et al. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):15178-83.
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003 Sep 15;63(18):5821-8.
Cournoyer S, Nyalendo C, Addioui A, Belounis A, Beaunoyer M, Aumont A, et al. Genotype analysis of tumor-initiating cells expressing CD133 in neuroblastoma. Genes Chromosomes Cancer. 2012 Aug;51(8):792-804.
Coulon A, Flahaut M, Muhlethaler-Mottet A, Meier R, Liberman J, Balmas-Bourloud K, et al. Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model. Neoplasia. 2011 Oct;13(10):991-1004.
Mao XG, Zhang X, Xue XY, Guo G, Wang P, Zhang W, et al. Brain Tumor Stem-Like Cells Identified by Neural Stem Cell Marker CD15. Transl Oncol. 2009 Dec;2(4):247-57.
Ward RJ, Lee L, Graham K, Satkunendran T, Yoshikawa K, Ling E, et al. Multipotent CD15+ cancer stem cells in patched-1-deficient mouse medulloblastoma. Cancer Res. 2009 Jun 1;69(11):4682-90.
Read TA, Fogarty MP, Markant SL, McLendon RE, Wei Z, Ellison DW, et al. Identification of CD15 as a marker for tumor-propagating cells in a mouse model of medulloblastoma. Cancer Cell. 2009 Feb 3;15(2):135-47.
Hansford LM, McKee AE, Zhang L, George RE, Gerstle JT, Thorner PS, et al. Neuroblastoma cells isolated from bone marrow metastases contain a naturally enriched tumor-initiating cell. Cancer Res. 2007 Dec 1;67(23):11234-43.
Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002 May 31;296(5573):1655-7.
Anderson DAMaJ. The Immune Response in Paediatric Cancer. The Open Pathology Journal. 2010;4:45-59.
Dahmane N, Sanchez P, Gitton Y, Palma V, Sun T, Beyna M, et al. The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development. 2001 Dec;128(24):5201-12.
Artavanis-Tsakonas S, Matsuno K, Fortini ME. Notch signaling. Science. 1995 Apr 14;268(5208):225-32.
Rehman AO, Wang CY. Notch signaling in the regulation of tumor angiogenesis. Trends Cell Biol. 2006 Jun;16(6):293-300.
van Amerongen R, Nusse R. Towards an integrated view of Wnt signaling in development. Development. 2009 Oct;136(19):3205-14.
Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005 Apr 14;434(7035):843-50.
Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, et al. Medulloblastoma comprises four distinct molecular variants. J Clin Oncol. 2011 Apr 10;29(11):1408-14.
Grinshtein N, Datti A, Fujitani M, Uehling D, Prakesch M, Isaac M, et al. Small molecule kinase inhibitor screen identifies polo-like kinase 1 as a target for neuroblastoma tumor-initiating cells. Cancer Res. 2011 Feb 15;71(4):1385-95.
Sunayama J, Sato A, Matsuda K, Tachibana K, Suzuki K, Narita Y, et al. Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells. Neuro Oncol. 2010 Dec;12(12):1205-19.
Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell. 2008 Jun;13(6):472-82.
Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009 May 22;324(5930):1029-33.
Dang CV. Links between metabolism and cancer. Genes Dev. 2012 May 1;26(9):877-90.
Schwab M, Alitalo K, Klempnauer KH, Varmus HE, Bishop JM, Gilbert F, et al. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature. 1983 Sep 15-21;305(5931):245-8.
Wise DR, DeBerardinis RJ, Mancuso A, Sayed N, Zhang XY, Pfeiffer HK, et al. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18782-7.
Yuneva M, Zamboni N, Oefner P, Sachidanandam R, Lazebnik Y. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells. J Cell Biol. 2007 Jul 2;178(1):93-105.
Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, et al. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature. 2009 Apr 9;458(7239):762-5.
Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell. 2009 Feb 6;136(3):521-34.
Land H, Parada LF, Weinberg RA. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18-24;304(5927):596-602.
Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J, et al. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature. 1997 Feb 6;385(6616):544-8.
Dang CV. MYC on the path to cancer. Cell. 2012 Mar 30;149(1):22-35.
Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC. PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev. 2008 Feb 15;22(4):436-48.
Marx J. Cancer biology. All in the stroma: cancer's Cosa Nostra. Science. 2008 Apr 4;320(5872):38-41.
Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet. 2001 Feb 17;357(9255):539-45.
Lin EY, Nguyen AV, Russell RG, Pollard JW. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med. 2001 Mar 19;193(6):727-40.
Duluc D, Corvaisier M, Blanchard S, Catala L, Descamps P, Gamelin E, et al. Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages. Int J Cancer. 2009 Jul 15;125(2):367-73.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74.
Hellstrom IE, Hellstrom KE, Pierce GE, Bill AH. Demonstration of cell-bound and humoral immunity against neuroblastoma cells. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1231-8.
Bill AH. The implications of immune reactions to neuroblastoma. Surgery. 1969 Aug;66(2):415-8.
Bill AH, Morgan A. Evidence for immune reactions to neuroblastoma and future possibilities for investigation. J Pediatr Surg. 1970 Apr;5(2):111-6.
D'Angio GJ, Evans AE, Koop CE. Special pattern of widespread neuroblastoma with a favourable prognosis. Lancet. 1971 May 22;1(7708):1046-9.
Evans AE, Gerson J, Schnaufer L. Spontaneous regression of neuroblastoma. Natl Cancer Inst Monogr. 1976 Nov;44:49-54.
Katz SC, Pillarisetty V, Bamboat ZM, Shia J, Hedvat C, Gonen M, et al. T cell infiltrate predicts long-term survival following resection of colorectal cancer liver metastases. Ann Surg Oncol. 2009 Sep;16(9):2524-30.
Hornychova H, Melichar B, Tomsova M, Mergancova J, Urminska H, Ryska A. Tumor-infiltrating lymphocytes predict response to neoadjuvant chemotherapy in patients with breast carcinoma. Cancer Invest. 2008 Dec;26(10):1024-31.
Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med. 2003 Jan 16;348(3):203-13.
Martin RF, Beckwith JB. Lymphoid infiltrates in neuroblastomas: their occurrence and prognostic significance. J Pediatr Surg. 1968 Feb;3(1):161-4.
Coughlin CM, Fleming MD, Carroll RG, Pawel BR, Hogarty MD, Shan X, et al. Immunosurveillance and survivin-specific T-cell immunity in children with high-risk neuroblastoma. J Clin Oncol. 2006 Dec 20;24(36):5725-34.
Fest S, Huebener N, Bleeke M, Durmus T, Stermann A, Woehler A, et al. Survivin minigene DNA vaccination is effective against neuroblastoma. Int J Cancer. 2009 Jul 1;125(1):104-14.
Huebener N, Fest S, Strandsby A, Michalsky E, Preissner R, Zeng Y, et al. A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity. Mol Cancer Ther. 2008 Jul;7(7):2241-51.
Huebener N, Fest S, Hilt K, Schramm A, Eggert A, Durmus T, et al. Xenogeneic immunization with human tyrosine hydroxylase DNA vaccines suppresses growth of established neuroblastoma. Mol Cancer Ther. 2009 Aug;8(8):2392-401.
Lampson LA, Fisher CA. Weak HLA and beta 2-microglobulin expression of neuronal cell lines can be modulated by interferon. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6476-80.
Reid GS, Shan X, Coughlin CM, Lassoued W, Pawel BR, Wexler LH, et al. Interferon-gamma-dependent infiltration of human T cells into neuroblastoma tumors in vivo. Clin Cancer Res. 2009 Nov 1;15(21):6602-8.
Raffaghello L, Prigione I, Airoldi I, Camoriano M, Morandi F, Bocca P, et al. Mechanisms of immune evasion of human neuroblastoma. Cancer Lett. 2005 Oct 18;228(1-2):155-61.
Raffaghello L, Prigione I, Bocca P, Morandi F, Camoriano M, Gambini C, et al. Multiple defects of the antigen-processing machinery components in human neuroblastoma: immunotherapeutic implications. Oncogene. 2005 Jul 7;24(29):4634-44.
Morandi F, Levreri I, Bocca P, Galleni B, Raffaghello L, Ferrone S, et al. Human neuroblastoma cells trigger an immunosuppressive program in monocytes by stimulating soluble HLA-G release. Cancer Res. 2007 Jul 1;67(13):6433-41.
Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L. NKT cells: what's in a name? Nat Rev Immunol. 2004 Mar;4(3):231-7.
Terabe M, Berzofsky JA. The role of NKT cells in tumor immunity. Adv Cancer Res. 2008;101:277-348.
Metelitsa LS, Wu HW, Wang H, Yang Y, Warsi Z, Asgharzadeh S, et al. Natural killer T cells infiltrate neuroblastomas expressing the chemokine CCL2. J Exp Med. 2004 May 3;199(9):1213-21.
Song L, Ara T, Wu HW, Woo CW, Reynolds CP, Seeger RC, et al. Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma. J Clin Invest. 2007 Sep;117(9):2702-12.
Truitt RL, Piaskowski V, Kirchner P, McOlash L, Camitta BM, Casper JT. Immunological evaluation of pediatric cancer patients receiving recombinant interleukin-2 in a phase I trial. J Immunother (1991). 1992 May;11(4):274-85.
Marti F, Pardo N, Peiro M, Bertran E, Amill B, Garcia J, et al. Progression of natural immunity during one-year treatment of residual disease in neuroblastoma patients with high doses of interleukin-2 after autologous bone marrow transplantation. Exp Hematol. 1995 Dec;23(14):1445-52.
Bartlett JB, Dredge K, Dalgleish AG. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat Rev Cancer. 2004 Apr;4(4):314-22.
Hayashi T, Hideshima T, Akiyama M, Podar K, Yasui H, Raje N, et al. Molecular mechanisms whereby immunomodulatory drugs activate natural killer cells: clinical application. Br J Haematol. 2005 Jan;128(2):192-203.
Berg SL, Cairo MS, Russell H, Ayello J, Ingle AM, Lau H, et al. Safety, pharmacokinetics, and immunomodulatory effects of lenalidomide in children and adolescents with relapsed/refractory solid tumors or myelodysplastic syndrome: a Children's Oncology Group Phase I Consortium report. J Clin Oncol. 2011 Jan 20;29(3):316-23.
Bartlett JB, Michael A, Clarke IA, Dredge K, Nicholson S, Kristeleit H, et al. Phase I study to determine the safety, tolerability and immunostimulatory activity of thalidomide analogue CC-5013 in patients with metastatic malignant melanoma and other advanced cancers. Br J Cancer. 2004 Mar 8;90(5):955-61.
Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004 Jan;4(1):71-8.
Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzesik DA, et al. Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res. 2006 Dec 1;66(23):11238-46.
Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005 Dec;5(12):953-64.
Mosser DM. The many faces of macrophage activation. J Leukoc Biol. 2003 Feb;73(2):209-12.
Mantovani A, Sica A, Locati M. Macrophage polarization comes of age. Immunity. 2005 Oct;23(4):344-6.
Sica A, Schioppa T, Mantovani A, Allavena P. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer. 2006 Apr;42(6):717-27.
Leek RD, Harris AL. Tumor-associated macrophages in breast cancer. J Mammary Gland Biol Neoplasia. 2002 Apr;7(2):177-89.
Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol. 2002 Mar;196(3):254-65.
Elgert KD, Alleva DG, Mullins DW. Tumor-induced immune dysfunction: the macrophage connection. J Leukoc Biol. 1998 Sep;64(3):275-90.
Gorelik L, Flavell RA. Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells. Nat Med. 2001 Oct;7(10):1118-22.
Chong H, Vodovotz Y, Cox GW, Barcellos-Hoff MH. Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages. J Cell Physiol. 1999 Mar;178(3):275-83.
Menetrier-Caux C, Montmain G, Dieu MC, Bain C, Favrot MC, Caux C, et al. Inhibition of the differentiation of dendritic cells from CD34(+) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. Blood. 1998 Dec 15;92(12):4778-91.
Giraudo E, Inoue M, Hanahan D. An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest. 2004 Sep;114(5):623-33.
Marconi C, Bianchini F, Mannini A, Mugnai G, Ruggieri S, Calorini L. Tumoral and macrophage uPAR and MMP-9 contribute to the invasiveness of B16 murine melanoma cells. Clin Exp Metastasis. 2008;25(3):225-31.
Schoppmann SF, Fenzl A, Nagy K, Unger S, Bayer G, Geleff S, et al. VEGF-C expressing tumor-associated macrophages in lymph node positive breast cancer: impact on lymphangiogenesis and survival. Surgery. 2006 Jun;139(6):839-46.
Nakao S, Kuwano T, Tsutsumi-Miyahara C, Ueda S, Kimura YN, Hamano S, et al. Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1 beta-induced neovascularization and tumor growth. J Clin Invest. 2005 Nov;115(11):2979-91.
DeNardo DG, Brennan DJ, Rexhepaj E, Ruffell B, Shiao SL, Madden SF, et al. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov. 2011 Jun;1(1):54-67.
Kurahara H, Shinchi H, Mataki Y, Maemura K, Noma H, Kubo F, et al. Significance of M2-polarized tumor-associated macrophage in pancreatic cancer. J Surg Res. 2011 May 15;167(2):e211-9.
Nakayama Y, Nagashima N, Minagawa N, Inoue Y, Katsuki T, Onitsuka K, et al. Relationships between tumor-associated macrophages and clinicopathological factors in patients with colorectal cancer. Anticancer Res. 2002 Nov-Dec;22(6C):4291-6.
Asgharzadeh S, Salo JA, Ji L, Oberthuer A, Fischer M, Berthold F, et al. Clinical significance of tumor-associated inflammatory cells in metastatic neuroblastoma. J Clin Oncol. 2012 Oct 1;30(28):3525-32.
Song L, Asgharzadeh S, Salo J, Engell K, Wu HW, Sposto R, et al. Valpha24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages. J Clin Invest. 2009 Jun;119(6):1524-36.
Cavill R, Kamburov A, Ellis JK, Athersuch TJ, Blagrove MS, Herwig R, et al. Consensus-phenotype integration of transcriptomic and metabolomic data implies a role for metabolism in the chemosensitivity of tumour cells. PLoS Comput Biol. 2011 Mar;7(3):e1001113.
Wei JS, Greer BT, Westermann F, Steinberg SM, Son CG, Chen QR, et al. Prediction of clinical outcome using gene expression profiling and artificial neural networks for patients with neuroblastoma. Cancer Res. 2004 Oct 1;64(19):6883-91.
Ohira M, Oba S, Nakamura Y, Isogai E, Kaneko S, Nakagawa A, et al. Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas. Cancer Cell. 2005 Apr;7(4):337-50.
Guo X, Chen QR, Song YK, Wei JS, Khan J. Exon array analysis reveals neuroblastoma tumors have distinct alternative splicing patterns according to stage and MYCN amplification status. BMC Med Genomics. 2011;4:35.
Mosse YP, Diskin SJ, Wasserman N, Rinaldi K, Attiyeh EF, Cole K, et al. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer. 2007 Oct;46(10):936-49.
Chen QR, Song YK, Yu LR, Wei JS, Chung JY, Hewitt SM, et al. Global genomic and proteomic analysis identifies biological pathways related to high-risk neuroblastoma. J Proteome Res. 2010 Jan;9(1):373-82.
Dettmer K, Hammock BD. Metabolomics--a new exciting field within the "omics" sciences. Environ Health Perspect. 2004 May;112(7):A396-7.
Griffin JL, Shockcor JP. Metabolic profiles of cancer cells. Nat Rev Cancer. 2004 Jul;4(7):551-61.
Florian CL, Preece NE, Bhakoo KK, Williams SR, Noble M. Characteristic metabolic profiles revealed by 1H NMR spectroscopy for three types of human brain and nervous system tumours. NMR Biomed. 1995 Sep;8(6):253-64.
Peet AC, McConville C, Wilson M, Levine BA, Reed M, Dyer SA, et al. 1H MRS identifies specific metabolite profiles associated with MYCN-amplified and non-amplified tumour subtypes of neuroblastoma cell lines. NMR Biomed. 2007 Nov;20(7):692-700.
Imperiale A, Elbayed K, Moussallieh FM, Neuville A, Piotto M, Bellocq JP, et al. Metabolomic pattern of childhood neuroblastoma obtained by (1)H-high-resolution magic angle spinning (HRMAS) NMR spectroscopy. Pediatr Blood Cancer. 2011 Jan;56(1):24-34.
Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol. 2001;19:275-90.
Amigorena S, Bonnerot C, Drake JR, Choquet D, Hunziker W, Guillet JG, et al. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science. 1992 Jun 26;256(5065):1808-12.
Van den Herik-Oudijk IE, Capel PJ, van der Bruggen T, Van de Winkel JG. Identification of signaling motifs within human Fc gamma RIIa and Fc gamma RIIb isoforms. Blood. 1995 Apr 15;85(8):2202-11.
Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008 Jan;8(1):34-47.
White CA, Weaver RL, Grillo-Lopez AJ. Antibody-targeted immunotherapy for treatment of malignancy. Annu Rev Med. 2001;52:125-45.
Safa MM, Foon KA. Adjuvant immunotherapy for melanoma and colorectal cancers. Semin Oncol. 2001 Feb;28(1):68-92.
Clynes R, Takechi Y, Moroi Y, Houghton A, Ravetch JV. Fc receptors are required in passive and active immunity to melanoma. Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):652-6.
van de Winkel JG, Bast B, de Gast GC. Immunotherapeutic potential of bispecific antibodies. Immunol Today. 1997 Dec;18(12):562-4.
Birkle S, Zeng G, Gao L, Yu RK, Aubry J. Role of tumor-associated gangliosides in cancer progression. Biochimie. 2003 Mar-Apr;85(3-4):455-63.
Modak S, Cheung NK. Disialoganglioside directed immunotherapy of neuroblastoma. Cancer Invest. 2007 Feb;25(1):67-77.
Gilman AL, Ozkaynak MF, Matthay KK, Krailo M, Yu AL, Gan J, et al. Phase I study of ch14.18 with granulocyte-macrophage colony-stimulating factor and interleukin-2 in children with neuroblastoma after autologous bone marrow transplantation or stem-cell rescue: a report from the Children's Oncology Group. J Clin Oncol. 2009 Jan 1;27(1):85-91.
Gillies SD, Lo KM, Wesolowski J. High-level expression of chimeric antibodies using adapted cDNA variable region cassettes. J Immunol Methods. 1989 Dec 20;125(1-2):191-202.
Mueller BM, Romerdahl CA, Gillies SD, Reisfeld RA. Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody. J Immunol. 1990 Feb 15;144(4):1382-6.
Yu AL, Uttenreuther-Fischer MM, Huang CS, Tsui CC, Gillies SD, Reisfeld RA, et al. Phase I trial of a human-mouse chimeric anti-disialoganglioside monoclonal antibody ch14.18 in patients with refractory neuroblastoma and osteosarcoma. J Clin Oncol. 1998 Jun;16(6):2169-80.
Handgretinger R, Anderson K, Lang P, Dopfer R, Klingebiel T, Schrappe M, et al. A phase I study of human/mouse chimeric antiganglioside GD2 antibody ch14.18 in patients with neuroblastoma. Eur J Cancer. 1995;31A(2):261-7.
Barker E, Mueller BM, Handgretinger R, Herter M, Yu AL, Reisfeld RA. Effect of a chimeric anti-ganglioside GD2 antibody on cell-mediated lysis of human neuroblastoma cells. Cancer Res. 1991 Jan 1;51(1):144-9.
Albertini MR, Hank JA, Schiller JH, Khorsand M, Borchert AA, Gan J, et al. Phase IB trial of chimeric antidisialoganglioside antibody plus interleukin 2 for melanoma patients. Clin Cancer Res. 1997 Aug;3(8):1277-88.
Kendra K, Malkovska V, Allen M, Guzman J, Albertini M. In vivo binding and antitumor activity of Ch14.18. J Immunother. 1999 Sep;22(5):423-30.
Ozkaynak MF, Sondel PM, Krailo MD, Gan J, Javorsky B, Reisfeld RA, et al. Phase I study of chimeric human/murine anti-ganglioside G(D2) monoclonal antibody (ch14.18) with granulocyte-macrophage colony-stimulating factor in children with neuroblastoma immediately after hematopoietic stem-cell transplantation: a Children's Cancer Group Study. J Clin Oncol. 2000 Dec 15;18(24):4077-85.
Yu AL, Gilman AL, Ozkaynak MF, London WB, Kreissman SG, Chen HX, et al. Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med. 2010 Sep 30;363(14):1324-34.
Sabzevari H, Gillies SD, Mueller BM, Pancook JD, Reisfeld RA. A recombinant antibody-interleukin 2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9626-30.
Becker JC, Varki N, Gillies SD, Furukawa K, Reisfeld RA. Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy. J Clin Invest. 1996 Dec 15;98(12):2801-4.
Osenga KL, Hank JA, Albertini MR, Gan J, Sternberg AG, Eickhoff J, et al. A phase I clinical trial of the hu14.18-IL2 (EMD 273063) as a treatment for children with refractory or recurrent neuroblastoma and melanoma: a study of the Children's Oncology Group. Clin Cancer Res. 2006 Mar 15;12(6):1750-9.
Shusterman S, London WB, Gillies SD, Hank JA, Voss SD, Seeger RC, et al. Antitumor activity of hu14.18-IL2 in patients with relapsed/refractory neuroblastoma: a Children's Oncology Group (COG) phase II study. J Clin Oncol. 2010 Nov 20;28(33):4969-75.