Journal of Neuro-Oncology 41: 159–166, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Clinical Study
Neuroblastoma as a neurobiological disease Nina Felice Schor Departments of Pediatrics, Neurology, and Pharmacology, University of Pittsburgh, Pittsburgh, PA 15213, USA
Key words: neuroblastoma, chemotherapy, apoptosis, dopamine receptors Summary While neuroscientists are often involved in the assessment and care of patients with central nervous system tumors, they are only rarely involved in the case of peripheral nervous system neoplasia. Neuroblastoma is a childhood tumor of the primitive sympathetic nervous system. It is at once one of the most common and one of the most deadly tumors of childhood. The prognosis for children with this tumor has not changed in the past two decades. Clearly, a fresh approach to neuroblastoma is needed. The neuroscientist has much to add to our understanding and treatment of neuroblastoma and its sequelae. Conversely, neuroblastoma has much to teach us regarding the normal development of the neural crest and the aberrant loss of neurons in this lineage. A neuroscientist’s approach to neuroblastoma, its biology and clinical features, is presented herein.
Introduction The term ‘neurooncology’ has generally conjured up the spectre of brain and spinal cord tumors. As such, tumors involving the peripheral nervous system, especially those not generally associated with a systemic genetic disorder of neurologic import, have been the exclusive province of the cancer biologist and oncologist, and not the neuroscientist. This artificial distinction is perhaps unfortunate; for there is much to be learned about normal and aberrant nervous system development from the study of these tumors, and the neuroscientist has a unique perspective to bring to the understanding and therapy of peripheral nervous system neoplasia. Neuroblastoma, the single most common tumor of childhood [1], is the best studied example of this principle. Despite major advances in the therapy of many other childhood oncological disorders, children with metastatic neuroblastoma (60% of patients at presentation) have only 5–15% chance of long-term survival [1]. Even ablative chemotherapy followed by bone marrow transplantation only increases longevity (and therefore, the short-term survival rate) without altering the ultimate survival rate [2]. This dismal
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long-term prognosis has not changed in the past two decades. Clearly, neuroblastoma is a tumor for which new approaches are needed. Neuroblastomas arise from the sympathetic ganglia. As such, they are peripheral neural crest derivatives [3]. For decades, cultured neuroblastoma cells have been used as models of the neural crest in which to study cell lineage, neurotrophin function and signal transduction, and other related basic phenomena [4]. Furthermore, in vivo studies involving the transposition and transplantation of neural crest tissue between and within animals have elucidated many of the critical interactions between the neural crest and adjacent mesenchyme [5,6]. Neuroscientists are uniquely positioned to apply this neurobiological knowledge base to the clinical problems presented by neuroblastoma.
Etiology and pathogenesis: a neurodevelopmental process Many tumors are thought to arise because of a somatic mutation in a cell already predisposed to neoplastic transformation by virtue of an initial ‘silent’ (i.e., recessive) mutation. The ‘second hit’, as it is often called,
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160 gives rise to the phenotypic and functional change in the cell that is associated with altered proliferation and malignant behavior [7]. In most tumors, this second event is hypothesized to occur after maturation of the cell has already taken place. That is, a normal cell is somehow transformed into a neoplastic one. In the case of neuroblastoma, many have proposed that tumors result, not from the transformation of a normal cell into a neoplastic one, but rather, from the persistence of an embryonic cell that would otherwise have either differentiated or, more likely, undergone developmentally programmed death or apoptosis. This suggestion has been further developed to hypothesize that in at least some instances of neuroblastoma, the normal chain of developmental events takes place, but is either delayed or arrested entirely at a point in its course [8]. The lines of evidence supporting this notion are severalfold. They include the finding that neuroblastic rests are more common in autopsy series of infants and young children than would be predicted from the incidence of neuroblastoma [9]. Furthermore, patients under the age of 2 years with small primary tumors associated with metastases only to the liver, skin, and bone marrow often demonstrate spontaneous regression of their complete tumor burden [10]. These observations have led some to speculate that neuroblastomas arise when neuroblastic rests evade developmental apoptosis [8]. Several genes have been discovered to play a role in regulating the occurrence of apoptosis, and alterations in the expression or sequence of these genes give rise to changes in the likelihood that a given cell will undergo apoptosis [11]. In the case of neuroblastoma, genes in the bcl-2 family and their respective proteins appear to play a major role both in the pathogenesis of the disorder and in the resistance of these tumors to chemoand radiation therapy [12,13]. Bcl-2 is one of a group of proteins that have been termed ‘anti-apoptosis’ proteins. Recently, Bcl-2 has been shown to prevent the release of cytochrome c from the mitochondrion, apparently a key step in the initiation of apoptosis in intact cells [14,15]. There is experimental evidence to support the notion that the expression of bcl-2 and related genes is developmentally regulated in normal neural crest cells, and that, ordinarily, cells become destined for apoptosis by down-regulation of this gene [16]. This basic neurobiological finding has led to the hypothesis that what goes awry in neuroblastoma is that these cells continue anomalously to express the anti-apoptosis genes. Recently, consistent with this hypothesis, it has
been found that artificially-induced overexpression of bcl-2 results in resistance of neuroblastoma cells to apoptosis induction [12], and that many neuroblastomas obtained from patients overproduce the Bcl-2like protein, Bcl-xL [13]. It remains to be seen whether the disappearance of congenital and stage IVS neuroblastomas results from a late turning-on of some pro-apoptosis or turning-off of some anti-apoptosis pathway [8]. In either case, the study of the clinical consequences of developmental regulation and aberrant expression of bcl-2 and related genes in neuroblastoma may yield important generalizable insights into the roles of such processes and their regulation in neurodegenerative and neurometabolic disorders. Unlike the case for glioblastomas [17,18], mutation of the pro-apoptosis gene, p53, and consequent upregulation of the waf1/cip1 gene, appear not to play a role in the genesis of neuroblastomas. This may be of relevance for the theory that neuroblastomas result from a developmental aberration or arrest (i.e., from abnormal regulation of gene expression during development), rather than a de novo mutation in a cell normally present in the adult. Cytogenetic aberrations of neural crest development have long been thought to play a role in the etiology of neuroblastoma. Partial monosomy for the short arm of chromosome 1 [19] and the long arms of chromosomes 11 and 14 [20] has been demonstrated alone and in various combinations for many neuroblastomas. This results in loss of heterozygosity for these alleles. As loss of heterozygosity for alleles on chromosome 1 or 11 has not been observed in Stage I or II tumors, the alternative possibilities have been raised of these mutations representing only one of several etiologic factors or of their being of prognostic rather than etiologic significance. Similar arguments could be mounted for the significance of duplication of the N-myc gene [21] in neuroblastomas.
Associated nervous system phenomena Among the most well-known and the least understood aspects of neuroblastoma is the paraneoplastic occurrence of opsoclonus–myoclonus. This neurological syndrome, characterized by conjugate ocular myoclonus and limb myoclonus [22], is the first manifestation of an occult neuroblastoma in approximately 5% of neuroblastoma patients [23]. The presence of opsoclonus–myoclonus in a child therefore mandates
161 a search for tumor. The tumor is found in more than a third of childhood opsoclonus–myoclonus patients [24], and it is postulated that some large fraction of the remaining children have or had a neuroblastoma that is either too small for detection at the present time, or has already disappeared spontaneously. Persistence of the opsoclonus and myoclonus for some time after removal of a neuroblastoma is the rule rather than the exception, so it is not surprising that some patients with involuted neuroblastomas might be found to have opsoclonus– myoclonus in the absence of detectable tumor [25]. Most of our understanding of opsoclonus– myoclonus as a paraneoplastic syndrome comes from studies in adult neurological patients, many of whom have turned out to have small cell lung, breast, or ovarian cancer [26,27]. It is clear in many of these patients that cross-reactive antibodies to the tumor attack cerebellar Purkinje cells and give rise to the neurological syndrome [28]. This is consistent with the observation that patients who develop opsoclonus– myoclonus on the basis of a defined anatomic lesion frequently have abnormalities either in the cerebellum or in cerebellar outflow tracts in the pons [29–32]. Despite many years of intensive study, no conclusive evidence has heen found for the role of similar antibodies in patients with neuroblastoma [32]. Similar anatomic localization of the disorder is, however, suggested by the incidence of paraneoplastic cerebellar ataxia with or without myoclonus in some children with neuroblastoma [33]. Further study of the substances responsible for this phenomenon in children with neuroblastoma must come from those with an understanding of neuroanatomy, neuropharmacology, and clinical neuroscience [34]. Such study may also lead to a better understanding of normal cerebellar control of motor programming [35]. From a clinical standpoint, the outlook for survival in children with neuroblastoma and opsoclonus– myoclonus is excellent. This syndrome tends to occur in children with low-stage tumor. However, the neurological outlook for these patients is far less uniformly favorable. Recovery from opsoclonus–myoclonus is slow, stuttering, and often incomplete even long after complete cure from its neoplastic precipitant. Exacerbations are common, and long-term neurobehavioral consequences, most often referrable to problems with subcortical motor control, attention, and social judgement [35]. Both ACTH and steroids of various types have symptomatic efficacy in opsoclonus–myoclonus in patients with and without neuroblastoma, perhaps
underscoring a neuroimmunologic mechanism for the etiology of this syndrome [34].
Prognosis Many neurodevelopmental factors have been found to influence prognosis for patients with neuroblastoma. The most robust of these is the age of the patient at the time of diagnosis, and in many cases, this factor appears to correlate with the expression and incidence of the various other putative arbiters of prognosis [21,36]. Children under the age of 2 years at first diagnosis, at all stages of disease, have a much better outlook than older children. It is not clear whether this relates to differential interaction of the host with the tumor or intrinsic differences in the tumors that develop in these two populations. Within the group of children that are under two years of age, it is clear that tumor cell ploidy influences the likelihood of long-term survival [36]. Diploidy almost always predicts early treatment failure with conventional methods, while hyperdiploidy bodes well for long-term disease-free survival. At any stage of disease, overexpression by the untreated tumor of the oncogene N-myc predicts poor prognosis [37]. The protein N-Myc is a transcription factor thought to be crucial during embryogenesis [38,39]. Proteins in the Myc family function, at least in part, as heterodimers with the protein Max, and, under various cellular conditions, can promote transformation, proliferation, or apoptosis [40]. There is some evidence to suggest that overexpression of N-myc results in the induction and secretion of autocrine growth factors [41]. It is tempting to hypothesize that this contributes to the autonomous growth of neuroblastomas. Other studies have defined a relationship between tumor cell expression of the tyrosine kinase-type neurotrophin receptors and prognosis [42–46]. While expression of the receptor genes trkB, trkC, the truncated form of trkB confer favorable outcome, expression of the full length transcript of trkB bodes poorly.
Treatment: a role for the neuroscientist The dismal prognosis for children whose neuroblastomas are not amenable to surgery alone places this tumor among the most therapeutically problematic neoplasms known. The fact that conventional
162 approaches that view neuroblastoma simply as a neoplasm are often ineffective suggests that something about the uniquely neuronal characteristics of these cells gives rise to treatment failure. Novel approaches might be aimed at exploiting these neuronal characteristics, and using what is known of the neurobiology of these tumor cells. Among the first attempts to do this were strategies that involved the use of the cytotoxic neurotransmitter analogue, 6-hydroxydopamine (OHDA). This compound gains selective entry to cells like neuroblastoma cells that have a catecholamine uptake system [47]. Once inside of these cells, it is a potent generator of reactive oxygen species like superoxide and hydroxyl radical. These oxygen radicals are highly toxic, and recently have been demonstrated to induce apoptosis in neural crest cells. Two fundamental observations have led to two distinct experimental approaches to the therapy of neuroblastoma. First, neuroblastoma cells are exquisitely sensitive to the toxicity of OHDA [48]. This implies that OHDA and other toxic neurotransmitter analogues might be used in targeted chemotherapy for neuroblastoma. Second, in a murine model, prior ablation of the normal sympathetic nervous system using OHDA results in slower growth of subsequently implanted neuroblastomas [49]. This suggests that there is some trophic effect of the normal sympathetic nervous system on neuroblastoma cells. The therapeutic strategies that have grown out of each of these basic neurobiological observations will be described in turn. Initial experimental attempts to use OHDA itself as a chemotherapeutic agent for neuroblastoma were predictably thwarted by the toxicity of OHDA for the normal sympathetic nervous system [50,51]. OHDA is sufficiently polar that it does not enter the central nervous system and is, therefore, not toxic to the brain [52]. Nonetheless, some means had to be devised to selectively protect normal sympathetic neurons from the toxicity of OHDA while leaving the tumor cells susceptible to attack. Several scavengers and detoxifiers of reactive oxygen species have been identified and shown to have selective activity in normal, and not neoplastic, cells [53,54]. Recent studies using the stable nitroxyl radical, Tempol, as a selective protectant of normal cells during OHDA treatment of neuroblastoma look extremely promising in a murine model [55], and are currently in the process of further development in human neuroblastoma xenograft models. The differential impact of this strategy on neoplastic and normal
neural cells and normal non-neural cells is illustrated in Figure 1. Another catecholamine analogue, metaiodobenzylguanidine (MIBG) has been used clinically in both radiologic detection and targeted radiotherapy of neuroblastoma. This compound, used as the 123 I or 131 I radioconjugate, demonstrates reliable detection of both primary disease and bone metastases of neuroblastoma [56], but yields both false positives and negatives in the case of hepatic disease [57]. In children with known metastatic neuroblastoma, MIBG scintigraphy may be
Figure 1. Differential protection of normal and neoplastic neural crest cells from the reactive oxygen species (ROS) generated intracellularly by 6-hydroxydopamine (OHDA). OHDA is only taken up by cells with a catecholamine uptake system, and is therefore excluded from non-neural cells. Its polarity precludes entry into the central nervous system. The ROS scavenger and superoxide dismutase mimic, Tempol, is preferentially active in normal cells relative to neoplastic cells, and therefore protects the normal sympathetic nervous system from ROS, leaving neuroblastoma cells susceptible to attack.
163 useful in following and predicting at mid-treatment the ultimate response to chemotherapy [58]. Furthermore, recent results suggest that 125 I-MIBG may be efficacious as targeted radiotherapy of neuroblastoma [59]. The suggestion that the normal sympathetic nervous system may be trophic for the neuroblastoma, coupled with the finding that substances such as nerve growth factor (NGF) can protect neuroblastomas from chemotherapeutic agent-induced apoptosis [60], has led to the hypothesis that neuroblastomas are in some measure chemoresistant because they are being ‘protected’ from drug-induced death by substances with which they are bathed both because of their proximity to normal, neurotrophin-secreting cells and, perhaps, their own secretion of such substances into their environment [61]. This, in turn, has led to a search for which of the many neurotrophin receptors known to be expressed on the surface of neuroblastoma cells is responsible for these protective effects. These studies are being pursued in hopes of ultimately designing specific antagonists of this tumor-protective activity. Several recent developments have put this within the realm of possibility. Two distinct NGF receptors have been identified [62], and their binding sites on the NGF molecule are not only unique, but at opposite ends of the molecule, making it possible to produce agonists and antagonists that bind only to one or the other of these receptors [63–65]. Further, it appears that, for some neuroblastomas, only one of these receptors, the one that is not independently involved in the differentiation-inducing effects of NGF, is necessary and sufficient for the protective effects of NGF against chemotherapeutic-induced death [66]. This may mean that, at least for a subset of neuroblastomas, antagonists of NGF that bind only to this receptor may overcome chemotherapeutic resistance (Figure 2A) without interfering with the effects of NGF at the other, differentiation-inducing receptor. The discovery that neural crest cells developmentally express antiapoptosis proteins of the bcl-2 family, and that the expression of such proteins appears to correlate with chemoresistance of neuroblastomas, has led to efforts to overcome bcl-2-related resistance. One such effort depends upon the finding that neurons that overexpress bcl-2 have unusually high levels of reducing species such as reduced glutathione (GSH) [67]. This strategy exploits these high GSH levels by using as chemotherapy a drug precursor that becomes effective only when activated by GSH or other sulfhydryl compounds. Neocarzinostatin and other antimitotic drugs
Figure 2. Overcoming chemotherapeutic resistance in neuroblastoma. (A) Endogenous nerve growth factor (NGF) is thought to prevent chemotherapeutic agent-induced apoptosis in neuroblastoma cells via its binding to the low-affinity p75 NGF receptor. Antagonists of NGF binding at this site may prevent NGF-mediated protection of neuroblastoma cells and facilitate chemotherapeutic efficacy. (B) Neuroblastomas overexpress antiapoptosis proteins in the Bcl-2 family. Bcl-2 has been shown to increase the reduced glutathione (GSH) content of neural cells [67]. By using the GSH-activated antimitotic agent precursor, neocarzinostatin (NCS), as chemotherapy, neuroblastoma cells may be targeted for attack. Most normal cells do not contain such high levels of anti-apoptosis proteins.
of the enediyne class exhibit this characteristic [68]. Unlike all other chemotherapeutic agents so studied, neocarzinostatin actually works better in neural crest tumor cells that overexpress bcl-2 than in cells with native levels of bcl-2 expression [69]. This differential efficacy may well make these agents both effective and relatively non-toxic, since many normal tissues do not produce levels of Bcl-2 as high as those found in the tumor (Figure 2B). One other proposed therapeutic approach that has shown some promise in tissue culture studies combines exploitation of the catecholamine uptake system on neuroblastoma cells with the need for sulfhydryl activation of antimitotic agents such as neocarzinostatin. This approach involves the adjunctive use of 6-mercaptodopamine, a sulfhydryl adduct of a catecholamine, with neocarzinostatin [70]. As is diagrammed in Figure 3, preferential uptake of
164 A neuroscientist’s perspective It has become increasingly clear that neuroblastomas have much to teach us about the development and function of the neural crest. These cells have already proven themselves to be useful model systems for the study of growth factors, signal transduction pathways, apoptosis, and differentiation in neural lineage cells. Only recently has it been realized that perhaps the neuroscientist has a novel and useful perspective from which to improve the therapy of neuroblastoma. Given the high incidence and poor prognosis of this tumor, aspects that have remained essentially unchanged despite maximal application conventional antineoplastic approaches, this new perspective may prove critical to these patients. Acknowledgement Thanks are due to Dr. Robert H. Schor for assistance with computer generation of the figures in this review. Many of the studies described in this review were funded by grants from the National Institutes of Health (CA74289) and the American Cancer Society (DHP-128). References Figure 3. Selective activation of the antimitotic agent, neocarzinostatin (NCS), in neural crest cells. In order for NCS to be maximally effective, it needs to be activated by intracellular sulfhydryl compounds and its target cell needs to be dividing. Selective loading of neural crest (i.e., catecholamine-concentrating) cells with sulfhydryl groups can be effected by incubation with the sulfhydryl adduct of dopamine, 6-mercaptodopamine (SHDA). Theoretically, the only cells that should be both dividing and capable of concentrating SHDA are the neuroblastoma cells. This therapeutic approach is currently in early preclinical development.
6-mercaptodopamine by dopamine uptake systemcontaining cells outside of the blood–brain barrier would theoretically result in preferential activation of neocarzinostatin in cells like those of the neuroblastoma, and less toxicity to other rapidly dividing tissues. The fact that most sympathetic neurons are not dividing should mean that activated neocarzinostatin will be less toxic to these cells than to the rapidly dividing neuroblastoma cells. Testing of this hypothesis awaits in vivo studies of this strategy.
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Address for correspondence and offprints: Dr. Nina Felice Schor, Division of Child Neurology, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. Tel.: 412-692-6471; Fax: 412-692-7824; E-mail:
[email protected].
