Neuroblastoma is the most common solid extracranial tumor in children. Although the outcome of patients with localized disease has improved substantially, the prognosis for those with advanced disease is still poor, despite multimodality therapeutic efforts of increasing intensity over the last 20 years. Dr. Matthay provides an excellent overview review of the biology and treatment of this devastating but fascinating childhood malignancy.
Many controversies about neuroblastoma abound. These center not only on the clinical management of this disease but also on the molecular and cellular biological aspects as they relate to disease pathophysiology, prognosis, and therapy. Since no one review can touch on all these issues in depth, further discussion of some of these issues, particularly the biological ones, will be continued here.
Prognostic Significance of MYCN Amplification in Low-Stage Disease
MYCN gene amplification, one of the first oncogene abnormalities identified in this malignancy, continues to be a source of controversy both clinically and biologically. Dr. Matthay states that amplification of MYCN has now been shown to correlate very closely with advanced-stage disease and, within each stage, to predict outcome. Data from the Childrens Cancer Group (CCG), Pediatric Oncology Group (POG), and other European cooperative groups all concur that MYCN is an important independent prognostic factor in advanced-stage disease (stage 3/4, International Neuroblastoma Staging System [INSS]/CCG staging; or stage C/D, POG staging).[1-3] However, the prognostic value of MYCN DNA amplification in localized disease and stage 4S (DS) is still unclear. In particular, it is not absolutely clear that every child with either localized disease or 4S disease containing MYCN amplification requires intensive multimodality therapy (surgery, chemotherapy, radiation, and/or bone marrow transplantation [BMT]) in order to have a chance of surviving.
In a recent POG review, six patients with localized neuroblastoma harboring MYCN gene amplification were described in detail. Four patients have remained disease-free. Two of the six samples had tissue available for MYCN protein expression, and neither expressed measurable MYCN protein, suggesting that the MYCN amplification did not result in functional protein. Data from an Italian study of six stage 4s tumors with MYCN amplification suggest that MYCN is not prognostic in stage 4S tumors.
As a result of these studies, the next localized disease treatment protocol (a CCG-POG cooperative effort) acknowledges the possibility that MYCN amplification may not be an independent prognostic factor in patients with stage 1 or 2 disease. These patients will be treated on the intermediate-risk protocol, which takes Shimada classification and diploidy into account as prognostic factors instead of MYCN amplification.
Need for Novel Biotherapies in Advanced-Stage Disease
Unlike adult cancer patients, more than 50% of pediatric oncology patients survive their malignancies. The glaring exceptions to this rule are patients with advanced-stage neuroblastoma, who, despite intensive combinations of surgery, myeloablative chemotherapy with BMT and hematopoietic growth factor support, and radiation, still cannot expect better than a 30% to 40% 5-year survival rate. Many experts now feel that more than just another new chemotherapeutic agent or combination of agents is needed to overcome this resistance point of 30% to 40%. Numerous recent advances in neuroblastoma molecular and cellular biology may provide novel avenues of treatment, and are in various stages of clinical development. Some of these approaches will be discussed below.
Agents Reversing mdr and mrp ExpressionThe first gene associated with multidrug resistance, mdr, has not proven to be a good molecular target for therapy in neuroblastoma, since its expression is found in all stages of disease and therefore is not predictive of outcome.[5-7] Nonetheless, the high prevalence of expression of this gene in neuroblastoma suggests a molecular basis for the relative chemosensitivity of this disease.
Clinical trials using mdr-reversing agents are in various phases in neuroblastoma patients. However, the expression of another gene, MRP (multidrug resistance associated-protein), has been shown to be strongly correlated to MYCN amplification, but is independent of MYCN expression in predicting reductions in event-free survival. Treatment options that target the expression of this gene, either directly through gene therapy using antisense constructs or indirectly through pharmacologic agents that interfere with the signaling pathways that regulate MRP expression, may override the multidrug resistance that characterizes the advanced-stage patients and thereby improve outcome.
Growth and Survival FactorsGrowth factors have been shown to regulate growth in various cell systems, including those of neural crest origin (such as neuroblasts). More importantly, withdrawal of these factors can induce apoptosis (programmed cell death) even if the cell is malignant. Many studies have focused on the role of three neurotrophins (NGF [nerve growth factor], brain-derived growth factor (BDNF], and NT-3 [neurotrophin-3]) and their receptors (TrkA, TrkB, and TrkC, respectively) in the control of growth and survival in neuroblastoma.
Dr. Matthay briefly discusses the positive correlation between TrkA expression, favorable outcome, and single-copy MYCN. Other work looking at TrkB expression and its ligand BDNF has revealed various interesting findings. Coexpression of BDNF and TrkB affects survival, differentiation, and invasiveness of human neuroblastoma cells in vivo. TrkB receptors may also enhance chemoresistance through a non-mdr mechanism. Coexpression of TrkC receptors and NT-3 also suggests a role for TrkC and its preferred ligand NT-3 in neuroblastoma differentiation and/or regression.
From a therapeutic standpoint, targeted gene therapy incorporating an agent to activate or upregulate TrkA and/or TrkC expression with a neuroblastoma-specific target (eg, GD2) may help decrease cell growth Conversely, direct downregulation of TrkB-receptor activity may help induce apoptosis and decrease cell survival. The hematopoietic receptor c-kit and its ligand stem-cell factor have also been shown to be coexpressed in neuroblastoma and to serve as a survival factor, which, if removed, induces apoptosis. Lastly, one might imagine the use of multiple trophin disrupters to induce apoptosis, either in concert or sequentially with standard therapeutic modalities.
Direct Targeting of Antiapoptosis GenesThe gene bcl-2 has been shown to be a powerful protector of survival in neuronal cells. Downregulation of bcl-2 is associated with retinoic acid-induced differentiation and growth arrest and/or death, and overexpression of bcl-2 can prevent NGF-withdrawal-induced apoptosis. Thus, another potential target of therapy, either genetic or pharmacologic, would be downregulation of bcl-2 expression. In addition, both bcl-2 and the related gene bcl-x can inhibit chemotherapy-induced apoptosis[16,17], and, thus, targeting bcl-2/bcl-x would be another way of overcoming multidrug resistance.
In experiments in which neuroblastoma cells were infected with a genetically engineered vector designed to disrupt bcl-2 and bcl-x expression, a rapid loss of cell viability, DNA fragmentation, and morphologic features of apoptosis were seen even in neuroblastoma cells transfected to overexpress bcl-2 and bcl-xL. Another approach to regulation of bcl-2 expression may be through growth or survival factor withdrawal. Downregulation of c-kit-receptor expression using antisense oligonucleotides induced apoptosis and simultaneously downregulated bcl-2 expression [Cohen PS, Thiele CJ, and Chan JS, unpublished data].
The great strides made in our understanding of neuroblastoma biology during the last decade suggest that we have much to look forward to in the near future. To achieve an improved outlook for children with advanced-stage disease, the tools of biotechnology and gene therapy should be seriously considered. In addition to the novel therapeutic approaches discussed above, other strategies involving antiangiogenesis agents, tumor vaccines, and immunomodulation, among others, are being investigated. However, to realize the promise of this work, funding priorities for this orphan disease, the incidence of which is only 500 cases per year, must change.