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There is considerable interest in the role of the TRK family of neurotrophin receptors in regulating the survival, growth and differentiation of normal and neoplastic nerve cells. Indeed, there is increasing evidence that TRK genes play an important role in the biology and clinical behavior of neuroblastomas, tumors of the peripheral nervous system. Evidence from several independent studies suggests that high expression of TrkA is an indicator of favorable outcome, and there is an inverse correlation between TrkA expression and N-myc amplification. In addition, some primary neuroblastomas differentiate in vitro in the presence of NGF but die in its absence. We have evidence that coexpression of full-length TrkB and BDNF is associated with N-myc amplification and may represent an autocrine survival pathway. Conversely, truncated TrkB is expressed predominantly in differentiated tumors. Finally, Trk-C is expressed in favorable neuroblastomas, essentially all of which also express TrkA. In summary, the study of neurotrophin receptor expression and function in neuroblastomas may provide important insights into the role that these pathways play in the pathogenesis and clinical behavior of this tumor. Ultimately, these pathways may provide attractive targets for the development of therapy aimed at inducing differentiation or programmed cell death in these tumors.

Nerve growth factor (NGF) is known to play a critical role in the differentiation and survival of normal sympathetic neurons through its interaction with a specific cell surface receptor. We analyzed ten well-characterized neuroblastoma cell lines for the expression and function of endogenous and exogenous p140TRK-A, and p75LNGFR. Exogenous LNGFR or TRK-A (or both) were introduced by transfection into three neuroblastoma cell lines. Transfected and untransfected neuroblastoma cell lines were analyzed by Northern analysis as well as tyrosine phosphorylation studies. Results indicate that endogenous TRK-A is expressed and/or p140TRK-A is phosphorylated in 10 of 10 cell lines. However, no other downstream responses to NGF stimulation (such as tyrosine phosphorylation of PLC gamma 1, PI-3 kinase, ERK1 and ERK2, induction of FOS and NGFI-A mRNAs, and neurite extension) were observed in the unresponsive cell lines. Transfection with p75LNGFR alone had no effect on responses to NGF stimulation. Three cell lines stably transfected with TRK-A exhibited early responses to NGF stimulation, but neurite extension was not observed. Our results indicate that endogenous TRK-A in non-responsive cell lines is either defective, or present in amounts below a threshold level required to elicit measurable responses to NGF. Furthermore, even after transfection with exogenous TRK-A, early responses were restored but later events such as neurite outgrowth did not occur, suggesting that downstream responsiveness is blocked as well.

There is considerable interest in the role of the TRK family of neuotrophin receptors in regulating growth and differentiation in normal and neoplastic nerve cells. A neuroblastoma is a common pediatric tumor derived from the neural crest, and the majority of favorable neuroblastomas express a high level of TRK-A mRNA. However, little is known about the expression or function of TRK-B in these tumors. TRK-B encodes a tyrosine kinase that binds to brain-derived neuotrophic factor (BDNF), as well as neurotrophin-3 (NT-3) and NT-4/5. We have studied the N-myc-amplified human neuroblastoma cell line, SMS-KCN, which expresses both TRK-B and BDNF. Exogenous BDNF induces tyrosine phosphorylation of TRK-B as well as phosphorylation of phospholipase C-gamma 1, the extracellular signal-regulated kinases 1 and 2, and phosphatidylinositol-3 kinase. BDNF also induces expression of the immediate-early genes c-FOS and NGFI-A but not NGFI-B or NGFI-C. In addition, BDNF appears to promote cell survival and neurite outgrowth. SMS-KCN cells also express TRK-A, which is phosphorylated in response to nerve growth factor. However, the downstream TRK-A signaling is apparently defective. Finally, we determined that in a series of 74 primary neuroblastomas, 36% express TRK-B mRNA, 68% express BDNF mRNA, and 31% express both. Truncated TRK-B appears to be preferentially expressed in more-differentiated tumors (ganglioneuromas and ganglioneuroblastomas), whereas full-length TRK-B is expressed almost exclusively in immature neuroblastomas with N-myc amplification. Our findings suggest that in TRK-B-expressing human neuroblastomas, BDNF promotes survival and induces neurite outgrowth in an autocrine or paracrine manner. The BDNF/TRK-B pathway may be particularly important for growth and differentiation of neuroblastomas with N-myc amplification.

Background and Methods: The nerve growth factor receptor is expressed in some neuroblastomas, in which its primary component is encoded by the TRK protooncogene. To determine the relation of the expression of TRK messenger RNA in neuroblastomas to other clinical and laboratory variables, we studied frozen tumor samples from 77 patients. In addition, we tested two primary neuroblastomas that expressed TRK for responsiveness to nerve growth factor., Results: TRK expression strongly correlated with favorable tumor stage (I, II, and IVS vs. III and IV), younger age (< 1 year vs. > or = 1 year), normal N-myc copy number, and low level of N-myc expression. N-myc amplification (indicated by a high copy number) correlated with advanced tumor stage, older age, an adrenal site of the primary tumor, low level of expression of TRK, and high level of expression of N-myc. Analysis of five-year cumulative-survival rates demonstrated an association of a very favorable outcome with a high level of TRK expression (86 percent vs. 14 percent) and with normal N-myc copy number (84 percent vs. 0 percent). Univariate analysis showed that these two variables were the most powerful predictors of outcome (chi-square = 51.30, P < 0.001; and chi-square = 93.61, P < 0.001, respectively). TRK expression still had significant prognostic value when the analysis was restricted to tumors without N-myc amplification. In primary cultures of neuroblastoma cells expressing TRK, exposure to nerve growth factor induced early gene expression and neurite outgrowth, but deprivation of nerve growth factor led to neuronal cell death., Conclusions: A high level of expression of the TRK proto-oncogene in a neuroblastoma is strongly predictive of a favorable outcome. A tumor with a functional nerve growth factor receptor may be dependent on the neurotrophin nerve growth factor for survival and may regress in its absence, allowing a new approach to the treatment of certain patients with neuroblastoma.

BACKGROUND AND METHODS. Genetic analysis of tumor tissue has provided considerable insight into mechanisms of malignant transformation and progression. Neuroblastomas have been studied by cytogenetics, flow cytometry, and molecular genetic techniques, and these studies have identified several specific abnormalities that allow subclassification of these tumors into genetic/clinical subtypes. RESULTS AND DISCUSSION. Four genetic abnormalities have been identified that are characteristic of certain neuroblastomas. These include: (1) loss of heterozygosity (LOH) for the short arm of chromosome 1, including band 1p36; (2) amplification of the N-myc protooncogene; (3) hyperdiploidy, or near triploidy; and (4) defects in expression or function of the nerve growth factor receptor (NGFR). Abnormalities of the NGFR are found in virtually all neuroblastoma cell lines, and some primary tumors. The latter have not been studied extensively. Hyperdiploidy is associated with lower stages of disease and with a favorable outcome in infants. LOH for chromomors. The latter have not been studied extensively. Hyperdiploidy is associated with lower stages of disease and with a favorable outcome in infants. LOH for chromosome 1, band p36, and N-myc amplification are more common in patients older than 1 year of age with advanced stages of disease. The latter two genetic abnormalities may be related, and LOH for 1p36 may precede the development of amplification. When these abnormalities are combined with assessment of DNA content, three distinct genetic subsets of neuroblastomas can be identified. The first is characterized by a hyperdiploid or near-triploid modal karyotype, with few if any cytogenetic rearrangements. These patients generally are younger than 1 year of age with localized disease and a good prognosis. The second has a near-diploid karyotype, with no consistent abnormality identified currently. These patients generally are older with more advanced stages of disease that progress slowly and are often fatal. The third group has a near-diploid or tetraploid karyotype, with deletions or LOH for 1p36, amplification of N-myc, or both. These patients generally are older with advanced stages of disease that rapidly are progressive. Thus, genetic analysis of neuroblastoma cells provides information that has prognostic significance and can direct a more appropriate choice of treatment.

We examined the expression of the trk protooncogene in a series of 82 neuroblastomas to determine its relationship to N-myc amplification and expression, disease stage, patient age, and survival. We found that virtually all stage I, II, and IV-S patients had moderate to high levels of trk expression, whereas most advanced stage neuroblastomas had low or absent levels. All but one tumor with N-myc amplification had low or absent trk expression, and the one exception was regressing at the time it was resected. Conversely, all neuroblastomas identified by mass screening had moderate to high expression of trk, and all these patients are surviving. Thus, trk expression was associated with an absence of N-myc amplification, lower disease stage, lower patient age, and favorable outcome. Tumors with high trk expression may be more likely to differentiate, regress spontaneously, or respond well to therapy.

We hypothesized that defects in the nerve growth factor receptor (NGFR) pathway may play a role in maintenance of the undifferentiated state of neuroblastomas. To test this hypothesis, we examined the structure and function of the NGFR in a panel of 10 neuroblastoma cell lines. Southern blot analysis showed that all 10 cell lines possess apparently normal NGFR genes. Northern blot and ligand binding/immunoprecipitation assays revealed four receptor-positive cell lines (NGP, NLF, SK-N-SH and, LA-N-6), with NGFR mRNA and protein of expected sizes (3.8 kb and approximately 75 kD respectively). NGF binding assays and Scatchard analyses were performed on these four lines. The NGP line possesses only low affinity receptor (Kd approximately 3.5 x 10(-9] while the other three lines express both low- and high-affinity forms (Kd approximately 10(-9) and Kd approximately 10(-11), respectively). However, none of the 10 lines exhibited an early or late response to NGF treatment as assayed by c-fos mRNA induction (45 min) and neurite extension (8-10 days). These results demonstrate at least three distinct defects in the NGFR pathway in these tumor lines: 1) absence of NGFR mRNA or protein expression, 2) expression of only low-affinity receptor, and 3) inability of high affinity receptor to mediate a response to NGF. Such defects may play an important role in the initiation or maintenance of the undifferentiated state of neuroblastoma.

Neuroblastoma is a tumor of postganglionic sympathetic origin, and nerve growth factor (NGF) is normally required for the survival and differentiation of sympathetic neuroblasts. Since the biological activity of NGF is mediated by the NGF receptor (NGFR), we hypothesized that defects in the NGF/NGFR pathway may play a role in maintenance of the undifferentiated state of neuroblastomas. To test this hypothesis, we examined the structure of the NGFR at the DNA, RNA, and protein levels in a panel of 10 neuroblastoma cell lines. In addition, we examined the function of the NGFR in these lines by analysis of NGF binding kinetics, as well as by the ability of NGF to induce c-fos expression and neurite outgrowth. Southern blot analysis showed that all 10 cell lines possess apparently normal NGFR genes. Northern blot and ligand binding/immunoprecipitation assays revealed four receptor-positive cell lines (NGP, NLF, SK-N-SH, and LA-N-6), with NGFR mRNA and protein of expected sizes (3.8 kilobases and Mr approximately 75,000, respectively). NGF binding assays and Scatchard analyses were performed on the four NGFR-positive lines. The NGP line possesses only low-affinity receptor (Kd approximately 3.5 x 10(-9)), whereas the other three lines express both low- and high-affinity forms (Kd approximately 10(-9) and Kd approximately 10(-11), respectively). However, none of the 10 lines exhibited a response to NGF treatment as assayed by c-fos mRNA induction and neurite extension.(ABSTRACT TRUNCATED AT 250 WORDS)