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© 2000 American Society for Clinical Oncology MYCN Expression Is Not Prognostic of Adverse Outcome in Advanced-Stage Neuroblastoma With Nonamplified MYCNFrom the Department of Pediatrics, Northwestern University Medical School; Children’s Memorial Hospital and Robert H. Lurie Comprehensive Cancer Center, Chicago, IL; Department of Statistics, University of Florida, and Pediatric Oncology Group Statistical Office, Gainesville, FL; and Children’s Cancer Institute Australia for Medical Research, University of New South Wales, and Sydney Children’s Hospital, Sydney, Australia. Address reprint requests to Susan L. Cohn, MD, Division of Hematology/Oncology, Box 30, Children’s Memorial Hospital, 2300 Children’s Plaza, Chicago, IL 60614; email scohn{at}northwestern.edu
PURPOSE: The clinical significance of MYCN expression in children with neuroblastoma (NB) remains controversial. To determine the prognostic significance of MYCN expression in the absence of MYCN amplification, we analyzed MYCN mRNA and protein expression in tumors from 69 patients. PATIENTS AND METHODS: Sixty-nine NB tumor samples with nonamplified MYCN from patients with stage C or D disease were obtained from the Pediatric Oncology Group Neuroblastoma Tumor Bank. MYCN mRNA was analyzed using a real-time reverse transcriptase polymerase chain reaction assay, and MYCN protein was examined by Western blot analyses.
RESULTS: The estimated 5-year event-free survival (EFS) and survival (S) rates plus SE for the cohort were 57% ± 17% and 60% ± 16%, respectively. Infants younger than 1 year had significantly higher rates of EFS and S than children CONCLUSION: High levels of MYCN expression are not prognostic of adverse outcome in patients with advanced-stage NB with nonamplified MYCN. A trend associating high levels of MYCN expression with improved outcome was observed.
N EUROBLASTOMA (NB), a pediatric malignancy that arises in cells of the neural crest, has a broad range of clinical behavior. Outcome is strongly associated with tumor stage and the age of the child at diagnosis.1-3 Infants younger than 1 year, as well as children  1 year of age with localized disease have a high probability of cure with surgery alone.4-7 The prognosis is also good for most infants with advanced-stage NB after treatment with surgery and chemotherapy.8-10 In contrast, long-term survival (S) rates for children older than 1 year with widely disseminated disease remain at less than 20%, despite treatment with intensive multimodality therapy.11 In addition to stage and age, tumor biology has also been shown to strongly impact prognosis. Amplification of the MYCN oncogene is one of the most powerful adverse prognostic factors in NB.12,13 Other indicators of poor outcome include deletion of the short arm of chromosome 1,14,15 17q gain,16 diploid DNA content,17 low-level TRKA expression,18,19 and unfavorable histology.20,21 MYCN is thought to play an important role in determining the biologic behavior of NB, as genomic amplification of the MYCN oncogene is strongly associated with advanced-stage disease, rapid tumor progression, and poor outcome.12,13 In laboratory studies, enhanced expression of MYCN has been shown to confer growth potential to cells in vitro as well as in vivo.22,23 Furthermore, MYCN antisense experiments have shown that downregulation of MYCN is associated with decreased proliferation and an inhibition in anchorage-independent growth.24,25 Recently, Weiss et al26 have shown that targeted expression of MYCN in neuroectodermal cells of transgenic mice results in the development of NB. Furthermore, compared with heterozygous mice, mice homozygous for the MYCN transgene exhibited an increase in incidence and a decrease in latency of tumor formation. Although high levels of MYCN expression clearly enhance malignant NB growth in laboratory studies, the clinical significance of MYCN expression in children with NB remains controversial.18,19,27-33 The reason for the discordant results reported in the published clinical studies may, in part, result from disparities in patient populations, as the proportion of infants younger than 1 year, patients with advanced-stage disease, and children with MYCN-amplified tumors differs in the various series. To determine specifically the prognostic significance of MYCN expression in the absence of MYCN amplification, we used a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay and Western blot analyses to quantify MYCN mRNA and protein expression, respectively, in NB tumors that lacked MYCN amplification from 69 patients. We further limited our study cohort to patients with regional or disseminated disease (Pediatric Oncology Group [POG] stage C or D),4 because unlike the excellent prognosis associated with localized NBs, outcome for children with advanced-stage disease and normal MYCN copy number is variable. Additional prognostic indicators would be of value for this group of patients to ensure that therapy is appropriately tailored according to risk.
Patients and Tumor Specimens NB tumors from 71 patients were obtained from the POG NB Tumor Bank. The criteria for inclusion of patients in this analysis were as follows: (a) that the tumor sample was obtained at the time of diagnosis, before the administration of chemotherapy; (b) that the child was known to have regional or disseminated disease (POG stage C or D)4; and (c) that the tumor lacked MYCN amplification. Subsequently, it was determined that one sample had not been obtained at the time of diagnosis and that one tumor was from a patient with stage A disease. These two patients were excluded from the analysis, which resulted in a final cohort of 69 patients. None of the samples analyzed in this study was included in our previous report of MYCN expression in NB.33 Because patients with bone marrow disease can frequently be diagnosed with a bone marrow aspirate and elevated catecholamines, not all patients with stage D disease undergo a surgical biopsy at the time of diagnosis. Therefore, tumor samples from patients with stage D disease are not always available for banking. For this reason, the tumors in the POG NB Tumor Bank do not reflect the expected distribution of patient stage. In this study, approximately equal numbers of tumors from patients with stages C and D disease were analyzed. Samples were sent to the investigators for MYCN expression analysis after the project was reviewed and approved by the POG Neuroblastoma Biology Subcommittee. Sixty-four of the patients from whom samples were obtained were registered on the POG biologic protocol #9047 between October 1990 and May 1997. Five children were diagnosed before 1990, and these patients were registered on the POG therapeutic protocol #8104 or #8743.8,34,35 The protocols were approved by individual institutional review boards, and informed consent was obtained for every patient registered onto the study. The diagnosis of NB was based on histologic examination of tumor specimens. In addition to at least a single bone marrow aspiration and single bone marrow biopsy, extent of disease for all patients was evaluated with computed tomography scan and/or magnetic resonance imaging, Tc99 bone scan, and skeletal radiographs.
Treatment
DNA Flow Cytometry and Genomic MYCN Analysis
Real-Time RT-PCR
Western Blot Analysis To confirm integrity of short-lived proteins, duplicate blots were probed with Ab-6 (Calbiochem, CN Biosciences, Inc, San Diego, CA), an anti-p53 antibody. The membranes were incubated for 2 hours with a 1:2,000 dilution of primary antibody and 2 hours with a 1:5,000 dilution of horseradish peroxidase—conjugated goat antimouse immunoglobulin G. Signals for both MYCN (62 to 64 kd) and p53 (53 kd) protein were detected in every sample scored as positive for MYCN protein expression. Samples in which the MYCN signal was not visualized but in which a signal for p53 was detected were scored as negative for MYCN protein. Short-lived proteins were considered degraded in the samples in which neither MYCN nor p53 signals were detected, and these samples were excluded from further analysis.
Statistical Analysis
Clinical Characteristics of Study Cohort The clinical features of the 69 patients with advanced-stage NB are listed in Table 1. All patients had tumors that lacked MYCN amplification. The estimated 5-year event-free survival (EFS) and S rates for the entire cohort were 57% ± 17% and 60% ± 16%, respectively (Fig 1A). As expected, age was predictive of outcome, and infants had significantly higher rates of EFS and S than children older than 1 year (P = .003 and P < .001, respectively) (Fig 1B). In addition, patients with stage C disease had better outcome than patients with stage D NB (P < .001) (Fig 1C). Tumor cell ploidy was also prognostic in this cohort of patients (P < .001) (Fig 1D). Regardless of whether the children were younger or older than 1 year, the outcome for children with hyperdiploid NB tumors was significantly better than that seen in patients with diploid tumors (Table 1).
Validation of the Standard Curve and Dynamic Range of Real-Time RT-PCR The dynamic range of the real-time PCR methodology for determining MYCN expression was assessed using the MYCN-amplified NB cell line BE(2)-C. A standard curve was constructed with a starting point of a cDNA equivalent of 50 ng of BE(2)-C RNA. A strong linear relationship (R = .99) over four orders of magnitude was demonstrated between the Ct and the log of the starting cDNA quantity, which represented a wide dynamic range with samples that contained as much as 50 ng or as little as 5 pg of equivalent total cDNA.
Expression of MYCN mRNA and Protein
Western blot analysis was used to examine MYCN protein expression because it is considered to be a more quantitative assay than immunohistochemistry. Of the 69 tumor samples analyzed, 52 expressed intact p53 and were considered assessable for MYCN protein expression (Fig 3). MYCN protein was detected in 13 of the 52 tumors. Seven of the tumors positive for MYCN protein were from infants, and six were from children older than 1 year. Using an analysis of variance model, an association between MYCN protein expression and high levels of MYCN mRNA was observed (P = .013) (Table 2). All but two of the tumors in which MYCN protein was detected expressed MYCN mRNA at levels higher than the mean value.
MYCN Expression and Outcome Surprisingly, the 4-year EFS was slightly better for patients with high versus low levels of MYCN expression, although the difference was not statistically significant (64% ± 13% and 50% ± 16%, respectively; P = .337) (Fig 4A, Table 3). Similarly, better S was associated with high levels of MYCN expression, but again, the difference was not statistically significant (70% ± 13% and 50% ± 14%; P = .290) (Fig 4B, Table 3). EFS and S analyses were also performed with dichotomization of the level of expression around the mean and upper quartile MYCN PCR values. Regardless of the value used, there was insufficient evidence to support a difference in EFS or S for patients with high versus low levels of MYCN mRNA expression. Four-year EFS and S rates were also not statistically different for patients with tumors with and without detectable MYCN protein expression (Table 3). However, a trend of improved outcome associated with detectable MYCN protein expression was observed.
The clinical significance of MYCN expression was also analyzed in subsets of patients older and younger than 1 year (Table 3). The trend of high MYCN expression associated with better outcome was observed in the infant subset. Four-year EFS for patients younger than 1 year with high MYCN expression was 88% ± 12%, compared with 75% ± 22% for those with low-level expression (P = .238). Similarly, enhanced S was observed for the group of infants with high-level MYCN expression, although the difference was not significantly different (P = .228) (Table 3). We found no statistical difference in EFS or S in children older than 1 year with high versus low levels of MYCN expression (Table 3). Furthermore, MYCN protein expression did not affect outcome in infants or in children older than 1 year, although again, a trend of better outcome associated with detectable levels of expression was observed. In the multivariate Cox proportional hazards models for EFS and patient S, after adjustment for the statistically significantly prognostic effects of stage, age, and ploidy, neither MYCN mRNA nor protein expression was found to be prognostic.
The adverse prognostic significance of MYCN amplification in NB has been well established.12,13,46 The aggressive tumor phenotype that is characteristic of MYCN–amplified NB, is thought to result, at least in part, from high levels of MYCN expression.27,47-50 However, the clinical significance of MYCN expression in NB remains controversial.18,19,27-33 In this study, we measured MYCN mRNA and protein in 69 tumors with normal MYCN copy number to determine specifically whether the level of MYCN mRNA and/or protein expression is prognostic in patients with tumors that lack MYCN amplification. Because patients with localized disease have high cure rates with surgery alone, even if biologic prognostic factors are unfavorable,5-7,51,52 we further limited the study cohort to patients with regional and disseminated disease. Outcome for patients with advanced-stage NB with normal MYCN copy number is variable, and additional prognostic factors for this group of patients are needed so that therapy can be tailored appropriately. In this population of patients, high levels of MYCN expression were not predictive of worse outcome. Rather, we observed a trend of high levels of MYCN associated with improved outcome, although the differences in EFS and S rates did not reach statistical significance. The S rates of the infants and older children included in this study paralleled those of patients with similar clinical and biologic features reported in other clinical trials,8,10,35-37,53,54 which suggests that our cohort was representative. However, the 4-year EFS rate of the infants with stage D disease in our cohort was lower than the 3-year EFS rate recently reported in a Children’s Cancer Group study of infants with stage IV disease without MYCN amplification.9 It is not clear why the infants in the Children’s Cancer Group study had a more favorable outcome, but differences in therapy and length of follow-up may have contributed to the disparate results. MYCN mRNA and protein have been detected in NB tumors from patients with all stages of disease, and several previous studies have shown that the level of MYCN mRNA and protein is not predictive of outcome in patients with NB.30-32,55 However, the clinical significance of MYCN expression remains controversial, as strong correlations between high levels of MYCN expression and poor S have been demonstrated in other series.18,19,27-29 The reason for the discrepancies in the literature remains unclear, but the conflicting results may reflect differences in study cohorts. The proportion of infants and patients with localized disease in the series can dramatically affect outcome.3,17,56 Most importantly, because MYCN amplification is associated with high levels of MYCN expression,47 the number of patients with MYCN amplification will significantly affect the association between S and MYCN expression. In a previous study, we reported that high-level MYCN expression was strongly predictive of poor outcome in children with NB.33 Interestingly, MYCN expression did not affect outcome for the subset of infants included in that study, whereas high-level expression was associated with significantly worse S in children older than 1. Unlike the current cohort, that series included patients with all stages of disease, and 13 of the 60 patients had MYCN-amplified tumors. As in other reports, a strong correlation between high levels of MYCN gene expression and the presence of MYCN gene amplification was observed.31,47 However, even after exclusion of patients with MYCN amplification, high levels of MYCN expression retained prognostic significance. In our previous study, clinical stage was also highly correlated with MYCN gene expression, and patients with favorable-stage disease had tumors with low-level expression. Thus, the disparity in results between our current series and the previously reported study is most likely a result of the lack of patients with localized disease in the current patient cohort. To date there have been two other studies in which multivariate analyses were performed to determine the prognostic impact of MYCN gene expression after stratification for the effect of MYCN gene amplification. In a series reported by Nakagawara et al18 that included patients with all stages of disease, MYCN gene expression had no predictive value independent of MYCN amplification. In contrast, Chan et al27 reported a highly significant correlation between MYCN protein expression and poor outcome in 23 patients with stages III, IV, and IV-S tumors that lacked MYCN amplification. However, age and MYCN gene copy number were not predictive of outcome in the series of patients in the study by Chan et al, which suggests that the cohort may not be representative. Interestingly, high levels of MYC expression recently have been shown to be predictive of favorable outcome in adult cancer studies. Bieche et al57 recently reported a trend that links MYC gene overexpression and favorable outcome in patients with breast cancer. In that series of patients with breast cancer, the worst outcome was seen in patients with the lowest level of MYC mRNA expression. Similarly, Smith and Goh58 reported a more favorable prognosis among a large series of patients with colorectal cancer whose tumors overexpressed MYC mRNA. The MYC family of proteins are known to participate in the regulation of both cell proliferation and apoptosis,59 and MYCN has been reported to accelerate progression to S phase60 and to induce apoptosis.61 Furthermore, ectopic MYCN expression also has been shown to cooperate with cytotoxic drugs to induce apoptosis in NB cells.62 Thus, the apparently better prognosis of patients with high-level MYC- or MYCN-expressing tumors may result from either higher rates of apoptosis or higher levels of cell proliferation and, thus, greater chemosensitivity. The results of our study indicate that high levels of MYCN expression are not prognostic of adverse outcome in patients with advanced-stage NB tumors that lack MYCN amplification. The outcome for infants was favorable regardless of the level of MYCN expression. In contrast, S rates for the older children with either high or low levels of MYCN expression was poor, which indicates that additional factors must contribute to the aggressive tumor growth in this cohort. Further studies that investigate the function of MYCN in this cohort of patients are needed to elucidate whether the trend that links improved outcome to higher levels of MYCN expression results from enhanced drug-induced apoptosis.
Supported in part by the Pediatric Oncology Group Grant CA 29139 and grant no. CA74824 from the National Cancer Institute, National Institutes of Health, Bethesda, MD; the Elise Anderson Neuroblastoma Research Fund; the Neuroblastoma Children’s Cancer Society; Bears Care; VisionTek Foundation; gifts from Ezra Schaffer and Dennis Drescher; the Robert H. Lurie Comprehensive Cancer Center, National Institutes of Health, National Cancer Institute Core Grant 5P30CA60553; and the National Health and Medical Research Council of Australia and the New South Wales Cancer Council. We thank the Pediatric Oncology Group Neuroblastoma Biology Committee for reviewing and approving this research project and for providing neuroblastoma tumor samples. We also thank Susan Rowe and Michael Nash from the Pediatric Oncology Group Neuroblastoma Reference Laboratory for performing the MYCN and ploidy studies.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
represents children who remain alive.
