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© 2000 American Society for Clinical Oncology
MYCN Expression in Neuroblastoma: A Mixed Message?University of California School of Medicine San Francisco, CA MYCN GENE amplification clearly identifies a subset of neuroblastomas with extremely malignant behavior. However, whether relative overexpression of MYCN RNA and/or MycN protein, in the absence of genomic amplification, is biologically or clinically significant is controversial, as demonstrated by the number of contradictory publications on this subject.1-7 The study by Cohn et al8 in this issue of the Journal of Clinical Oncology examines tumors, all lacking MYCN gene amplification, from a group of 69 patients with International Neuroblastoma Staging System (INSS)9 stage 3 and 4 neuroblastoma for MYCN RNA and protein expression. The results show that the level of MYCN expression in tumors from patients without gene amplification does not predict outcome; this result is seemingly at odds with a previous Journal of Clinical Oncology publication by some of the same authors.1 The reasons for the discrepant results may be due to interactions with other tumor genetic factors, the laboratory methods used, or to selection in the patient population with regard to stage, age, tumor MYCN gene amplification, or the treatments given. The MYCN oncogene was originally cloned in 1983 by identifying an amplified DNA sequence with partial homology to the MYC proto-oncogene (c-myc) in neuroblastoma cell lines with double minute chromosomes or homogeneously staining regions.10,11 The MYCN gene is located on the distal short arm of chromosome 2(2p24).10 Subsequent studies showed that amplification was also seen in approximately one third of untreated human neuroblastoma tumors, significantly associated with advanced stage12 and associated with rapid tumor progression, independent of both stage and age.13 Innumerable retrospective and prospective studies have confirmed these original observations, as well as showing frequent association of MYCN amplification with other unfavorable biologic tumor characteristics, such as chromosomal deletions of 1p,14 low expression of TRKA,6,7 and undifferentiated and stroma-poor histology.7,15 The pathways activated by MYCN that result in cell proliferation and tumor progression have only been partly characterized. The MYCN gene product is a nuclear protein with 38% amino acid identity to c-myc and a short half-life.16 Like all MYC family proteins, MycN contains an N- terminal transactivation domain (Myc box) and a C-terminal region containing a basic helix-loop-helix/leucine zipper (bHLH-LZ) motif. The bHLH-LZ region mediates DNA binding and interactions with other HLH-LZ proteins, such as Max and Mad. With increased production of MycN, as with entrance into the cell cycle or as a result of genomic amplification, heterodimerization of MycN and Max occurs, which leads to transcriptional activation of an as yet undefined series of growth-promoting genes. Several genes thought to be upregulated by MYC proteins have been identified, but the exact target genes affected by MYCN overexpression in neuroblastomas have not yet been defined.11 Like Myc, MycN is thought to have dual biologic functions and drive either cellular proliferation or commit a cell to an apoptotic death.17 Thus abolition of the apoptotic signaling resulting from MycN overexpression is likely to be essential for MycN-related oncogenesis. It is of interest, therefore, that a recent study showed that the caspase 8 gene (CASP8) is deleted or preferentially silenced by methylation in MYCN-amplified tumors, thus potentially inactivating the Fas apoptotic pathway.18 It is also possible that MycN protein downregulates inhibitors of endothelial cell proliferation (recently shown to be the case for activin A), thus contributing to tumor angiogenesis.19 Nonetheless, other biologic markers have also emerged that may have a role in cell proliferation or metastasis independent of MYCN gene amplification, such as the unfavorable prognostic implications of 17q gains20,21 or high telomerase activity,22-24 each of which were found to be independent of MYCN amplification in multivariate analyses. Furthermore, although patients with single-copy tumors often have a more favorable outcome than those with MYCN amplification, patients who are older than 1 year at diagnosis and have stage 4 disease still have only 40% estimated survival with maximal therapy.25 Although this may be explained in part by other genetic lesions, such as unbalanced gain of 17q or loss of heterozygosity at 1p or 11q,11,26,27 it has been hypothesized that relatively increased MYCN expression in the absence of amplification may account for the poor outcome in some patients. Forced expression of MYCN can transform normal mammalian cells, but only in cooperation with another oncogenic stimulus, such as mutated ras.28,29 Addition of antisense RNA to MYCN-overexpressing neuroblastoma cell lines can decrease proliferation and/or induce differentiation.30,31 Some neuroblastoma cell lines have been characterized that have a single copy of MYCN but have relative overexpression of MYCN RNA relative to nonneuroblastoma lines.32,33 More recently, Weiss et al34 have shown that targeting overexpression of MYCN to the mouse neuroectoderm using a tyrosine hydroxylase promoter results in formation of tumors resembling neuroblastoma, with a penetrance related to the transgene dosage. Not only do these tumors resemble neuroblastoma histologically, with ganglionic differentiation, neurosecretory granules, synaptophysin, and neuron-specific enolase expression, but they exhibit consistent chromosomal gains and losses in many regions syntenic with those observed in human neuroblastoma.35 However, there continues to be controversy in reports of clinical correlative studies of MYCN expression in human tumors, as recently summarized by Bordow et al.1 Initial studies of MYCN expression clearly showed that tumors with MYCN gene amplification had markedly increased MYCN mRNA7,36,37 and protein38 expression compared with those without amplification. However, a fairly wide range of expression by Northern blot was seen in tumors that had a single copy of MYCN, extending from low or undetectable up through the rare instance of expression levels equivalent to those seen in tumors with multiple copies of MYCN.1,36 Multiple studies have attempted to define the prognostic significance of MYCN expression. This has been reported both anecdotally in small groups of patients as well as in at least seven larger studies allowing for meaningful statistical analyses. These prior studies comprised 35 to 80 patients and included tumors both with and without MYCN gene amplification. In contrast to the report of Cohn et al8 in this issue of the Journal of Clinical Oncology, each of these previous reports showed that the patients whose tumors had a relatively high expression of MYCN had a significantly less favorable disease outcome.1-7 However, one of the strengths of the Cohn study8 was the restriction of the analysis to 69 tumors with single-copy MYCN to have a uniform population in which to examine the prognostic value of expression. Gene expression as a prognostic factor was statistically significant even when the analysis was restricted to single-copy tumors only in the previous studies of Chan et al2 and Bordow et al.1 Expression was not independent of MYCN amplification in the study of Nakagawara et al,6 which included 66 tumor with a single copy of MYCN. The analysis of MYCN expression restricted to single-copy tumors was not reported in the four other studies.3-5,7 Interestingly, as in the report of Nakagawara et al,6 there was no significance to higher MYCN expression levels in tumors without gene amplification in the report in this issue of the Journal of Clinical Oncology.8 The disagreement among these reports may be due to methodological, clinical, or biologic differences. The methods used include three studies in which expression was quantified with Northern blots,5-7 three studies using immunohistochemistry,2-4 and two using reverse transcriptase polymerase chain reaction (RT-PCR).1-8 A previous study of 126 patients showed excellent concordance between Northern blot mRNA expression and immunohistochemistry for MycN protein.38 The current study by Cohn et al8 also used Western blot in addition to real-time quantitative PCR to measure expression. Protein expression by Western blot analysis was only seen in 25% of patients, with a significant association to relatively high levels of expression of mRNA. It is possible that the dichotomization of the RT-PCR expression ratio using only MYCN single-copy tumors lowered the median value, potentially blunting the differences between very high and intermediate MYCN expression levels. The real-time TaqMan RT-PCR technology used in the report by Cohn et al (TaqMan Universal PCR MasterMix; Perkin-Elmer Applied Biosystems, Foster City, CA) may be more sensitive than the RT-PCR method in the previous contradictory report from Bordow et al.1 However, all of these methods have been widely used and are fairly standardized. Thus one is left with the more likely possibility that the discrepancies regarding the significance of overexpression in single-copy tumors are due rather to other clinical or biologic factors. An important variable in these studies was the clinical factors, including stage, age, and treatment protocol. The Cohn et al study8 attempted to standardize this by restricting the study to patients with INSS9 stage 3 or 4 neuroblastoma. Unfortunately, the INSS stage 3 patients are quite heterogeneous with regard to prognosis compared with the INSS stage 4 patients, depending on other biologic factors such as histology, DNA index, and serum ferritin.39,40 Thus although the previous studies may have been obscured by inclusion of varying proportions of infants or patients with low-stage disease, as noted in the current report, the same criticism could still apply here. There was an unusually high proportion of INSS stage 3 disease in the current study (50%) as opposed to the usual 20% expected in a group of advanced-stage patients, probably due to the likelihood of obtaining specimens for analysis. In fact, if one examines biologic risk factors, INSS stage 3 patients usually consist of at least two thirds good-risk patients, with greater than 90% survival. Thus the skewed nature of this group may have made it difficult to see significance for MYCN expression. Infants also have an excellent prognosis, even with advanced-stage disease, with greater than 90% survival in MYCN single-copy patients, unlike similar patients who are older than 1 year at diagnosis.39 In the study by Cohn et al, infants whose tumors had higher MYCN expression actually had superior survival (although not significantly so) than those with lower expression. The possible adverse biologic effects of the increased expression may have been counterbalanced by some other mitigating factor, such as higher TRK expression.7 Treatment for neuroblastoma has been gradually intensifying in high-risk disease and may be eradicating some of the influence of MYCN expression. Thus in the recently reported Children’s Cancer Group randomized study of patients with high-risk neuroblastoma, there was a significant improvement in event-free survival for those with MYCN-amplified tumors treated with high-dose therapy and autologous bone marrow transplantation compared with standard-dose chemotherapy.25 In summary, although MYCN expression is certainly associated with worse outcome at the very high levels resulting from genomic amplification, the biologic consequence of relative overexpression in the absence of amplification is less clear. However, the article by Cohn et al8 in this issue of the Journal of Clinical Oncology demonstrates that analysis of MycN expression is unlikely to provide prognostically relevant information for patients with neuroblastoma who have single-copy MYCN tumors. REFERENCES 1. Bordow SB, Norris MD, Haber PS, et al: Prognostic significance of MYCN oncogene expression in childhood neuroblastoma. J Clin Oncol 16: 3286-3294, 1998[Abstract] 2. Chan HS, Gallie BL, DeBoer G, et al: MYCN protein expression as a predictor of neuroblastoma prognosis. Clin Cancer Res 3: 1699-1706, 1997[Abstract] 3. Hiyama E, Hiyama K, Yokoyama T, et al: Immunohistochemical analysis of N-myc protein expression in neuroblastoma: Correlation with prognosis of patients. J Pediatr Surg 26: 838-843, 1991[Medline]
4.
Nakada K, Fujioka T, Kitagawa H, et al: Expressions of N-myc and ras oncogene products in neuroblastoma and their correlations with prognosis. Jpn J Clin Oncol 23: 149-155, 1993
5.
Nakagawara A, Kadomatsu K, Sato S, et al: Inverse correlation between expression of multidrug resistance gene and N-myc oncogene in human neuroblastomas. Cancer Res 50: 3043-3047, 1990
6.
Nakagawara A, Arima Nakagawara M, Scavarda NJ, et al: Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma. N Engl J Med 328: 847-854, 1993
7.
Suzuki T, Bogenmann E, Shimada H, et al: Lack of high-affinity nerve growth factor receptors in aggressive neuroblastomas [see comments]. J Natl Cancer Inst 85: 377-384, 1993
8.
Cohn SL, London WB, Huang D, et al: MYCN expression is not prognostic of adverse outcome in advanced-stage neuroblastoma with non-amplified MYCN. J Clin Oncol 18: 3604-3613, 2000
9.
Brodeur GM, Pritchard J, Berthold F, et al: Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment [see comments]. J Clin Oncol 11: 1466-1477, 1993 10. Schwab M, Alitalo K, Klempnauer KH, et al: Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature 305: 245-248, 1983[Medline]
11.
Maris JM, Matthay KK: Molecular biology of neuroblastoma. J Clin Oncol 17: 2264-2279, 1999
12.
Brodeur GM, Seeger RC, Schwab M, et al: Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224: 1121-1124, 1984 13. Seeger RC, Brodeur GM, Sather H, et al: Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 313: 1111-1116, 1985[Abstract]
14.
Fong CT, Dracopoli NC, White PS, et al: Loss of heterozygosity for the short arm of chromosome 1 in human neuroblastomas: Correlation with N-myc amplification. Proc Natl Acad Sci U S A 86: 3753-3757, 1989
15.
Shimada H, Stram DO, Chatten J, et al: Identification of subsets of neuroblastomas by combined histopathologic and N-myc analysis. J Natl Cancer Inst 87: 1470-1476, 1995
16.
Slamon DJ, Boone TC, Seeger RC, et al: Identification and characterization of the protein encoded by the human N-myc oncogene. Science 232: 768-772, 1986 17. Prendergast GC: Mechanisms of apoptosis by c-Myc. Oncogene 18: 2967-2987, 1999[Medline] 18. Teitz T, Wei T, Valentine MB, et al: Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN [see comments]. Nat Med 6: 529-535, 2000[Medline]
19.
Breit S, Ashman K, Wilting J, et al: The N-myc oncogene in human neuroblastoma cells: Down-regulation of an angiogenesis inhibitor identified as activin A. Cancer Res 60: 4596-4601, 2000 20. Plantaz D, Mohapatra G, Matthay KK, et al: Gain of chromosome 17 is the most frequent abnormality detected in neuroblastoma by comparative genomic hybridization. Am J Pathol 150: 81-89, 1997[Abstract]
21.
Bown N, Cotterill S, Lastowska M, et al: Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma [see comments]. N Engl J Med 340: 1954-1961, 1999 22. Hiyama E, Hiyama K, Yokoyama T, et al: Correlating telomerase activity levels with human neuroblastoma outcomes [see comments]. Nat Med 1: 249-255, 1995[Medline]
23.
Poremba C, Scheel C, Hero B, et al: Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: A molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues. J Clin Oncol 18: 2582-2592, 2000 24. Reynolds CP, Zuo JJ, Kim NW, et al: Telomerase expression in primary neuroblastomas. Eur J Cancer 33: 1929-1931, 1997
25.
Matthay KK, Villablanca JG, Seeger RC, et al: Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis- retinoic acid: Children’s Cancer Group. N Engl J Med 341: 1165-1173, 1999
26.
Maris JM, Weiss MJ, Guo C, et al: Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: A Children’s Cancer Group study. J Clin Oncol 18: 1888-1899, 2000 27. Guo C, White PS, Weiss MJ, et al: Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas. Oncogene 18: 4948-4957, 1999[Medline] 28. Schwab M, Varmus HE, Bishop JM: Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature 316: 160-162, 1985[Medline]
29.
Yancopoulos GD, Nisen PD, Tesfaye A, et al: N-myc can cooperate with ras to transform normal cells in culture. Proc Natl Acad Sci U S A 82: 5455-5459, 1985 30. Negroni A, Scarpa S, Romeo A, et al: Decrease of proliferation rate and induction of differentiation by a MYCN antisense DNA oligomer in a human neuroblastoma cell line. Cell Growth Differ 2: 511-518, 1991[Abstract] 31. Schmidt ML, Salwen HR, Manohar CF, et al: The biological effects of antisense N-myc expression in human neuroblastoma. Cell Growth Differ 5: 171-178, 1994[Abstract] 32. Wada RK, Seeger RC, Brodeur GM, et al: Characterization of human neuroblastoma cell lines that lack N-myc gene amplification. Prog Clin Biol Res 271: 57-69, 1988[Medline] 33. Cohn SL, Salwen H, Quasney MW, et al: Prolonged N-myc protein half-life in a neuroblastoma cell line lacking N-myc amplification. Oncogene 5: 1821-1827, 1990[Medline] 34. Weiss WA, Aldape K, Mohapatra G, et al: Targeted expression of MYCN causes neuroblastoma in transgenic mice. EMBO J 16: 2985-2995, 1997[Medline]
35.
Weiss WA, Godfrey T, Francisco C, et al: Genome-wide screen for allelic imbalance in a mouse model for neuroblastoma. Cancer Res 60: 2483-2487, 2000
36.
Nisen PD, Waber PG, Rich MA, et al: N-myc oncogene RNA expression in neuroblastoma. J Natl Cancer Inst 80: 1633-1637, 1988
37.
Slavc I, Ellenbogen R, Jung WH, et al: myc gene amplification and expression in primary human neuroblastoma. Cancer Res 50: 1459-1463, 1990 38. Seeger RC, Wada R, Brodeur GM, et al: Expression of N-myc by neuroblastomas with one or multiple copies of the oncogene. Prog Clin Biol Res 271: 41-49, 1988[Medline]
39.
Matthay KK, Perez C, Seeger RC, et al: Successful treatment of stage III neuroblastoma based on prospective biologic staging: A Children’s Cancer Group study. J Clin Oncol 16: 1256-1264, 1998
40.
Bowman LC, Castleberry RP, Cantor A, et al: Genetic staging of unresectable or metastatic neuroblastoma in infants: A Pediatric Oncology Group study. J Natl Cancer Inst 89: 373-380, 1997
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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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