[CANCER RESEARCH 50, 1459-1463, March 1, 1990] myc Gene Amplification and Expression in Primary Human Neuroblastoma1 Irene Slave, Richard Ellenbogen, Woo-Hee Jung, Gordon F. Vawter, Cynthia Kretschmar, Holcombe Grier, and Bruce R. Korf Division of Genetics [I. S., B. R. K.J, and Departments of Neurosurgery [R. E.J, Pathology ¡W.-H.J., G. F. Y.J, and Neurology [B. R. K.J. The Children's Hospital, and Department of Pediatrie Oncology, Dana-Farber Cancer Institute/C. K., H. G.J, Boston, Massachusetts 02115 ABSTRACT Although N-myc amplification in neuroblastomas correlates with poor prognosis, not all neuroblastomas which fail to respond to therapy have N-myc amplification. To determine whether other modes of myc gene activation underlie progression of some neuroblastomas, 45 were analyzed for amplification of N-wiyc, c-myc and L-myc and 26 were studied for transcription of these oncogenes. N-myc amplification was found in 6 of 45 tumors; no tumor had amplification of c-myc or L-myc. Transcription of both N-myr and c-myc occurred in 21 of 26 neuroblastomas. No tumor without \-myc amplification had a level of \-myc expression near that of a tumor or cell line with amplification. One tumor with \-myc amplification was the only specimen with N-myc but not c-myc expres sion. Five samples had c-myc but not \-myc expression; all had histolog- ical features of ganglioneuroma. DNA index did not correlate with myc gene amplification or expression. It is concluded that N-ni.vc and c-myc are commonly expressed in primary untreated neuroblastomas, but in the absence of N-myc amplification, expression of these genes does not appear to correlate with disease progression. INTRODUCTION Amplification of the protooncogene N-myc occurs in approx imately one-third of neuroblastomas and correlates with poor prognosis (1, 2). Yet the absence of N-myc amplification does not necessarily predict a favorable outcome since a high pro portion of children over 1 year of age with stage III or IV neuroblastomas eventually die from their disease (3, 4). Al though nonaneuploid DNA content (5-7) and deletion of the short arm of chromosome 1 (8) have been found to correlate with advanced disease, no molecular genetic markers have been identified in the advanced neuroblastomas lacking N-myc am plification which might explain their poor outcome. The association of N-myc amplification with poor prognosis suggests two hypotheses to explain the progression of tumors without amplification. First, N-myc is one member of a "family" of nuclear oncogenes (9), which also includes c-myc, L-myc, B- myc etc. In small cell lung carcinomas, amplification of N-myc, c-myc, or L-myc occurs in various tumors (10, 11), and c-myc amplification has been found in other neoplasms (12-15). It is therefore possible that some neuroblastomas might have am plification of one of these other myc family oncogenes. A second possibility is that some tumors might achieve high levels of myc gene expression by means other than amplification. This phe nomenon has been observed in some Wilms' tumors, for ex ample, which do not have N-myc amplification but may have levels of N-wiyc expression approximating those seen in neu roblastomas with amplification (16). In this paper we present results of the study of 45 primary untreated neuroblastomas for amplification of N-myc, c-myc, and L-myc. In addition, 26 tumors were analyzed for expression of these myc family oncogenes. The data are compared with Received 6/28/89; revised 11/9/89; accepted 11/14/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This investigation received support from a Clinical Investigator Development Award NS01097 from the National Institute of Neurological Disorders and Stroke (B. R. K.) and the Max Kade Foundation (I. S.). clinical outcome of the patients, histological features of the tumors, and DNA histograms obtained by flow cytometry. MATERIALS AND METHODS Tumor Specimens. Tumor samples were obtained either in the oper ating room or the pathology laboratory, immediately frozen in liquid nitrogen, and stored at —¿ 70°C. The neuroblastoma cell lines LAN-1 and IMR-32 were used as controls for N-myc amplification. Pathological Examination. Portions of tumors were fixed and stained by conventional histopathological means. Samples studied for oncogene expression were examined by a pathologist for areas of differentiation and classified according to the criteria of Shimada ( 17). The histological analysis was performed without knowledge of the results of RNA studies. Flow Cytometry. Nuclei were isolated and harvested from 50-/im- thick sections of formalin-fixed paraffin blocks by deparaffinization, rehydration, digestion in pepsin, disaggregation by vortex, digestion in RNase, and filtration through SO-^m nylon mesh (18). Papanicolaou- stained smears were used to assess proportion of tumor nuclei and adequacy of disaggregation. The nuclei were stained with propidium iodide and processed through a Becton-Dickinson fluorescence-acti vated cell sorter flow cytometer. Data on 5000 nuclei were analyzed with an EPICS multiparameter data acquisition and display system single parameter computer. The proliferative compartment of the cells of the tumor samples was estimated as the percentage of cells in S phase plus G2 plus M phase by cytographic pattern. DNA and RNA Analysis. DNA and RNA were prepared as described previously (19). DNA samples were digested with £coRIand quantified by DAPI (4'-6-diamidino-2-phenylindole) fluorescence. Three-Mg sam ples were loaded onto a 0.7% agarose gel, subjected to electrophoresis, and blotted onto Hybond-N (Amersham). Total cellular RNA was quantified by spectrophotometry and 25-^g samples were electropho- resed in formaldehyde-agarose gels and then blotted onto Biotrans (ICN) membranes. Hybridization was carried out sequentially using N-myc (20), c-myc (21), and L-myc (10) DNA probes labeled with 32P by the random primer method (22). RNA blots were also hybridized with a chick a- tubulin sequence (23) as a control for amount of RNA loaded into the gel. Hybridizations were carried out in 50% formamide at 42°Cand washed at high stringency in 0.1 x standard (1 x = 0.15 M NaCl-0.015 M sodium citrate) saline-citrate at 65°C.Autoradiography was done using X-Omat AR film and an intensifying screen. Hybridization intensity was measured using an Ultrascan XL laser densitometer. N-myc copy number was determined by comparison of the hybridization intensity of the N-myc band between the tumor, a normal control sample, and DNA from the neuroblastoma cell line IMR-32, which has 25-fold N-myc amplification (24). A single copy sequence (D13S28) hybridized to the same blots was used to correct for amount of DNA loaded into each lane. For RNA analysis, in order to compare intensity of hybridization between samples on the same blot, and between samples on different blots, densitometric readings were normalized to the intensity of the a-tubulin hybridization for each sample. Statistical Analysis. Levels of N-myc and c-myc expression were tested for correlation with age at diagnosis, stage, site of tumor, Shimada classification, degree of differentiation, and DNA index using the Mann-Whitney nonparametric method (25). Correlation of levels of expression with proliferative compartment was tested using the Spearman rank correlation coefficient (25). 1459 Research. on December 29, 2021. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from