[CANCER RESEARCH 48, 4307-4311, August 1, 1988] Indistinguishable Patterns of Protooncogene Expression in Two Distinct but Closely Related Tumors: Ewing's Sarcoma and Neuroepithelioma Catherine McKeon, Carol J. Thiele, Robert A. Ross, Michael Kwan, Timothy J. Triche, James S. Miser, and Mark A. Israel1 Pediatrie Branch, Division of Cancer Treatment [C.M., C.J.T., M.K., J.S.M., MA.l.J; the Laboratory of Pathology, Division of Cancer Biology and Diagnosis, [T.J.TJ National Cancer Institute, NIH, Bethesda, Maryland 20892; and the Department of Biology, Fordham University, New York, New York 10023[Rjl.R.] ABSTRACT Genetic characterization of human tumors promises new insights of biological importance and clinical relevance. We have found that two solid tumors, peripheral neuroepithelioma and Ewing's sarcoma of bone, which share a common cytogenetic rearrangement, are characterized by an indistinguishable and highly reproducible pattern of protooncogene expression, c-myc, N-myc, c-myb, and c-mll/raf-1 are all expressed at similar levels in these tumors, c-fes and c-sis expression was not detected in any specimens of either tumor. In contrast, the protooncogene ort.v-1, located near the breakpoint of the chromosomal translocation in these tumors, is variable in its expression. We also detected high levels of choline acetyltransferase in these tumors, which suggests a common neural origin. Since it is likely that the clinical behavior and therapeutic responsiveness of tumors relate closely to their biological and genetic features, the pattern of protooncogene expression of individual tumors may provide a novel basis for their characterization. INTRODUCTION The practice of medicine is anchored in the classification of disease into homogeneous disorders that make possible individ ualized approaches to therapeutic intervention and prognosti cation. It is ironic that while cancer is perhaps the most heter ogeneous of diseases, we can rarely discern homogeneous bio logical entities among the various solid tumors. Furthermore, current tumor nosologies are based almost exclusively on the appearance of tumor tissues and the cytological features of tumor cells. Such an approach to classification greatly limits the possibility of recognizing tumors that may be biologically and even pathologically closely related if they cannot be recog nized as arising in the same organ or in closely related tissues. We investigated the possibility that patterns of protoonco gene expression may identify genetically and biologically related malignancies. Protooncogenes are the cellular homologues of viral transforming genes and are differentially expressed in a highly restricted manner during normal growth and develop ment (for review, see Refs. 1 and 2). The expression of a protooncogene in a specific tissue seems to be determined by a number of cellular characteristics, such as tissue lineage, mat urational stage, and growth rate (1, 3, 4). Since these same features are important in determining the behavior of malignant tumors, the pattern of protooncogene expression in individual tumors might reflect clinically important features and identify biologically homogeneous tumor groups. In pursuit of this idea, we investigated the pattern of pro tooncogene expression in two histopathologically distinct ma lignancies, Ewing's sarcoma and neuroepithelioma, that are both characterized by the cytogenetic rearrangement t(ll;22) (q24;ql2). Ewing's sarcoma is the second most common child- Received11/4/87;revised3/11/88;accepted4/26/88. 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. 1To whom requests for reprints should be addressed, at Building 10, Room 13N240, Pediatrie Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD 20892. hood tumor of bone. Since the first description of a chromo somal rearrangement in this tumor in 1983, this translocation or a complex variant has been recognized as a consistent cyto genetic finding in Ewing's sarcoma tumors (5-9). Neuroepithe lioma is a rare tumor of the peripheral nervous system (S). A t(l 1;22) (q24;ql2) chromosomal translocation is also found in this tumor (9, 10). Despite intensive cytogenetic and molecular analyses of the chromosomal rearrangement in these two tu mors, it has not been possible to distinguish them from one another (11-14). In contrast, histológica!, ultrastructural, and immunohistochemical studies reveal many markers of neural differentiation in neuroepithelioma but not in Ewing's sarcoma (15-17). Indeed, the cell of origin of Ewing's sarcoma has been a topic of considerable speculation largely because of the ab sence of recognizable markers of tissue-specific differentiation in this tumor. Despite their histológica! and cytological differences, we found that the pattern of protooncogene expression in Ewing's sarcoma and neuroepithelioma are highly predictable and in distinguishable. We also found that these two tumors share at least one common neuron-specific marker, namely high levels of choline acetyltransferase activity. These similarities suggest that Ewing's sarcoma and neuroepithelioma arise from cells of a common lineage and that the determination of patterns of protooncogene expression may be a useful approach to the characterization and classification of tumors. MATERIALS AND METHODS Tissues and Cell Lines. Tissue samples were obtained either during surgery or at autopsy from tumors diagnosed as either ES2 of bone or NE on the basis of standard clinical and pathological evaluation. Only tumor samples with a classic ES or NE pathology were used for this study. Extraosseous ES tumors were excluded from this analysis, since they do not show a t(ll;22) and may represent a separate entity (9). Most specimens were frozen in liquid nitrogen and stored at — 70°C before isolation of RNA. Specimens of normal human brain were obtained in the same manner and were studied to determine protoon cogene patterns in a normal neuronal tissue. Most tumor samples were karyotyped by examination of material cultured overnight. All tumor specimens that could be evaluated had demonstrable t(ll;22) marker chromosomes (see Table 1). Tissue culture lines were started from tumor specimens by dispersing cells in 0.02% trypsin and culturing in RPMI 1640 medium supplemented with 15% fetal calf serum. (The NE lines N1000, N1008, N1016, N1043, N1046, and TC32 and the ES lines N1001, N1002, N1050, TC71, TC106, 5838,6647, and A4573 are continuous cell lines established at NIH; CHP100 was obtained from Children's Hospital, Philadelphia, PA and SK-N-MC from Dr. June Biedler, Sloan-Kettering Memorial Hospital, New York, NY.) We used all the ES and NE tissue culture lines available to us which had been karyotyped (9)3 (see Table 1). In addition, fibroblasts and a lymphocyte (IM-9) cell line were studied for mRNA expression. Neurotransmitter Enzyme Assays. Tissue culture cells were grown in 2 The abbreviations used are: ES, Ewing's sarcoma; NE, neuroepithelioma; TH, tyrosine hydroxylase; CAT, choline acetyltransferase; DBH, dopamine ,¡ hydroxylase; poly(A)*, polyadenylated; kb, kilobase. 3J. Whang-Peng and M. A. Israel, unpublished results. 4307 Research. on September 27, 2021. © 1988 American Association for Cancer cancerres.aacrjournals.org Downloaded from