Identification of Novel Regions of Deletion in Familial Wilms' Tumor by Comparative Genomic Hybridization1

Wilms' tumor, an embryonic renal neoplasm diagnosed primarily in young children, can occur in either a noninheritable (sporadic) or a familial form, with the latter presenting earlier and more often at bilateral sites. Although familial Wilms' tumor is thought to develop through inherited and acquired mutational inactivation of the two alÃ-elesof pre disposing tumor suppressor genes, only a small percentage of cases can be accounted for by mutations affecting the H77 gene or linkage to the Beckwith-Weidemann syndrome of the BWS region on the short arm of chromosome 11. To find chromosomal regions that might contain genes important in the development of this disease, we used comparative genomic hybridization to analyze tumor specimens from familial cases for chromosomal regions that were consistently lost. Although inherited le sions of tumor suppressors are most often inactivating point mutations, accompanying somatic lesions in the malignant clones are often chromo somal deletions; therefore, consensus regions of loss in familial tumors are likely to harbor genes linked to familial predisposition. There were exten sive genomic aberrations among the eight familial cases studied, with an average of 6.5 changes/tumor (range, 0-22). The most consistent findings with likely biological relevance were deletions of chromosomes 4 (consen sus, 4q21-qter), 9 (consensus, 9p21-pter), 20p, and 3 (consensus, 3ql2q21). These regions have not been previously implicated in Wilms' tumor and may harbor novel genes that could aid attempts to understand the familial predisposition as well as the development and progression of these tumors. INTRODUCTION Wilms' tumor affects approximately 1 of every 10,000 children worldwide, making it the most common renal neoplasm of childhood (1, 2). Like retinoblastoma, it can occur in either a sporadic (nonhereditary) or familial form. Although the latter is estimated to comprise only 1.5% of all Wilms' tumors, the percentage of individuals carry ing a germline mutation in a gene predisposing to this tumor is probably much higher (3, 4). The molecular pathogenesis of familial Wilms' tumor remains unclear, but it is thought to follow a "two-hit" model, in which an initial mutation affects a tumor suppressor locus in the germline, but does not become transforming until a second (somatic) mutation inactivates the wild-type alÃ-elein a target progen itor cell (5). In contrast, sporadic Wilms' tumors may develop through mechanisms involving two sequential somatic mutations that affect tumor suppressor loci (5). Recent findings have implicated more than one tumor susceptibility locus in the genesis of Wilms' tumor. Overall, 10-15% of patients have mutations in the WT1 gene on chromosome 11, band pi3 (6-8). Some of these mutations occur somatically, whereas others are inherReceived 4/1/96; accepted 6/18/96. 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. 1Supported in part by Grant CA 23099 and Cancer Center CORE Grant CA 21765 from the National Cancer Institute. NIH. and by the American Lebanese Syrian Associ ated Charities. St. Jude Children's Research Hospital. 2 To whom requests for reprints should be addressed, at St. Jude Children's Research Hospital, Department of Experimental Oncology. 332 North Lauderdale. Memphis, TN 38105. Phone: (901 ) 495-3500; Fax: (901) 495-2032. ited or are new germline alterations. Deletions that affect WTJ, generally acquired as new germline lesions, often result in the WAGR syndrome (Wilms' tumor, aniridia, genitourinary malformations, and mental retardation; Ref. 9). Dominantly acting point mutations of this locus result in the Denys-Drash syndrome (nephropathy, Wilms' tumor, and genital anomalies; Refs. 10 and 11). A second gene involved in the pathogenesis of Wilms' tumor resides at Ilpl5.5(12), near or coincident with the locus for the Beckwith-Weidemann syn drome, characterized by macroglossia, visceromegaly, hypoglycemia, and abdominal wall defects. Most of these cases are sporadic, but approximately 15% are familial or occur in association with recog nized chromosomal abnormalities (13). LOH3 studies have recently implicated acquired deletions of specific regions on chromosome arms Ip and 16q in the development of sporadic Wilms' tumor lacking associated congenital anomalies (14). Both of these regions appear to carry prognostic significance (14), but linkage was excluded with these regions in families with Wilms' tumor (15). Similarly, gene analysis and linkage studies indicate that only a small percentage of familial cases can be attributed to mutations of WT1 or the BeckwithWeidemann syndrome-associated genes on the short arm of chromo some 11 (16, 17). Thus, other chromosomal regions are likely to harbor genes that are important in the familial transmission of this disease (3). To gain further insight into the pathogenesis of familial Wilms' tumor, we have used CGH to analyze the cellular DNA of eight tumors with documented familial origins. This method, based on the detection of differences between tumonnormal fluorescence intensity ratios, has distinct advantages over LOH because it allows one to analyze the entire genome (rather than a single chromosomal site per informative probe) for DNA sequence losses and gains. Regions frequently identified with decreased copy number are likely to harbor tumor suppressor genes, whereas regions with clearly increased copy numbers indicate either chromosomal duplication or gene amplifica tion, commensurate with overexpression of a cellular proto-oncogene (18). Our study is based on the fact that inherited mutations of one alÃ-eleof a tumor suppressor are frequently accompanied by deletion or chromosomal loss of the other alÃ-eleas the somatic tumor-specific inactivating event, which will be detectable using CGH analysis. Here, we describe the detection of several regions of nonrandom chromosomal loss that may harbor novel antioncogenes with critical roles in familial Wilms' tumor pathogenesis. MATERIALS AND METHODS Tumor Specimens. Eight familial Wilms' tumor specimens (three stage V, one stage IV, one stage III, one stage II, and two unknown stages) were obtained from St. Jude Children's Research Hospital and the Pediatrie Oncol ogy Group (Table 1). All were classified as having favorable histology. The samples were collected at diagnosis, before administration of chemotherapy or radiation. DNA was extracted from the frozen tumor tissue according to -' The abbreviations used are: LOH, loss of heterozygosity; CGH, comparative genomic hybridization; FISH, fluorescence in situ hybridization; DAPI, 4',6,diamidino2-phenylindole.