[CANCER RESEARCH 62, 6481– 6484, November 15, 2002] Advances in Brief No Evidence for the Presence of an Imprinted Neuroblastoma Suppressor Gene within Chromosome Sub-Band 1p36.3 1 Michael D. Hogarty, 2 Cynthia L. Winter, Xueyuan Liu, Chun Guo, Peter S. White, A. Thomas Look, Garrett M. Brodeur, and John M. Maris Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318 [M. D. H., C. L. W., X. L., C. G., P. S. W., G. M. B., J. M. M.]; Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [M. D. H., P. S. W., G. M. B., J. M. M.]; and Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, Massachusetts 02115 [A. T. L.] Abstract Deletion of the distal short arm of chromosome 1 occurs in 35% of primary neuroblastomas (NBs). These deletions tend to be large and extend to the telomere, but a common region within sub-band 1p36.3 is consistently lost. Despite intensive investigation, no candidate tumor sup- pressor gene within this region has been shown to undergo tumor-specific mutation consistent with biallelic inactivation. In addition, initial studies demonstrated preferential loss of the maternally inherited 1p homologue in NBs with 1p loss of heterozygosity (LOH) without MYCN amplification. This has led to the widely accepted hypothesis that a genomically im- printed NB suppressor gene is the target of 1p deletion in this subset. To test this hypothesis we have studied 293 primary NBs for LOH within 1p36.3 and determined the parental origin of the deleted 1p homologue. LOH within 1p36.3 was demonstrated in 55 NBs (19%). Of these, 29 occurred in tumors without MYCN amplification: 13 had deletion of the maternally inherited 1p, whereas 16 had deletion of the paternally inher- ited 1p (P  0.58). These data strongly refute a parent-of-origin effect for 1p deletions in NB and exclude the existence of an imprinted NB suppres- sor locus in this region. Introduction Cytogeneticists first identified alterations of the distal short arm of chromosome 1 (1p) in primary NBs and tumor-derived cell lines 20 years ago (1). Molecular analyses have subsequently confirmed and extended these initial findings with uniform results. Deletions within 1p occur in 35% of primary NBs, and these deletions tend to be large and terminal, often extending from 1p32 to the telomere (2– 4). Interstitial deletions are infrequent, and a SRO 3 has been identified consistently within 1p36.3 (5–7). Constitutional genomic alterations within this region have also been characterized in patients with NB, and may predispose to the genesis of this tumor (8, 9). Finally, transfer of 1p chromosomal material into a NB-derived cell line has induced phenotypic changes consistent with loss of malignant potential and reversion to tumorigenicity correlated with loss of the transferred 1p homologue (10). Taken together, these data strongly support the presence of one or more NB suppressor genes within chromosome sub-band 1p36.3. However, tumor-specific biallelic in- activation has not been demonstrated for any candidate 1p36 suppres- sor gene in NB despite intensive investigation. Thus, alternate models of tumor-suppressor inactivation such as genomic imprinting, somatic epigenetic silencing, haploinsufficiency, and dominant inhibitor effect have been postulated. Reports documenting preferential deletion of the maternally inherited 1p in NBs without MYCN amplification led to the hypothesis that an imprinted suppressor gene is targeted for disruption in this subset (11, 12). Deletion of the normally expressed homologue (inherited through the maternal genome in this instance) of an imprinted suppressor gene would result in biallelic inactivation because the paternally inherited homologue would be epigenetically silenced. Additional support arose from the observation that 1p dele- tions in NBs without MYCN amplification tend to be smaller and define a more telomeric SRO than those in NBs with MYCN ampli- fication (12). These findings have supported speculation that a more telomeric 1p NB suppressor gene that is regulated by genomic im- printing is targeted for disruption in NBs without MYCN amplifica- tion, whereas a more proximal nonimprinted suppressor gene is tar- geted by deletions in MYCN-amplified NBs. To test this hypothesis we have determined the parental origin of the deleted 1p allele in a large series of NBs, both with and without MYCN amplification, and show that no parent-of-origin effect for 1p36 deletion is apparent to support the presence of an imprinted NB suppressor gene in this region. Materials and Methods Patients and Tissue Samples. A total of 293 NB cases were ascertained in which tumor tissue, constitutional tissue (blood or bone marrow without tumor infiltration), and blood from one or both parents were available for DNA extraction and PCR amplification. Patients had been registered and tissue specimens banked through the following United States cooperative group biology studies: Pediatric Oncology Group-9047, CCG-B947, and CCG-B974. Eligibility criteria required that tumor samples contain 80% NB cells. Tumor MYCN status was determined by fluorescence in situ hybridization with amplification defined as 10 MYCN signals within a tumor cell and was performed in the cooperative group reference laboratory as described previ- ously (13). Tumor histopathology was classified according to the system of Shimada et al. (14), and stage was assigned using the INSS (15). DNA was extracted from tumors, blood, and bone marrow samples using Qiagen anion- exchange chromatography (Qiagen Inc., Valencia, CA). All of the patients and/or legal guardians signed informed consent, and this study was approved by the Children’s Hospital of Philadelphia Institutional Review Board. LOH and Parental Origin of Deleted Allele Determination Studies. LOH was assessed after PCR amplification of matched tumor/constitutional DNA pairs. Fluorescence-labeled primers for the highly polymorphic markers D1S468, D1S2660, and D1S214 (which map within 1p36.3) were obtained from ABI and used in a triplex PCR reaction to screen for LOH. Briefly, PCR was performed in a 15 l volume with a final concentration of 1 M for each of the primers, 1 mM of each deoxynucleoside triphosphate, 2.5 mM MgCl 2 , 1 GeneAmp PCR Buffer II, 0.6 units AmpliTaq Gold (Perkin-Elmer Applied Received 9/3/02; accepted 9/27/02. 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. 1 Supported by NIH Grants RO1CA78745 and U1078966 (to J. M. M.), and by a Career Development Award from the American Society of Clinical Oncology (to M. D. H.), The Richard and Sheila Sanford Chair in Pediatric Oncology, and grant U01-CA30969 (to the Children’s Oncology Group). 2 To whom requests for reprints should be addressed, at Children’s Hospital of Philadelphia, Division of Oncology, 9 North ARC, 3615 Civic Center Boulevard, Phila- delphia, PA 19104-4318. E-mail: hogartym@email.chop.edu. 3 The abbreviations used are: SRO, smallest region of overlapping deletion; NB, neuroblastoma; INPC, International Neuroblastoma Pathology Committee; INSS, Inter- national Neuroblastoma Staging System; LOH, loss of heterozygosity; CCG, Children’s Cancer Group. 6481 Research. on March 7, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from