[CANCER RESEARCH 55, 5366-5369, November 15, 19951 ABSTRACT Human neuroblastoma cells often have deletions of the distal short arm of chromosome 1 (ip). Earlier studies using chromosome analysis had suggested that the ip deletion is correlated with a poor survival chance for the patient. We have reevaluated this possibility by analyzing 51 neuro blastomas for loss of heterozygosity (LOH) at lp We detected LOH in 32% of the cases. LOH did not correlate with the age of the patients at diagnosis or with tumor stage but was correlated significantly with am plification of the MYCN proto-oncogene. Nine of 10 MYCN-amplifled tumors had deletions in ip (P < 0.001). Survival chances of patients with tumors carrying MYCN amplification together with the deletion at ip were decreased significantly (eight of nine affected patients died) corn pared with a patient group without any of these aberrations (P < 0.001). However, the deletion of ip alone without MYCN amplification was not associated with a poor outcome compared with patients who had neither deletion nor amplification (only two of eight affected patients died; P = 0.803). From these data we conclude that lp deletions are not reliable markers to determine a patient's prognosis. They may, however, identify a subgroup of neuroblastomas in which MYCN is amplified readily, resulting in rapid tumor progression. INTRODUCTION Neuroblastoma is the most frequent type of solid tumor in children, with an annual incidence of 1 per 100,000 in Germany (1). Prognostic parameters for neuroblastoma patients are both tumor stage and age of the patient at the time of diagnosis. Localized tumors (stage I or II), stage IVs tumors, and tumors in children younger than 1 year of age at diagnosis are associated with a good survival prognosis, whereas advanced or metastatic tumors (stage III or IV) or tumors in children older than 1 year of age are correlated with a poor prognosis. Cyto genetic studies (2—4)have identified partial monosomy of ip as the most consistent chromosomal aberration in neuroblastoma cells. On the basis of RFLP markers, the incidence of LOH3 at ip varied in different studies between 22 and 89% (5—9).Most of the deletions overlap at lp36, which indicates that loss of genetic information from this band may contribute to neuroblastoma. Some of these studies (10, 11) have suggested on the basis of cytogenetic analyses a prognostic value of ip deletion with a correlation to shorter survival time, but the clinical significance has remained a matter of controversy. Part of this controversy has been due to the fact that ip deletions have been evaluated together with another genetic alteration, MYCN amplifica tion. The amplification of the gene MYCN has been shown to be correlated strongly with advanced clinical stages and low survival chances (12, 13). Neuroblastoma has been the first tumor in which Received 6/5/95; accepted 9/19/95. 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. I This study was financially supported by the Dr. Mildred Scheel Stiftung Fund, the Deutsche Krebshilfe, the Wilhelm Sander-Stiftung Fund, the Deutsche Forschungsge meinschaft, and the Heidelberg-Mannheim Comprehensive Cancer Center and is inte grated into the frame of the European Communities' concerted action, Molecular Cyto genetics of Solid Tumors (Grant BMH1-CT92-156). 2 Two whom requests for reprints should be addressed. 3 The abbreviation used is: LOH, loss of heterozygosity. gene amplification as a molecular aberration turned out to be of clinical significance (for review, see Ref. 14). In this study, we have analyzed 51 neuroblastomas for both dde tions at ip with RFLP and (CA),, microsatellite markers and the status of MYCN to delineate the significance of ip deletions. Statistical analysis revealed no significant correlation of ip deletions alone with stage, age at diagnosis, or poor outcome, whereas MYCN amplifica tion together with ip deletions was associated with poor survival chances. These data show that only MYCN amplification, not ip deletion, is an independent marker that can be used to assess the prognoses of patients with neuroblastoma. MATERIALS AND METHODS Tumor and Control DNA. Fifty-one neuroblastoma samples were ob tamed from different hospitals cooperating within the framework of the Ger man Neuroblastoma Study Group. Tumors were staged at diagnosis according to the system of Evans et a!. (15). Briefly, stage I and II tumors are localized; stage III tumors are regional; stage IV tumors are widely metastatic; and stage IVs tumors are found in infants younger than 1 year with a high rate of spontaneous regression. Reference DNA was isolated from peripheral blood lymphocytes or from EBV-transformed lymphoblastoid cell lines of the cor responding patient. RFLP Analysis Preparation, digestion, and Southernanalysis were done as described (8). DNA probes to detect LOH on ip were used: pl—79(16), pi—3i(17), LMS1 (18), and YNZ2 (19, 20), which are located on the loci DJV, DISJJ2, DJS7, and D1S57, respectively. MYCN amplification was determined by using the 1.0-kb insert of the plasmid Nb-i (21). (CA),, Microsatellite Analysis. PCR analysis was done as described (22). Four microsatellite loci, D1S243, D1S199, D1S216, and D1S305 from ip, and the locus D1S249 from lq were analyzed (22). Statistical Analysis. The significance of the correlation between ip dde tion and clinical stage or patient's age at diagnosis, respectively, was examined by the Fisher's exact test (23, 24). The survival curves were estimated by the Kaplan-Meier method (25). RESULTS Detection of ip Deletions by RFLP and (CA),, Polymorphism. Fifty-one tumors and the cell line HD-MG-1, established from one of these tumors, were analyzed by PCR amplification of five polymor phic (CA),, repeats. Twenty-one of these tumors were analyzed addi tionally with four RFLP markers. All 51 patients showed a heterozy gous genotype in the reference DNA from peripheral blood lymphocytes at one or more boci on ip. A total of 17 tumors (32%) had ip deletions, which varied in size but seemed to include the distal end of ip (data summarized in Table 1). Correlation between ip Deletions, Tumor Stage, and Patient's Age at Diagnosis. Three patients had tumors according to Evans' stage IVs; another three patients had stage I tumors. Six patients had stage II tumors; nine patients stage III tumors; and 30 patients stage Iv tumors.Twelveof 30 stage IV, 2 of 9 stage III, 2 of 3 stage I, and 1 of 3 stage IVs tumors had deletions in ip. In none of the six stage II tumors was a deletion detected. For the statistical analysis, the prognostically favorable stages I, II, and IVs were combined and compared with the prognostically unfa 5366 The ip Deletion Is Not a Reliable Marker for the Prognosis of Patients with Neuroblastoma1 Manuela Gehring, Frank Berthold, Lutz Edler, Manfred Schwab, and Lukas C. Am1er@ Departments of Cytogenetics (M. G., M. S.. L C. A.] and Biostatistics IL El, German Cancer Research Center, Im Neuenheimer Feld 280, D-69124 Heidelberg, and University Children ‘s Hospital Kdln, Josef-Stelzmann-Strasse 9, D-50924 Köln(F. B.], Germany Research. on September 9, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from