[CANCER RESEARCH54, 2848-2851, June 1, 19941 Advances in Brief Chromosome 9 Allelic Losses and Microsatellite Alterations in Human Bladder Tumors' Irene Orlow, Pilar Lianes, Louis Lacombe, Guido Dalbagni, Victor E. Reuter, and Carlos Cordon-Cardo2 Department of Pathology [I. 0., P. L., V. E. R., C. C-C.] and Urology Service [L L., G. D.], Memorial Sloan-Kettering Cancer Center, New York New York 10021 Abstract Chromosome 9 allelic losses have been reported as a frequent and early eventoccurringinbladdercancer.Ithasbeenpostulatedthata candidate tumor suppressor gene may reside on this chromosome, alterations of which may lead to the development of a subset of superficial bladder tumors.Morerecently,theinvolvementoftwodifferentregionsharboring suppressor loci, one on each of both chromosome 9 arms, has been proposed. We undertook the present study with the objectives of better defining the deleted regions of chromosome 9 in bladder tumors, as well as evaluatingthefrequencyof microsateffitealterationsaffectingcertain loci on this chromosome in urothelial neoplasia. Seventy-three primary bladdertumorswereanalyzedusingasetofhighlypolymorphicmarkers, andresultswerecorrelatedwithpathologicalparametersassociatedwith poor Clinical outcome. We observed that, overall, 77% of the tumors studied showed either loss of heterozygosity for one or more chromosome 9 markers and/or microsteffite abnormalities at chromosome9 loci. De tailed analyses showed that two regions, one on 9p at the interferon cluster, and the other on 9q associated with the q34.1—2bands, had the highest frequencies of allelic losses. Furthermore, T@lesions appeared to present mainly with 9q abnormalities, while T1 tumors displayed a mix tare of aberrant9p and 9q genotypes.Theseobservationsindicatethat loss of heterozygosity of 9p may be associated with the development of superficial tumors with a more aggressive biological behavior or, alter natively, they may be related to early disease progression. In addition, microsatellite alterations were documented in over 40% of amplified cases. Taken together, these data suggest that two different tumor sup pressor gene loci on chromosome 9 are involved as tumorigenic events in bladder cancer and that chromosome 9 microsatellite alterations are frequent events occurring in urothelial neoplasia. Introduction Cytogenetic and molecular genetic analyses of bladder cancer have identified abnormalities in a number of chromosomes which appear to be involved in the development and progression of these tumors (1—4).Loss of heterozygosity of chromosome 9 is a frequent mutation occurring in both superficial and muscle invasive lesions. Allelic losses in 9p (5—7)and 9q (3, 8, 9) have been reported, estimating the area of deletions between 9p22 and 9q34.1. The involvement of two different regions containing suppressor loci in bladder tumors has been recently proposed (10). In addition, somatic instability at mic rosatellite repeats, detected in low stage bladder tumors, has been reported as an early alteration associated with tumorigenic events in such neoplasias (11). The microsatellite instability observed in the hereditary nonpolyposis colorectal cancers (Lynch syndrome) sug gested that these tumors may arise through a mechanism different than Received 2/28/94; accepted 4/21/94. Thecostsof publicationof thisarticleweredefrayedin partbythepaymentof 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 work was in part supported by NCI Grant CA-47538 (to C. C. C.). P. L is the recipient of a Postdoctoral Fellowship from the Spanish Government (P155 93/5052) and an NCI/EORTC Award. L L is the recipient of a Fellowship Award from the Kidney Foundation of Canada. 2 To whom requests for reprints should be addressed, at Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. the inactivation of a tumor suppressor gene (12—14).The inherited basis of these neoplasia appears to be due to an inactivation of DNA damage recognition/repair pathways. However, even in hereditary nonpolyposis colorectal cancers, multiple hits are necessary for the transformed cells to acquire the malignant phenotype. The objectives of this study were to better define the deleted regions of the chromo some 9 in bladder tumors, to delineate the possible role of the loss of heterozygosity (LOH3) in 9p and 9q arms, and to evaluate the exis tence and frequency of microsatellite alterations in urothelial neopla sias. We examined genetic alterations occurring on chromosome 9 using a well-characterized cohort of patients affected with superficial and muscle invasive bladder tumors. We centered our study on the analyses of RFLP, VNTR, and microsatellite markers assigned to both arms of chromosome 9. Materials and Methods Tissues. Seventy-three primary bladder tumors were obtained from rad ical cystectomy samples (n = 64) and transurethral resection specimens (n 9). Tissues were embedded in tissue TEK OCI' cryopreservative (Miles, Inc., Elkhart, IN) and stored frozen at —70°C. Normal tissue was obtained from the same bladder in an area free of tumor or, in 5 cases, from other normal tissue (i.e., peripheral blood or prostate). Five-p@msections were used for hematoxylin and eosin staining. The sections were examined by a pathologist (V. E. R.) to confirm the presence of tumor, evaluation of tissue morphology, and pathology staging. The 73 cases selected showed at least 50% of tumor cells on the sample and were included in the study. All specimens were graded by WHO classification and staged according to the ThM pathological staging system. DNAIsolationandSouthernBlotting.Twentyto30consecutive30-g.irn thick sections were cut from each tissue block. The DNA was isolated, digested, and transferred to nylon membrane as described previously (2, 3). The DNA was fixed to the membrane using a UV linker (UV Stratalink 1800; Stratagene, La Jolla, CA). Probes. The following probes with chromosome map positions, loci, and restriction enzymes were obtained from the ATCC (Rockville, MD) and were used for RFLP analysis in this study: p08782 (9q34—qter; Assgl; HindIII); pabl K2 (9q34; v-nb!; TaqI); pMCOA12 (9q; D9S28; MspI); p04580 (9q34.3; D9SJO; TaqI) VNTR; pEFD126.3 (9q34; D9S7; TaqI) VN'FR; DR6 (9p21 —ter;D9S3; Hind!!!) (provided by Jane Fountain, Massachusetts Insti tate of Technology-Center for Cancer Research, Cambridge, MA); pINIF A3 (9p22; interferon a-2; MspI) (provided by Dr.P.M. Pitha Rove, The John Hopkins University-Oncology Center, Baltimore, MD). Primers. The following primers were used for the present study: IFNA cluster (5'-TGCGCGTFAAGTTAA11XIG1T-3' and 5'-AGTGGGGGlTf C- CACCITAC-3', provided by Dr. Manuel Diaz, Loyola University School of Medicine, Chicago, IL); D9S54 (ATCC); ABL 1 (9q34.1) (ATCC); and Ass (9q34.1) (ATCC). Hybridization, PCR Amplification, and Autoradiography. The probes were labeled to a high specific activity with [a-32P]dCTP by the random oligolabeling technique using the Primed DNA Labeling kit (Boehringer Mannheim, Indianapolis, IN), and membranes were pretreated and hybridized 3 The abbreviations used are: LOH, loss of heterozygosity; RFLP, restriction fragment length polymorphism; VNTR, variable number of tandem repeats; ATCC, American Type Culture Collection; PCR, polymerase chain reaction. 2848 on June 20, 2015. © 1994 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from