[CANCER RESEARCH 63, 455– 457, January 15, 2003] Deletion of Chromosome 3p14.2-p25 Involving the VHL and FHIT Genes in Conventional Renal Cell Carcinoma 1 Farkas Su ¨ko ¨sd, 2 Naoto Kuroda, 2 Tamas Beothe, Amrit Pal Kaur, and Gyula Kovacs 3 Laboratory of Molecular Oncology, Department of Urology, Ruprecht-Karls-University, D-69120 Heidelberg, Germany [F. S., N. K., A. P. K., G. K.]; Department of Pathology, University of Szeged, 6701 Szeged, Hungary [F. S.]; and Department of Urology, University of Pecs, 7643 Pecs, Hungary [T. B.] ABSTRACT Loss of heterozygosity (LOH) at chromosome 3p and inactivation of the VHL gene are associated with the development of conventional renal cell carcinomas (RCCs). Recently, it was suggested that LOH at the FHIT gene at 3p14.2 is an early event in the development of RCC and is characteristic for all types of RCC. We have analyzed 88 conventional, 30 papillary, and 22 chromophobe RCCs for LOH at the VHL and FHIT regions and at other loci on chromosome 3p. A continuous deletion of 3p14.2-p25 harboring the VHL and FHIT genes occurred in 96% of the conventional RCCs but only in 10% of the papillary RCCs and 18% of the chromophobe RCCs. Our data indicate that LOH at chromosome 3p14.2- p25 is specific for conventional RCC and that loss of one allele of both the VHL and FHIT genes occurs in early stage of tumorigenesis. INTRODUCTION Deletion of chromosome 3p is the most frequent genetic change in sporadic and VHL 4 disease-associated conventional RCCs, whereas alteration of this region is rarely seen in papillary and chromophobe RCCs (1). Comprehensive chromosome and DNA studies detected allelic loss at chromosome 3p in 96 –100% of the series of conven- tional RCCs, indicating that this genetic change is a crucial event in tumor development (2, 3). A germ-line mutation of the VHL gene at chromosome 3p25 is associated with the development conventional RCCs (4). Inactivation of the VHL gene by mutation or methylation, however, occurs in only 60 – 65% of the sporadic conventional RCCs, suggesting the existence of another tumor suppressor gene (5, 6). Deletion mapping and chromosomal replacement studies have iden- tified several nonoverlapping regions along the entire chromosome 3p (for review, see Ref. 3). Although some genes were cloned from these regions, none of them has been proved to be a tumor suppressor gene (7–10). The fragile histidine triad (FHIT) gene at the most common fragile site FRA3B at chromosome 3p14.2 has also been suggested to be involved in the genetics of RCCs (7). Recently, Velickovic et al. (11, 12) reported that LOH occurs selectively at the FHIT gene and suggested that LOH at the FHIT locus is an early event in the development of “clear” cell RCC. Moreover, they suggested that LOH at chromosome 3p is a universal phenomenon in all “morphotypes” of kidney cancer. These data are in strong disagreement with our previ- ous chromosomal and LOH studies. To reconcile this contradiction, we have also analyzed the three major types of RCCs for allelic changes at chromosome 3p including the VHL and FHIT regions. MATERIALS AND METHODS Samples and DNA Extraction. Fresh tumor and normal kidney parenchy- mal tissues were obtained by nephrectomy at the Departments of Urology, Hannover Medical School and Heidelberg University, Germany between 1986 and 1988 and between 1993 and 1997, respectively. We have included 11 small conventional RCCs obtained from a patient with VHL disease. This study comprised 43, 15, 36, and 2 conventional RCCs of pathological stage T1–2, T3a, T3b, and T4, respectively. Grade 1, 2 and 3 disease was identified in 38, 38, and 20 cases, respectively. The size of conventional RCCs varied between 0.4 and 14 cm. Six tumors were smaller than 1 cm, four tumors displayed a size between 1 and 2 cm, and seven tumors were between 2 and 3 cm. Altogether, 67 conventional RCCs were smaller than 7 cm in diameter. Thus, all stages of tumor development are represented in this series of con- ventional RCCs. The experiment was approved by the Ethical Committee of the Heidelberg University. A piece of tumor was immediately processed for short-term cultures. Another piece of tumor and corresponding normal kidney tissue were imme- diately snap-frozen in liquid nitrogen and stored at – 80°C, whereas the remaining tissue was fixed in 4% buffered formaldehyde for histological report. The diagnosis was established according to the Heidelberg Classifica- tion (13). Based on the histological analysis, the type of some of the renal cell tumors cannot be determined with complete certainty. Therefore, to establish the diagnosis, we have previously analyzed the chromosomal regions specif- ically involved in the genetics of conventional, papillary, and chromophobe RCCs each by two or three microsatellites (14 –18). Primary cell cultures were established by a combined collagenase and mechanical treatment of tumor tissues as described previously (2). Cells from primary cultures or first passages containing exclusively tumor cells were trypsinized and pelleted. In 6 of the 96 conventional RCCs, 7 of the 30 papillary RCCs, and all cases of chromophobe RCCs, only frozen tissues were available. A frozen tumor sample was placed in a plastic Petri dish, covered with 2 ml of TE9 buffer, and allowed to thaw. The tumor cells were then carefully scraped or pushed out to separate them from stromal tissue under an inverted microscope by a pathologist (G. K.) experienced in this technique. The stromal tissue rests were then discarded. Tumor cells from cell culture or tissues afterward were resuspended in 3–5 ml of TE9 buffer with 1% SDS and 0.2 mg/ml proteinase K and incubated for 5 h at 55°C. DNA was extracted by phenol-chloroform and dissolved in TE buffer after ethanol precipitation. Normal control DNA was extracted from corresponding kidney parenchymal specimens by the same method. The concentration of DNA samples was adjusted to 50 ng/l. Microsatellite Analysis. Microsatellite markers and their approximate po- sitions are shown in Fig. 1. The sequences and location of the markers were obtained from the Genome Sequencing Project. 5 The FHIT gene is mapped between 492 and 758 kb within the sequence segment NT_005607.11, whereas the loci D3S1540, D3S1234, and D3S1300 are mapped to 346, 865, and 1268 kb, respectively. The VHL gene is located between 10.648 and 10.661 kb within the sequence segment NT_005927.11, whereas the flanking microsat- ellite loci D3S1597, D3S1317, and D3S1038 are assigned to 9.831, 10.669, and 10.978 kb, respectively. Matched normal and tumor DNA samples were amplified in 10 l reactions with 50 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 200 M each deoxynucleoside triphosphate, 5 pmol of Cy5-labeled forward primer, 5 pmol of reverse primer, and 0.5 unit of Taq DNA polymerase (Life Technologies, Inc., Eggenstein, Germany). After 2 min of denaturation at 94°C, the PCR mixes were subjected to the following conditions: 40 s at 94°C, 30 s at 55°C, and 40 s at 72°C for 28 cycles followed by a step for 10 min at 72°C in a PTC200 thermal cycler (MJ Received 7/22/02; accepted 11/13/02. 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 Grant Ko 841/8-1 from the German Research Council. 2 Both authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Laboratory of Molecular Oncology, Department of Urology, Ruprecht-Karls University, Im Neuenheimer Feld 325, Room 002, D-69120 Heidelberg, Germany. Phone: 49-6221-566519; Fax: 49-6221- 564634; E-mail: gyula.kovacs@urz.uni-heidelberg.de. 4 The abbreviations used are: VHL, von Hippel-Lindau; RCC, renal cell carcinoma; LOH, loss of heterozygosity; AI, allelic imbalance. 5 http://www.ncbi.nlm.nih.gov/mapview/. 455 Research. on October 17, 2021. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from