Int. J. Cancer: 77, 494–497 (1998) r 1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer GENETIC ANALYSIS OF 2 CASES OF CLEAR CELL RENAL CANCER IN 2 SISTERS Siebe D. BOS1*, Eva VAN DEN BERG2, Trynie DIJKHUIZEN2, Anke VAN DEN BERG2, Tineke G. DRAAIJERS2 and Han J.A. MENSINK3 1Department of Urology, Medical Centre Alkmaar, Alkmaar, The Netherlands 2Department of Medical Genetics of the University of Groningen, Groningen, The Netherlands 3Department of Urology, University Hospital Groningen, Groningen, The Netherlands Two sisters affected with renal cell carcinoma (RCC) is an extremely rare finding, and may indicate a hereditary pattern or the presence of other predisposing factors. We describe here 2 sisters presenting with clear cell renal cell cancer. Examination for von Hippel-Lindau (VHL)-related features and tuberous sclerosis (M. Bourneville) was negative and both had a normal constitutional karyotype. Cytogenetic analysis of the tumor tissue of both patients showed a translocation involving chromosomes 3 and 5, resulting in loss of 3p sequences and gain of part of 5q. The 5q breakpoints were similar, but the breakpoints at 3p appeared to differ. Allelic imbalance analysis supported our observations. Microsatellite analysis revealed that both sisters inherited different chromosome 3 parental alleles. For chromosome 5, 3 different haplotypes could be deduced, but the chromosome 5 alleles overrepresented in the different tumor tissues were from different parental origin. The development of the 2 RCCs in these 2 sisters thus cannot be explained by the inheritance of a mutated VHL gene located at 3p25, nor by the inheritance of other gene defects at chromosomes 3p or 5q. Although the chance that 2 sisters develop sporadic RCC is very low, in the presented case it is probably coincidental or related to another genetic predisposition. Int. J. Cancer 77:494– 497, 1998. r 1998 Wiley-Liss, Inc. Renal cell cancer (RCC) accounts for 85% of all malignant neoplasms of the kidney. Of all adult malignancies, 2–3% are RCCs. The annual incidence in the general population is 7.5 per 100,000 and there is a male to female ratio of 2:1. Clear cell RCC, responsible for 75% of RCC and originating in the proximal tubule of the nephron, is characterized by deletions of the short arm of chromosome 3 (Yamakawa et al., 1991; Foster et al., 1994; Lubinski et al., 1994; van den Berg et al., 1996). Evidence is accumulating that different regions at 3p are involved in the development of these tumors (van den Berg et al., 1997). Trisomy of 5q is the second most common aberration in clear cell RCC and often arises concurrent with loss of 3p sequences by the formation of an unbalanced t(3;5). Gain of the smallest overlapping region at 5q22 is important in the development of these tumors as well (Kenck et al., 1997). Most cases of RCC are sporadic in origin. Familial or inherited forms of RCC account for 1–2% of RCC cases (Griffin et al., 1967). These are characterized by a frequent bilateral and/or multicentric appearance and an early age of onset. The most common hereditary form of RCC is associated with the dominantly inherited von Hippel Lindau (VHL) disease. The VHL gene, located at 3p25, has been cloned, mutations of which have been detected in VHL patients as well as in sporadic clear cell RCC (Gnarra et al., 1994, 1996; Shuin et al., 1994; Foster et al., 1994; Latif et al., 1993). RCC families with constitutionally balanced translocations between chromosomes 3 and 6 or 8 have also been described (Cohen et al., 1979; Kovacs et al., 1989). The incidence of ‘‘pure’’ familial RCC with no VHL or other predisposing genetic syndromes is extremely low. Until now, only 39 family aggregates of RCC have been reported (Yao and Shuin, 1995), but the true incidence probably should be higher, due to the fact that not all cases are reported. Environmental factors contributing to the development of RCC are poorly understood but smoking, particularly in men, and obesity, especially in women, have been related to RCC development (Mellemgaard et al., 1995; McLaughlin et al., 1995). Occupational exposure to various hydrocarbons and asbestos also increases the risk of having this disease (Mandel et al., 1995). A higher incidence of RCC is also found in patients with tuberous sclerosis, acquired cystic kidney disease and endstage renal disease. We describe here 2 sisters presenting with clear cell RCC. Both patients were examined for VHL-related features and tuberous sclerosis. Genetic analysis was performed on tumor and normal kidney tissue of both patients in an attempt to find a hereditary cause. MATERIAL AND METHODS A 51-year-old woman, without antecedents of smoking or obesity, was referred to our outpatient department with a right kidney tumor. The tumor was found during an abdominal hysterectomy for menorrhagia and dysmenorrhoea due to adenomyosis. The kidney tumor was asymptomatic. Laboratory findings were normal. A radical transabdominal nephrectomy with regional lymph node dissection was performed and reconvalescence was uneventful. On pathological examination, a RCC of the clear cell type was found with one unifocal lesion, stage pT2N0M0 (Harmen and Sobin, 1992). After a follow-up of 60 months, there is no evidence of disease. Her sister, 59 years old, also non-smoking and not obese, was evaluated for nausea and vomiting and, on ultrasound, a tumor in the left kidney was found. Laboratory findings were normal. We performed a transabdominal nephrectomy with regional lymph node dissection. Reconvalescence was uneventful. A unifocal RCC of the clear cell type was found on pathological investigation, stage pT2N0M0. After a follow-up of 16 months, there is no evidence of disease. Because of the rarity of such spontaneous development of RCC in 2 sisters, both were examined for features of VHL disease, i.e., retinal hemangiomas, cerebellar, medullary or spinal hemangioblastomas, and pheochromocytoma. Neither had any of these features. Screening for signs of tuberous sclerosis was also negative. No further cases of RCC, up to the third degree of relatives, or other VHL-associated tumors, were found in this family. Fresh representative samples of normal and tumor tissue of both patients were submitted to cytogenetic investigation. One part of the tissue was cultured for 5–7 days in RPMI 1640 supplemented with fetal calf serum (FCS) (16%), glutamine and antibiotics. The cultures were harvested and chromosome preparations were made according to standard cytogenetic techniques. The chromosomes were G-banded using pancreatin, and karyotypes were described according to the ISCN ’95 guidelines for cancer cytogenetics (Mitelman, 1995). Allelic imbalance analysis was carried out as described previously (van den Berg et al., 1996). Primer sequences and polymer*Correspondence to: Department of Urology, Medical Centre Alkmaar, Wilhelminalaan 12, 1815 JD Alkmaar, The Netherlands. Fax: (31) 72-5482605. E-mail: [email protected] Received 1 October 1997; Revised 13 February 1998 GENETICS OF CLEAR CELL RCC ase chain reaction (PCR) conditions were retrieved from the Genome Data Base, Johns Hopkins University, Baltimore, MD. RESULTS Cytogenetic analysis of the tumor tissue of both patients was carried out. Case 1 revealed a 44,47,XX,1X, del(1)(p36),der(3)t(3; 5)(p11;q22)[cp3]/82,84,idemx2,-X,-8,-16[cp3] chromosomal pattern (Fig. 1). The composite karyotype of case 2 was 46,47,XX, der(3)t(3;5)(p12;q22),1der(7)t(7;10)(p13;p11),add(13)(q34)[cp3] (Fig. 2). Both patients had a normal 46,XX constitution. The RCC of case 1 showed allelic imbalance for 9 informative markers, mapping in the 3p11-pter region (Table I). Retention of alleles was detected for D3S1101 mapping more proximal in 3p11. The markers D3S1317, D3S1007, D3S1029, D3S1480, D3S1210, D3S1511, D3S1577, D3S1254, D3S276, D3S1595 and D3S1251 were all not informative. The RCC of case 2 showed allelic imbalance for 7 informative markers, mapping in the 3p12-pter region (Table I). Retention of alleles was detected for 2 markers, D3S1552 and D3S1101, mapping more proximal. The same 11 markers, not informative for case 1, and D3S1217, were not informative in case 2. Cytogenetic analysis indicated the presence of one apparently normal copy of chromosome 3 and one derivative chromosome 3 with loss of the distal part. This indicated that the allelic losses as detected upon microsatellite analysis all involved the same parental allele. Haplotypes thus can be deduced based on these loss of heterozygosity (LOH) analyses; alleles that were retained in the tumor most likely originated from one parental allele and alleles that were lost in the tumor most likely originated from the other parental allele. The haplotypes for both sisters are shown in Table II. 495 Cytogenetic analysis has indicated the presence of an extra copy of the q-arm of chromosome 5 for both RCC samples. Allelic imbalance analysis using 3 5q markers confirmed the cytogenetically observed gain (Table I). These results were used to deduce the chromosome 5 haplotypes assuming that the overrepresented marker alleles originated from one parental allele (Table II). DISCUSSION RCC is a heterogeneous group of tumors that constitutes 2–3% of all cancers in adults. The chance that 2 sisters develop RCC without hereditary or other predisposing factors is therefore extremely rare. We encountered 2 sisters affected with clear cell RCC. Neither the patients nor their family history revealed signs or symptoms of VHL disease or tuberous sclerosis. In an attempt to find evidence for a hereditary predisposition we performed a genetic analysis on normal and tumor tissue of both patients. Hereditary RCC is associated with mutations of the VHL gene or with the presence of a constitutionally balanced translocation involving chromosome 3. The constitutional karyotype of our 2 patients revealed a normal 46,XX chromosomal pattern. Karyotypes of tumor cells of both patients showed a t(3;5). The breakpoints at chromosome 5 were similar but the breakpoints at chromosome 3 slightly differed. For the latter, allelic imbalance analysis with markers mapping all over the p-arm indicated loss of the distal part of 3p in both tumors. In case 1, loss of markers was found in 3p11-pter and in case 2, loss of markers was found in 3p12-pter, confirming the cytogenetically observed differences in chromosome 3 breakpoints. To investigate whether or not both sisters inherited the same chromosome 3 and/or 5 parental alleles, including a putative mutation, allelic imbalance analysis for both chromosomes was FIGURE 1 – Representative karyotype of case 1 showing a 47,XX,1X,del(1)(p36),der(3)t(3;5)(p11;q22) chromosomal pattern. Structural rearrangements are indicated by arrows. BOS ET AL. 496 FIGURE 2 – Representative karyotype of case 2 showing a 47,XX,der(3)t(3;5)(p12;q22),1der(7)t(7;10)(p13;p11),add(13)(q34) chromosomal pattern. Structural rearrangements are indicated by arrows. TABLE I – RESULTS OF CHROMOSOME 3- AND 5-SPECIFIC MICROSATELLITE ANALYSIS1 Chromosome band 3p25 3p21 3p14 3p13 3p12 3p11 5q22-q23 5q31.1-33.3 5q33.2-33.3 Marker D3S1038 D3S643 D3S1481 D3S1233 D3S1217 D3S1284 D3S1274 D3S1776 D3S1552 D3S1101 D5S421 D5S210 C5F1R Case 1 1 1 1 1 1 1 1 1 2 2 1 1 1 (1*/2) (1/2*) (2*/3) (2*/3) (1/2*) (1/2*) (1*/2) (1*/2) (1/2) (1/2) (1*/3) (2*/3) (1*/2) Case 2 1 1 1 1 1 1 1 1 1 2 1 1 1 (1/2*) (1*/2) (1*/2) (1*/3) (n.i.) (1*/2) (1/2*) (1/2*) (1/2*) (1/2) (2/3*) (1/3*) (1/2*) 1Markers have been placed in their most likely order according to published mapping data (for chromosome 3, see Naylor et al., 1996; chromosome 5 markers are according to the Genome Data Base, Johns Hopkins University, Baltimore, MD). 1: allelic imbalance; 2: no allelic imbalance; *: alleles under/overrepresented in the tumor; n.i.: not interpretable. performed. Haplotypes were determined, assuming that imbalances of marker alleles were confined to the same parental chromosomes. For chromosome 3, 4 different haplotypes were observed, indicating that both sisters inherited different paternal and maternal chromosome 3 alleles. It is thus very unlikely that these tumors developed as part of a hereditary VHL disease or other gene defects at 3p. Surprisingly, taking into account the reported heterozygosity frequencies for the 3p markers used in our study, for both sisters, about half of the markers were homozygous for the same allele. In TABLE II – DEDUCED CHROMOSOME 3 AND 5 HAPLOTYPES FOR THE TWO SISTERS1 Marker D3S1038 D3S643 D3S1481 D3S1233 D3S1217 D3S1284 D3S1274 D3S1776 D3S1552 D5S421 D5S210 C5F1R 1*: Chromosome band 3p25 3p21 3p14 3p13 3p12 5q22-q23 5q31.1-5q33.3 5q33.2-5q33.3 Case 1 Case 2 Allele 1* Allele 2 Allele 1* Allele 2 1 2 2 2 2 2 1 1 2 1 3 3 1 1 2 2 1 2 1 3 3 2 2 1 1 3 1 1 2 2 2 3 3 2 1 2 2 1 1 2 1 1 1 2 1 1 alleles over/underrepresented in the tumor. addition, a higher degree of resemblance between the marker alleles was observed than may be expected. Genealogical investigation, however, failed to reveal consanguinity for at least 5 generations. For chromosome 5, 3 different haplotypes could be observed, indicating that both sisters had inherited one different allele and one identical allele (allele 2 of case 1 and allele 1 of case 2). The chromosome 5 alleles overrepresented in the tumors were of different parental origin. Therefore, most likely, chromosome 5q also does not carry a heritable mutation responsible for the development of clear cell RCC in these patients. Taken together, the finding of clear cell RCC in the present 2 sisters is probably coincidental. Furthermore, neither the 2 patients, nor their family history, revealed signs or symptoms of VHL GENETICS OF CLEAR CELL RCC disease or tuberous sclerosis. There were no known environmental risk factors such as smoking or obesity in either sister. We cannot, however, rule out the presence of other environmental risk factors 497 for these 2 sisters, who are living in the same rural area, since the knowledge of the influence of environmental factors on the development of (clear cell) RCC is limited. REFERENCES COHEN, A.J., LI, F.P., BERG, S., MARCHETTO, D.J., TSAI, S., JACOBS, S.C. and BROWN, R.S., Hereditary renal-cell carcinoma associated with a chromosomal translocation. N. Eng. J. Med., 301, 592–595 (1979). FOSTER, K., CROSSEY, P.A., CAIRNS, P., HETHERINGTON, J.W., RICHARDS, F.M., JONES, M.H., BENTLEY, E., AFFARA, N.A., FERGUSON-SMITH, M.A. and MAHER, E.R., Molecular genetic investigation of sporadic renal cell carcinoma: analysis of allele loss on chromosome 3p, 5q, 11p, 17 and 22. Brit. J. Cancer, 69, 230–234 (1994). GNARRA, J.R. and 19 OTHERS, Molecular cloning of the von Hippel-Lindau tumor suppressor gene and its role in renal carcinoma. Biochim. biophys. Acta, 1242, 201–210 (1996). GNARRA, J.R. and 23 OTHERS, Mutations of the VHL tumour suppressor gene in renal carcinoma. Nature (Genet.), 7, 85–90 (1994). GRIFFIN, J.P., HUGHES, G.V. and PEELING, W.B., A survey of the familial incidence of the adenocarcinoma of the kidney. Brit. J. Urol., 9, 63–66 (1967). HARMEN, M. and SOBIN, L. (eds.), TNM classification of malignant tumors. UICC, Geneva (1992). KENCK, C., BUGERT, P., WILHELM, M. and KOVACS, G., Duplication of an approximately 1.5 Mb DNA segment at chromosome 5q22 indicates the locus of a new tumour gene in nonpapillary renal cell carcinomas. Oncogene, 14, 1093–1098 (1997). KOVACS, G., BRUSA, P. and DE RIESE, W., Tissue-specific expression of a constitutional 3;6 translocation: development of multiple bilateral renal-cell carcinomas. Int. J. Cancer, 43, 422–427 (1989). LATIF, F. and 32 OTHERS, Identification of the von Hippel-Lindau disease tumor suppressor gene. Science, 260, 1317–1320 (1993). LUBINSKI, J., HADACZEK, P., PODOLSKI, J., TOLOCZKO, A., SIKORSKI, A., MCCUE, P., DRUCK, T. and HUEBNER, K., Common regions of deletions in chromosome regions 3p12 and 3p14.2 in primary clear cell renal carcinomas. Cancer Res., 54, 3710–3713 (1994). MANDEL, J.S., MCLAUGHLIN, J.K., SCHLEHOFER, B., MELLEMGAARD, A., HELMERT, U., LINDBLAD, P., MCCREDIE, M. and ADAMI, H.O., International renal-cell cancer study. IV. Occupation. Int. J. Cancer, 61, 601–605 (1995). MCLAUGHLIN, J.K., LINDBLAD, P., MELLEMGAARD, A., MCCREDIE, M., MANDEL, J.S., SCHLEHOFER, B., POMMER, W. and ADAMI, H.O., International renal-cell cancer study. I. Tobacco use. Int. J. Cancer, 60, 194–198 (1995). MELLEMGAARD, A., LINDBLAD, P., SCHLEHOFER, B., BERGSTROM, R., MANDEL, J.S., MCCREDIE, M., MCLAUGHLIN, J.K., NIWA, S., ODAKA, N. and POMMER, W., International renal-cell cancer study. III. Role of weight, height, physical activity, and use of amphetamines. Int. J. Cancer, 60, 350–354 (1995). MITELMAN, F., Guidelines for cancer cytogenetics, supplement to an international system for human cytogenetic nomenclature. pp. 1–104. Karger, Basel (1995). NAYLOR, S.L. and 32 OTHERS, Report on the sixth international workshop on human chromosome 3 mapping 1995. Cytogenet. Cell Genet., 72, 255–270 (1996). SHUIN, T., KONDO, K., TOREGOE, S., KISHIDA, T., KUBOTA, M., NAGASHIMA, Y., KITAMURA, H., LATIF, F., ZBAR, B., LERMAN, M.I. and YAO, M., Frequent somatic mutations and loss of heterozygosity of the Von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res., 54, 2852–2855 (1994). VAN DEN BERG, A., DIJKHUIZEN, T., DRAAIJERS, T.G., HULSBEEK, M.M.F., MAHER, E.R., VAN DEN BERG, E., STÖRKEL, S. and BUYS, C.H.C.M., Analysis of multiple renal cell adenomas and carcinomas suggests allelic loss at 3p21 to be a prerequisite for malignant development. Genes Chromosomes Cancer, 19, 228–232 (1997). VAN DEN BERG, A., HULSBEEK, M.M.F., DE JONG, D., KOK, K., VELDHUIS, P.M.J.F., ROCHE, J. and BUYS, C.H.C.M., Major role for a 3p21 region and lack of involvement of the t(3;8) breakpoint region in the development of renal cell carcinoma suggested by loss of heterozygosity analysis. Genes Chromosomes Cancer, 15, 64–72 (1996). YAMAKAWA, K., MORITA, R., TAKAHASHI, E., HORI, T., ISHIKAWA, J. and NAKAMURA, Y., A detailed deletion mapping of the short arm of chromosome 3 in sporadic renal cell carcinoma. Cancer Res., 51, 4707–4711 (1991). YAO, M. and SHUIN, T., Familial renal cell carcinoma: review of recent molecular genetics. Int. J. Urol., 2, 61–70 (1995).