Cancer Genetics and Cytogenetics 128 (2001) 161–163

0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0165-4608(01)00413-7

Short communication

Allelic loss at 10q23.3 but lack of mutation of

PTEN/MMAC1

in chromophobe renal cell carcinoma

Farkas Sükösd, Boris Digon, Joachim Fischer, Torsten Pietsch, Gyula Kovacs*

Laboratory of Molecular Oncology, Department of Urology, Ruprecht-Karls-University, Heidelberg and Department of Neuropathology,University of Bonn, Bonn, Germany

Received 21 September 2000; accepted 15 January 2001

Abstract

Chromophobe renal cell carcinoma (RCC) is characterized by loss of multiple chromosomes in-cluding chromosome 10. This study was undertaken to determine the LOH at the

PTEN/MMAC1

locus (chromosome band 10q23.3) and to search for gene mutations in 15 chromophobe, 50 con-ventional, and 10 papillary RCCs as well as in 10 renal oncocytomas. Loss of heterozygosity(LOH) wa seen at all informative loci in all chromophobe RCCs and in two conventional RCCs.We did not find mutations by analyzing exon 1 to 9 of the

PTEN/MMAC1

gene using the PCR-

SSCP technique in tumors with LOH at 10q23.3. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction

Chromophobe renal cell carcinoma (RCC) was describedas a cytomorphological variant among experimental kidneytumors of the rat and later as a subtype of human renal celltumors [1,2]. The observation that over 90% of patients withchromophobe RCC are alive five years after surgery under-lines the importance of precise diagnosis of this unique typeof tumor [3]. Some of the chromophobe RCC, however,may show a “chromophilic” or granular/eosinophilic cellu-lar phenotype similar to those seen in renal oncocytomas andconventional RCCs, both having distinct clinical courses.Karyotyping, comparative genomic hybridization, and mic-rosatellite analyses of chromophobe RCCs have revealed aspecific combination of monosomies of chromosomes 1, 2,6, 10, 13, 17, and 21, each occurring in 75–100% of thecases [4–6]. The gene alterations at these chromosomal re-gions, with exception of the

P53

tumor suppressor gene,have not yet been identified. Contractor et al. [7] showedthat a constant loss of chromosome 17 is associated withmutation of the remaining allele of the

P53

tumor suppres-sor gene in 27% of the chromophobe RCCs. A recentlyidentified tumor suppressor gene, the

PTEN/MMAC1

ismapped to the 10q23.3 region [8]. Loss of heterozygosity(LOH) at the

PTEN

locus and mutation of the remaining al-lele were shown in distinct types of tumors, suggesting thatinactivation of this gene may play a crucial role in the

pathogenesis of a variety of human cancers. Therefore, wescreened 15 chromophobe RCCs for LOH at 10q23.3 andfor mutations of the

PTEN

gene. As some authors suggestedthat LOH at 10q occurs frequently in renal cell carcinomas,we have analyzed conventional and papillary RCCs as wellas renal oncocytomas for allelic changes at this region.

2. Materials and methods

2.1. Tumor samples

Fresh tumor and corresponding normal kidney tissueswere obtained from the Departments of Pathology, MedicalSchool Hannover, and Albert-Ludwigs-University Freiburg,and from the Department of Urology, Ruprecht-Karls-Uni-versity Heidelberg. One part of the tumor as well as a pieceof normal parenchymal tissue were snap-frozen in liquid ni-trogen immediately after nephrectomy and stored at

�

80C.Another part was processed for short-term culture as de-scribed earlier [9]; another piece of tumor tissue was fixedin buffered formaldehyde and embedded in paraffin, andH&E stained slides were used as a reference for molecularstudies. The histological diagnosis was established accordingto the Heidelberg Classification of Renal Cell Tumours [10].

2.2. DNA extraction

A small piece of frozen normal kidney and tumor tissuefrom chromophobe RCCs and ROs was placed on a plasticPetri dish, covered with 1 ml TE9 buffer (0.5M Tris-C1, pH9; 0.1M EDTA) and allowed to thaw up. Tumor cells werethen carefully scraped and pushed from the tissue and the

* Corresponding author. Tel.:

�

49-6221-566519; fax:

�

49-6221-564634.

E-mail address

: gyula.kovacs@urz.uni-heidelberg.de (G. Kovacs).

162

F. Sükösd et al. / Cancer Genetics and Cytogenetics 128 (2001) 161–163

remaining stroma was discarded. Tumor cells were then re-suspended in a final volume of 5 ml TE9 buffer with 1% so-dium dodecylsulfate (SDS) and 0.2 mg/ml proteinase K, andwere incubated for 3 hours at 56

�

C. DNA was extracted byphenol-chlorophorm and dissolved in a TE buffer after etha-nol precipitation. For analysis of conventional and papillaryRCCs, DNA was isolated form short-term cultures of tumorcells.

2.3. Microsatellite analysis

Microsatellite markers for loci D10S1744, D10S541,D10S1242, and D10S677, which are mapped between cM112.6 and 117 around the

PTEN

gene, were used in thisstudy (www.genome.wi.mit.edu). PCR reactions were per-formed in a total volume of 10

�

l with 50 ng genomicDNA, 200

�

M dNTPs, 2 pmol of each primer, 10 mM Tris-HCl pH 7.0, 50 mM KCl, 1.5 mM MgCl

2

, 0.5% Tween 20,0.1% bovine serum albumine, and 0.5 U Taq DNA poly-merase (GIBCO BRL, Eggenstein, Germany). All forwardprimers were labeled at the 5

�

end with Cy5 for fluores-cence detection. After 2 minutes of denaturation at 94

�

C,the PCR mixes were subjected to the following conditions:40 seconds at 94

�

C, 30 seconds at 55

�

C, and 40 seconds at72

�

C for 28 cycles with a delayed last step for 10 minutes at72

�

C in a PTC100 thermal cycler (MJ Research Inc. Water-town, Massachusetts). Prior to loading, 20

�

l stop solutionof 50 mM EDTA and 5 mg/ml DextranBlue 2000 in 100%deionized formamide were added. Cy5-labelled PCR prod-ucts of 100, 200, and 250 bp were used as an external stan-dard. Prior to loading, the samples were denaturated at 95

�

Cfor 2 minutes and immediately cooled on ice for 1 minute.

Analysis was performed on an automated DNA sequencer(ALFexpressII, Amersham/Pharmacia Biotech, Freiburg, Ger-many). The 6% denaturating polyacrylamide gels made ofacrylamide:bisacrylamide

�

19:1 were run at 400 V, 55 mA,and 30W in 1

�

TBE buffer at 55

�

C. The collected datawere evaluated using the Fragment Manager (FM 1.2) soft-ware (Amersham/Pharmacia Biotech).

2.4. SSCP analysis

Oligonucleotide primers for amplification of exons 1–9of the

PTEN

were published earlier [11]. PCR was per-formed in a volume of 10

�

l containing 10 ng of templateDNA, 50 mM KCl, 10 mM Tris-HCl, 200

�

M of eachdNTP, 0.1% gelatin, 10 pmol of each primer, 1.0 to 2.0 mMMgCl

2

and 0.5 U Taq polymerase. Initial denaturation at94

�

C for 3 minutes was followed by 30 cycles on an auto-mated thermal cycler (PTC 200, M.J. Research Inc, Water-town, MA). These included denaturation at 94

�

C for 30 sec-onds, annealing at 50

�

C to 55

�

C for 40 seconds, andextension at 72

�

C for 40 seconds. a final extension step at72

�

C for 10 minutes was added. Single-strand conformationpolymorphism (SSCP) analysis was performed on a se-quencing apparatus (Pokerface II, Hoefer, San Francisco,USA) using 10, 12, and 14% acrylamide gels, electrophore-

sis at 5–10 W, and variable temperatures for 14 hours. Sil-ver staining of the gels was performed as previously de-scribed [11].

3. Results and discussion

Each of the 15 chromophobe RCCs (100%) showed LOHat all informative loci around the

PTEN

gene. Examples areshown in Fig. 1. LOH was seen only in two (4%) of the 50conventional RCCs. None of the 10 renal oncocytomas andpapillary RCCs showed LOH at 10q23. We detected allelicimbalances at all informative loci in five (10%) of the 50conventional RCCs. As we used DNA extracted from short-term cultures of conventional RCCs for this study, a con-tamination with normal cells could be excluded. During pre-vious deletion mapping of 3p, all tumors showed completeloss of signal of one allele at 3p [12]. Thus, an allelic imbal-ance at 10q might be evaluated either as an LOH occurringin mosaic form or a trisomy of 10q. Our study showed thatLOH at 10q is a specific genetic alteration in chromophobeRCCs. Therefore, we screened the entire coding region ofthe

PTEN/MMAC1

for mutation by PCR-SSCP analysis intumors with LOH at this region. No altered band mobilitywas seen even after repeating the SSCP analysis under dif-ferent conditions. Thus, we can exclude the

PTEN

gene as a

Fig. 1. Examples of LOH at locus D10S677 in three chromophobe renalcell carcinomas. DNA from peripheral blood (N) and tumor tissue (T) wereused for amplification.

F. Sükösd et al. / Cancer Genetics and Cytogenetics 128 (2001) 161–163

163

target of allelic losses at chromosome 10 in chromophobeRCCs. Previous chromosome and CGH studies showed thatnot only the 10q23.3 region, where the PTEN is located, butthe entire chromosome 10 is lost in chromophobe RCCs.These findings suggest that another gene might be involvedin the highly specific alterations of 10q in chromophobeRCCs.

Interestingly, some authors showed alteration of chromo-some 10 in clear-cell RCCs and in “renal cell carcinomas”as well. RFLP studies revealed LOH at 10q in 28% of thetumors and the smallest overlapping region of deletion wasmapped to the 10q23 region [13,14]. It was also speculatedthat loss of chromosome 10 correlates with progression ofclear-cell RCCs [15]. However, most comprehensive chro-mosome, microsatellite, and FISH studies showed that alter-ation of chromosome 10 occurs only at the level of geneticbackground in conventional RCCs [9,16,17]. Gesk et al.[16] demonstrated by FISH analysis that trisomy and/or tet-rasomy occurred in 26% of conventional RCCs, probablydue to frequent hyperploidization of the originally near-dip-loid chromosome set. During our previous karyotypingstudies on more than 100 conventional RCCs, we also foundthat approximately 15% of the conventional RCCs display achromosome set in the triploid-tetraploid range, includingrandom trisomies of chromosome 10 as a consequence ofpolyploidization [9]. These findings may explain the allelicimbalance at chromosome 10 detected in 10% of conven-tional RCCs in the present study.

In summary, monosomy/LOH of chromosome 10 is ahighly specific genetic change in chromophobe RCCs. Thedeletional/mutational inactivation of the

PTEN

tumor sup-pressor gene on the 10q23.3 region can be excluded as aspecific genetic change in this type of tumor. Likely, an-other unknown gene is targeted by LOH at chromosome 10.Until such genes are cloned, the combination of LOH atchromosome 10 and other specific chromosomes can beused for precise diagnosis of chromophobe RCCs.

References

[1] Bannasch P, Schacht U, Storch E. Morphogenesis and micromorphologyof epithelial tumors of the kidney of nitrosomorpholine intoxicated rats. I.Induction and histology. Z Krebsforsch Klin Onkol 1974;81:311–31.

[2] Thoenes W, Störkel S, Rumpelt HJ. Human chromophobe cell renalcarcinoma. Virchows Arch 1985;48:207–17.

[3] Crotty TB, Farrow GM, Lieber MM. Chromophobe cell renal carci-noma: clinicopathological features of 50 cases. J Urol 1995;154:964–7.

[4] Kovacs A, Kovacs G. Low chromosome number in chromophobe re-nal cell carcinomas. Genes Chromosom Cancer 1992;4:267–8.

[5] Speicher MR, Schoell B, du Manoir S, Ried T, Kovacs A, Störkel S,Cremer T, Kovacs G. Specific loss of chromosomes 1, 2, 6, 10, 13, 17and 21 in chromophobe renal cell carcinomas revealed by compara-tive genomic hybridization. Am J Pathol 1994;145:356–64.

[6] Bugert P, Gaul C, Weber K, Akhtar M, Ljungberg B, Kovacs G. Spe-cific genetic changes of diagnostic importance in chromophobe renalcell carcinomas. Lab Invest 1997;76:203–8.

[7] Contractor H, Zariwala M, Bugert P, Zeiler J, Kovacs G. Mutation ofthe p53 tumor suppressor gene occurs preferentially in chromophobetype of renal cell tumors. J Pathol 1997;181:136–9.

[8] Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH,Langford LA, Baumgard ML, Hattier , Davis T, Frye C, Hu R, Swed-lund B, Teng DH, Tavtigian SV. Identification of a candidate tumoursuppressor gene, MMAC1, at chromosome 10q23.3 that is mutated inmultiple advanced cancers. Nat Genet 1997;15:356–62.

[9] Kovacs G, Frisch S. Clonal chromosome abnormalities in tumor cellsfrom patients with sporadic renal cell carcinomas. Cancer Res 1989;49:651–9.

[10] Kovacs G, Akhtar M, Beckwith BJ, Bugert P, Cooper CS, DelahuntB, Ritz E, Roos G, Schmidt D, Srigley JR, Störkel S, van den Berg E,Zbar B. The Heidelberg classification of renal cell tumors. J Pathol1997;183:131–3.

[11] Duerr EM, Rollbrocker B, Hayashi Y, Peters N, Meyer-Puttlitz B,Lous DN, Schramm J, Wiestler OD, Parsons R, Eng C, Deimling A.PTEN mutations in gliomas and glioneuronal tumors. Oncogene1998;16:2259–64.

[12] Chudek J, Wilhelm M, Bugert P, Herbers J, Kovacs G. Detailed mic-rosatellite analysis of chromosome 3p region in nonpapillary renalcell carcinomas. Int J Cancer 1997;73:225–9.

[13] Morita R, Ishikawa J, Tsutsumi M, Hikiji K, Tsukada Y, KamidonoS, Maeda S, Nakamura Y. Allelotype of renal cell carcinoma. CancerRes 1991;51:820–3.

[14] Morita R, Saito S, Ishikawa J, Ogawa O, Yoshida O, Yamakawa K,Nakamura Y (1991). Common regions of deletion on chromosome5q, 6q, and 10q in renal cell carcinoma. Cancer Res 1991;51:5817–20.

[15] van den Berg E, Dijkhuisen T, Oosterhuis JW, van Kessel AG, deJong B, Störkel S. Cytogenetic classification of renal cell cancer.Cancer Gent Cytognet 1997;95:103–7.

[16] Gesk S, Siebert R, Wacker HH, Nürnberg N, Harder L, Lehman J,Klöppel G, Grote W, Stöckle M, Schlegeberger B. Lack of deletionsof the PTEN/MMAC1 and MXI1 loci in renal cell carcinoma by in-terphase cytogenetics. Cancer Genet Cytogenet 2000;118:87–8.

[17] Trash-Bingham CA, Greenberg RE, Howard S, Bruzel A, Bremer M,Goll A, Salazar H, Freed JJ, Tartof KD. Comprehensive allelotypingof human renal cell carcinomas using microsatellite DNA probes.Proc Natl Acad Sci USA 1995;92:2854–8.