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Renal Cell Carcinoma Genetic Analysis by Comparative Genomic Hybridization and Restriction Fragment Length Polymorphism Analysis
0022-5347/96/1561-0281$03.00/0
THEJOURNAL OF UROLOGY
Vol. 156, 281-285, July 1996 Printed in U.S.A.
Copyright 0 1996 by AMERICAN UROL~GICAL ASSOCIATION, INC.
RENAL CELL CARCINOMA GENETIC ANALYSIS BY COMPARATIVE GENOMIC HYBRIDIZATION AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS JOSEPH C. PRESTI, JR.," HOLGER MOCH, VICTOR E. REUTER, CARLOS CORDON-CARD0 FREDERIC M. WALDMAN
AND
From the Department of Urology, Division of Molecular Cytometry and the Department of Laboratory Medicine, University of California, San Francisco and the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York
ABSTRACT
Purpose: To compare comparative genomic hybridization (CGH) with restriction fragment length polymorphism (RFLP) analysis in renal cell carcinoma (RCC). Materials and Methods: Fifteen RCC specimens were analyzed by both CGH and RFLP analysis a t 18 loci. Results: Restriction fragment length polymorphism analysis was informative on 90 chromosomal arms. Allelic imbalance was identified on 27 chromosomal arms by RFLP and on 26 arms by CGH. Data from CGH and RFLP demonstrated a high degree of concordance (p <0.001). Comparative genomic hybridization identified previously documented areas of interest in RCC as well as potential new areas of interest including loss of genetic material on chromosome 2 and gains of genetic material on chromosome 16p. Conclusions: Comparative genomic hybridization can successfully be performed in RCC specimens. As it surveys the entire genome simultaneously, it may be more efficient than conventional cytogenetics or RFLP analysis in analyzing RCC. KEY WORDS:kidney neoplasms, genetics Numerous reports have identified genetic alterations in the pathogenesis of renal cell carcinoma (RCC).1-12 These studies have used conventional cytogenetics based upon short-term cultures or molecular genetic techniques, including Southern or polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) analysis on primary tumor specimens. Conventional cytogenetic techniques are labor intensive, require the establishment of short-term cultures for each sample, and often result in unsatisfactory metaphase spreads for interpretation. Molecular genetic techniques may be successfully applied to the analysis of primary tumors, yet are limited in scope as specific probes to presumptive areas of interest must be used. In addition, while genetic alterations may be more readily identified, the differentiation between allelic deletions or amplifications is often difficult. The technique of comparative genomic hybridization (CGH) has recently been developed.13 In CGH, total tumor genomic DNA and normal human genomic DNA (detected in different colors) are simultaneously hybridized to normal metaphase spreads. The ratio of the colors along the normal chromosomes provides a quantitative map of the relative copy number of DNA sequences in the tumor and normal genomes. Thus the entire tumor genome can be surveyed in a single step without the need to select which genetic loci to test. In addition the distinction between amplification and deletion is more readily apparent. The objectives of the present study were: 1) to validate CGH results with RFLP results in a group of 15 RCC specimens; 2) to identify potentially new chromosomal regions that may be of importance in the pathogenesis of RCC.
MATERIALS A N D METHODS
Tissue and histopathological diagnosis. Tumor specimens were obtained from radical nephrectomy specimens from 15 patients. Normal kidney tissue from an area away from the tumor or peripheral blood from each patient served as normal controls for RFLP analysis. Adjacent sections were processed for histopathological evaluation. Diagnostic specimens were formalin fixed and paraffin embedded. Six-micron thick sections were stained with hematoxylin and eosin. Each tumor was characterized for cell type, histological growth pattern, pathological grade using the Fuhrmann System and pathological stage according to the tumor-node-metastasis classification system without prior knowledge of genetic analysis results.14 Deoxyribonucleic acid isolation, Southern blotting, hybridizations and autoradiography. High molecular weight DNA was isolated from tumor and normal tissue by the nonorganic method developed by Oncop (Gaithersburg, Maryland). Restriction digests of 7.5-pg. aliquots of paired tumor and normal samples were performed in parallel, fractionated on 0.7% agarose gels and transferred to nylon membranes (Sure blot; Oncor? as previously d e ~ c r i b e dMembranes .~ were washed, prehybridized and hybridized with 32P-dCTPlabeled probes as previously described.9 Washes were performed under stringent conditions (final wash a t 65C). Densitometry. Autoradiographs were analyzed with an u1trascan XL Laser Densitometer (Pharmacia LKB Biotechnology, Piscataway, New Jersey) as previously described.15 If normal tissue demonstrated heterozygosity for a particular probe, then the ratio of alleles in both tumor and normal samples was calculated with densitometry. Normalization of Accepted for publication January 19, 1996. * Requests for reprints: Department of Urology, University of Cal- the densitometric tracing was done against the remaining ifornia, San Francisco, 533 Parnassus, San Francisco, California nondeleted allele. This method could introduce error in the 94143-0738. event of amplification of the nondeleted allele. A decrease in Dr. Presti is a recipient of an American Cancer Society Clinical signal intensity in 1allele of a t least 25%in tumor tissue was Oncology Career Development Award (#93-62). 281
282
RCC GENETIC ANALYSIS
considered loss of heterozygosity (LOH). All RFLP scoring ances were scored by CGH only if both hybridizations (CGH and inverse CGH) demonstrated imbalances. All CGH scorwas performed without knowledge of CGH results. Probes. Restriction fragment length polymorphism probes ing was performed without knowledge of RFLP results. Statistical analysis. For each specimen, allelic imbalance to chromosomal arms frequently reported to be altered in several RCC studies were used. The following probes with for each chromosomal arm as detected by RFLP analysis was chromosome map position, locus and restriction enzyme were scored and compared with CGH results. Only tumors inforobtained from the American Type Culture Collection (Rock- mative a t a particular chromosomal arm by RFLP were inville, Maryland) for use in this study: p627 (3~24-25;c N 1 ; cluded in each analysis. Chi square analysis and the kappa Taq I), pH3H2 i3p21; DNF15S2; Hind 1111, pHF12-32 ( 3 ~ 1 4 - statistic were used to investigate the relationship between 21; D3S2; Msp I), pEFD134.7 (3q26.2-qter; D3S45; Msp I), RFLP and CGH results. pCfms-MLA (5q23-q31; IL3; Bgl 11), pTP5E (5q23-q31; D6S44; Taq I), pJCZ30 D5S70; Taq I), pYNZ132 (6~21-qterm; RESULTS (6; D6S37; Hind III), pJCZ67 (7q; D7S396; Msp I), pEFD175 Histopathologic characteristics of all specimens are shown (10q26; D10S25; Taq I), pbc-N1 (111115.5; H W 1 ; Taq I), pCMM65 i16p; D16S84; Msp I), pYNH37.3 (17~13.3; in table 1. Comparative genomic hybridization was perD17S28; Taq I), pYNZ22 (171313.3; D17S5; Taq I), php53B formed on 15 primary RCC specimens, and the results are (17~13.1; TP53; Bgl II), pEW301(17~11.1-2;D17S58; Taq I), shown in table 2. Losses on chromosomes 1,2,3p, 6,8p, 9,113, pTHH59 (17q23-q25.3; D17S4; Taq I), and 0s-4 (18q21.3- 13q, 17 and gains on chromosomes 5q, 7 and 16p were freqter; D18S5; Taq I). The results of 8 of these probes have been quently observed in our study. The average chromosomal arm loss was 5.4 per specimen; average gain was 2.1. No high published. 10 Comparative genomic hybridization analysis. Comparative level amplification was seen in any specimen. A total of 18 RFLP probes directed at 12 chromosomal genomic hybridization was done essentially as desc15bed.l~ Briefly, DNAs from tumor and a sex-matched normal control arms was used in this study. A comparison between CGH and were labeled by nick translation with biotin-14-dATP (tumor) RFLP on all specimens is shown in table 3. For comparison, or digoxigenin-11-dUTP (normal). They were mixed with un- each chromosomal arm was scored as being informative or labeled Cot-1 DNA to block binding to repetitive sequences, not on the basis of RFLP analysis. Of a potential 180 chrodenatured and hybridized to a normal lymphocyte meta- mosomal arms, 90 were informative by RFLP analysis and phase spread for 2 to 3 days a t 37C. After washing to remove constitute the basis of comparison with CGH analysis. Allelic unbound probe, DNA was stained with avidin-FITC (green) imbalance by RFLP was detected on 27 chromosomal arms to detect biotinylated tumor DNA, and with antidigoxigenin while no imbalance was observed on 63 chromosomal arms. rhodamine (red) to detect normal DNA. The relative binding Comparative genomic hybridization detected allelic imbalof tumor (green) and normal (red) DNAs along each meta- ance on 26 chromosomal arms while no imbalance was obphase chromosome reflects the relative abundance of DNA served on 64 chromosomal arms. sequences in the tumor. Thus, DNA sequences that were The two modalities agreed on 73 chromosomal arms and overrepresented in the tumor show relatively increased disagreed on 17 chromosomal arms (p <0.001; table 4). The green fluorescence, and those regions that were under- discordance between RFLP and CGH for each informative represented in the tumor appear with relatively decreased chromosomal arm was 4 of 10 on 3p, 1of 8 on 3q, 0 of 3 on 5q, green fluorescence. The labeled normal DNA served as a 1 of 7 on 6p, 0 of 5 on 6q, 4 of 10 on 7q, 0 of 8 on lOq, 1of 7 control for regional variations in the ability to hybridize to on l l p , 2 of 6 on 16p, 3 of 10 on 17p, 1of 9 on 17q and 0 of 7 the target chromosomes. This DNA does not have to come on 18q. Nine chromosomal arms demonstrated allelic imbalfrom the same person as the tumor DNA. Inverse labeling ance by RFLP yet were not scored as having imbalance by CGH was performed on all samples (DNA was stained with CGH. In 5 of these 9, CGH demonstrated an imbalance in 1 antidigoxigenin rhodamine (red) to detect biotinylated tumor hybridization t h a t could not be confirmed by the inverse DNA and with avidin-FITC (green) to detect normal DNA) hybridization. Eight chromosomal arms demonstrated imbalfor confirmation of all alterations. Chromosomes in the nor- ance by CGH yet were not scored as imbalance by RFLP. In mal metaphase spread were counterstained with DAPI, all of these cases, the imbalance observed by CGH was a gain which also produces a banding pattern used for chromosome of genetic material. One case demonstrated imbalance by identification. RFLP, which was identified as a gain of genetic material by Digital image analysis. A quantitative image processing CGH (specimen 1, chromosome 16p). The figure demonsystem (QUIPS) was used for the acquisition of sequential three-color images using filters for green (FITC), red (rhodamine), and blue (DAPI) fluorescence. QUIPS is based on a Zeiss Axioplan microscope equipped with multiband pass TABLE1. Histopathological data filters, excitation filter wheel, motorized stage and a PhotoSpecimen # Grade* P-stage? Cell type: Histological Patterns metrics cooled CCD camera interfaced to a SUN workstation. 1 3 3b clear/granular acinar Software programs based on the SCILimage software pack2 3 3a clear acinar age were used to display and overlay multicolor images in 3 3 3a clear/granular acinar pseudocolors and to contrast-stretch the images to enhance 4 2 3a granular acinar 5 3 3b clear acinadpapillary green and red color differences and the blue DAPI banding. 6 1 3a clear acinar Chromosome boundaries were defined by thresholding after I 2 3a clear/granular acinar background subtraction, and the medial axis of each chromo8 2 3a cleadgranular acinar some was defined on the basis of the DAPI image. The inten9 4 3a cleadgranular acinar 1 3a 10 clear acinar sity profiles of green and red fluorescence were then calcu11 3 3a granular/clear acinar lated by integrating fluorescence values across the 2 3b 12 gramladclear acinar chromosome width along the medial axis. Green-to-red ratios 13 3 3a granular/clear acinar of each chromosome were then plotted as a function of dis14 2 3b clear acinar 15 1 3b clear acinar tance from the p to the q terminus (left to right). Generally, * Fuhrmann grade. 4 ratio profiles were averaged for each chromosome (2 sepat N and M categories were zero in all specimens. rate metaphases) to reduce noise. Green-to-red ratios >1.20 1: Cell types listed in order of most prevalent to least prevalent in tumor. were considered as gains of genetic material while ratios B Histologic patterns listed in order of most prevalent to least prevalent in c0.8 were considered as loss& of genetic material. Imbal- tumor.
283
RCC GENETIC ANALYSIS TABLE2 . Comparative genomic hybridization results in I5 primary renal cell carcinomas -
Soccimen #
~-
CGH Losses
CGH Gains
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
TABLE3. RFLP and CGH Results Specimen # 1
2 3 4 5 6 7
3P 01. 01. c/o 01. 010 -I0 010 01. 01. -I0 -I0 -1. 01.
3q
5q
-1. -1. 01.
-1.
01.
1 -.
01.
-10
01.
-1.
.I
Chromosome Arm 7q 1oq
6P
6q
-1. -1. -I. 010 01. -I.
-1. -1.
01. -I.
-1. -I0 -1. -I. 010 -I0
01. 1 -.. -1. 01. -1. 01. -I. 01.
1lP
16P
17p
17q
~ l m -1. -1. -1.
-1. 01. -1. -10
01 .. -1. mlo
01.
01. -10
01. 01. mlo -1. -1. 01. -I0
01. -Im -1. -Im -1. 01.
010
-I0
-1. -1. 01. clm 010 01. 01.
.I 01.
-10 01. -1.0 010 *I* -im 1 -. -I0 -I0 -..I 9 -10 -1. -I. *I* -1. 10 01. -h .I ./. 01. 01. 11 -1. 1-. -1. .I *I. *I. 01. 12 .I -I. 010 010 01. -10 01. 13 mlo 010 010 010 01. -I0 01. 14 -10 -I. -1. 01. -h 01. -1. - /m 15 010 01. 01. -I. -1. 01 .. 01. .I Data presented for each specimen as RFLPICGH results RFLP key: 0 , informative without allelic imbalance; 0, informative with allelic imbalance: -, noninformative CGH key: 0 , no imbalance: 0 , relative loss: H, relative gain 8
-1.
-I. -10
TABLE4. Correlation between RFLP and CGH results on all chromosomal arms*
1-. -I. -1. -10
-I. *I. 1-. mlm 01. -h
mlo -1.
18q
010
mlm 010 010
01. -/m
Chromosome 3
Allelic Imbalance by RFLP yes no 18 8f Allelic Imbalance yes by CGH no 9: 55 Chi-square = 26.8, p <0.001 Kappa statistic = 0.55 ( 9 5 6 confidence interval; 0.34-0.75) * 90 chromosomal arms informative by RFLP used for comparison t All 8 demonstrated gains by CGH $ 5 of 9 demonstrated loss by CGH in 1 of 2 hybridizations yet was not confirmed in the second hybridization.
1
I
Chromosome 17 strates representative CGH profiles and RFLP blots from chromosomes 3 and 17. DISCUSSION
We report a combined application of CGH and RFLP analysis in the evaluation of primary renal cell carcinoma tumors. Comparative genomic hybridization is a relatively new technique that offers tremendous advantages over conventional cytogenetic and other molecular genetic techniques. It does not require short-term cultures nor interpretation of complex karyotypes as does cytogenetics. In addition, CGH screens the entire genome simultaneously, unlike RFLP techniques which require locus-specificscreening. Great enthusiasm has developed over the past few years for the identification of genetic events associated with tumor initiation and progression. Comparative genomic hybridization may prove to be the most efficient means of screening for chromosomal areas of interest in the analysis of solid tumors. Our CGH results confirm alterations on several chromosomal regions of interest which have previously been re-
Comparative genomic hybridization profile of chromosome 3 demonstrating loss of genetic material (vertical arrows) from chromosome 3p (top left) and loss of heterozygosity (horizontal arrow) by probe pHF12-32 (3~14-213(top right). Comparative genomic hybridization profile of chromosome 17 demonstrating loss of entire chromosome (vertical arrows)(bottom left) and loss of heterozygosity (horizontal arrow) by probe pYNZ22 (171313.3)(bottom right).
ported in RCC, including losses on chromosomes 1 , 3 , 6 , 8 , 9 , 10, 13, 17 and gains on chromosomes 5 and 7. Our observed frequency of these alterations was lower than that commonly reported in the literature. The von Hippel-Lindau gene on 3p has recently been cloned and appears to be involved in the pathogenesis of most sporadic RCC.16.*7 In the present study, CGH identified loss on 3p in only 8 of 15 cases. Of the 7 cases which did not demonstrate loss on 3p by CGH, 1 demon-
284
RCC GENETIC ANALYSIS
strated a gain on 3p (specimen 4), and 3 demonstrated loss of densitometric tracing in this fashion will erroneously result 3p by one hybridization which could not be confirmed by in the reporting of LOH when in fact no loss of genetic inverse hybridization (1,2,13). If these 4 specimens had been material has occurred but rather a gain of the allele used for scored as a 3p alteration by CGH, then 3p alterations were normalization. I t is for this reason that allelic imbalance observed in 12 of 15 (80%) specimens. This percentage is rather than LOH may be the more proper term when reportlower than that reported by Anglard and associates (89%) ing RFLP data. An alternative means to correct for differen. and Kovacs and Frisch (95%)who analyzed cell line^.^.'^ tial DNA loading among the lanes of a Southern blot has been Contamination of tumor DNA by normal DNA is a well- to use constant bands from other probe hybridizations using recognized problem in the analysis of primary tumor tissue the same membranes. This also may be problematic as one and will decrease the sensitivity of detection of genetic ab- cannot be sure that the locus of the “constant” band is indeed normalities by any modality. The DNA of lymphocytes and unaltered in the tumor tissue. Comparative genomic hybridstromal cells found in primary tumors results in the dilution ization provides more reliable information with respect to of tumor DNA. Of the remaining 3 cases, 1 did not demon- allelic imbalances and more readily distinguishes between strate allelic imbalance by RFLP (8), 1 was noninformative gains and losses. Comparative genomic hybridization can successfully be apfor all RFLP probes at 3p (12) and 1 demonstrated allelic imbalance by RFLP which was not observed by CGH (9). I t plied to the evaluation of RCC specimens. As it surveys the should be noted that this specimen (9) did not demonstrate entire genome simultaneously, it may be more efficient than conventional cytogenetics or RFLP analysis in understandany abnormalities on any chromosomes by CGH. The gain of chromosome 5q has been reported to occur in as ing the genetic events involved in the initiation and progresmany as 48% of RCC cell lines analyzed by cytogenetic anal- sion of RCC. y s k 3 We observed this abnormality in only 4 of 15 specimens (27%). This observation may be a result of the small sample REFERENCES size; however, normal cell DNA contamination may also be a contributing factor. 1. Kovacs, G., Szucs, S., DeRiese, W. and Baumgartel, H.: Specific A recent report has identified a region of interest on chromosome aberrations in human renal cell carcinoma. Int. J. Cancer, 40:171, 1987. 9p21-22 as a candidate tumor suppressor gene involved in 2. Path&, S. and Dhaliwal, M. K.: Cytogenetic lesions in renal cell RCC.12 We also have observed, by CGH, the loss of genetic carcinoma. Cancer Bull., 41: 324, 1989. material from chromosome 9 in 4 of our cases. Loss on chro3. Kovacs, G. and Frisch, S.: Clonal chromosome abnormalities in mosome 9 is considered an early event in the pathogenesis of tumor cells from patients with sporadic renal cell carcinomas, several tumors including bladder and nasopharyngeal carciCancer Res., 4 9 651, 1989. nomas.19.20 We have also identified potentially new chromo4. Bar, B., Brauch, H., Talmadge, C. and Linehan, M.: Loss of somal areas of interest, including losses on chromosome 2 alleles of loci on the short arm of chromosome 3 in renal cell and gains on chromosome 16p. An additional observation was carcinoma. Nature, 327: 721, 1987. the lack of high level amplification in any of our specimens, 5. Kovacs, G., Erlandsson, R., Boldog, F., Ingvarsson, S., Muller-Brechlin, R., Klein, G. and Sumegi, J.: Consistent chrounlike reports in CGH analysis of breast cancer specimens.21 mosome 3p deletion and loss of heterozygosity in renal cell Our study also validated the use of CGH in RCC by comcarcinoma. Proc. Natl. Acad. Sci. U.S.A., 8 5 1571, 1988. parison of CGH and RFLP analyses. The correlation between 6. van der Hout, A. H., Kok, K., van den Berg, A., Oosterhuis, J. W., the 2 modalities was high. The most common discrepancy Carritt, B. and Buys, C. H. C. M.: Direct molecular analysis of was when RFLP did not demonstrate allelic imbalance while a deletion of 3p in tumors from patients with sporadic renal CGH demonstrated a gain of genetic material (n = 8). There cell carcinoma. Cancer Genet. Cytogenet., 32: 281, 1988. may be several contributing factors: 1)the two techniques 7. Anglard, P., Tory,K., Brauch, H., Weiss, G. H., Latif, F., Merino, may differ in their sensitivity with respect to the detection of M. J., Lerman, M. I., mar, B. and Linehan, W. M.: Molecular gains and losses of genetic material; the cut-off value for analysis of genetic changes in the origin and development of renal cell carcinoma. Cancer Res., 51: 1071, 1991. scoring allelic imbalance for RFLP was 25% and 20% for 8. Morita, R., Ishikawa, J., Tsutsumi, M., Hikiji, K., Tsukada, Y., CGH (CGH ratios >1.2 for tumor to normal tissue were Kamidono, S., Maeda, S. and Nakamura, Y.: Allelotype of considered gains); 2) CGH analyses were performed in duplirenal cell carcinoma. Cancer Res., 51: 820, 1991. cate (inverse hybridizations were needed to confirm original 9. Presti, J. C., Jr., Rao, P. H., Chen, Q., Reuter, V. E., Li, F. P., findings in order to score the imbalance) while RFLP hybridFair, W. F. and Jhanwar, S. C.: Histopathological, cytogenetic, izations were performed only once. In the 9 cases that demand molecular characterization of renal cortical tumors. Canonstrated allelic imbalance by RFLP but not by CGH, 5 did cer Res., 51: 1544, 1991. show CGH abnormalities in 1 of 2 hybridizations, yet were 10. Presti, J. C., Jr., Reuter,V. E., Cordon-Cardo,C., Mazumdar, M., not confirmed in the second hybridization. Comparative Fair, W. R. and Jhanwar, S. C.: Allelic deletions in renal genomic hybridization should be considered as a screening tumors: histopathological correlations. Cancer Res., 53: 5780, 1993. tool to identify large alterations in the genome, usually on the order of megabase-pairs, while RFLP analysis can iden- 11. Bergerheim, U., Nordenskjold, M. and Collins, P.: Deletion mapping in human renal cell carcinoma. Cancer Res., 4 9 1390, tify alterations on the order of kilobase-pairs. While CGH is 1989. valuable in identifying gains or losses of genetic material, it P., Tokino, K., Eby, Y. and Sidransky,D.: Localization of will not be able to detect reciprocal translocations or other 12. Cairns, tumor suppressor loci on chromosome 9 in primary human rearrangements resulting in no net change in genetic materenal cell carcinomas. Cancer Res., 55: 224, 1995. rial. With RFLP analysis such rearrangements will be de- 13. Kallioniemi, A., Kallioniemi, O., Sudar, D., Rutovitz, D., Smith, tected if they cause a change in size of the restriction fragH., Gray, J., Waldman, F. and Pinkel, D.: Comparative ment to which the probe binds. In this setting no LOH is genomic hybridization: a powerful new method for cytogenetic analysis of solid tumors. Science, 258 818, 1992. observed, but rather, a novel allele (different size) is detected. As mentioned previously, one case (specimen 1, chromosome 14. American Joint Committee on Cancer: Kidney. In: Manual for Staging of Cancer. 3rd edition. Edited by 0. H. Beahrs, D. E. 16p) demonstrated an imbalance by RFLP which was a gain Henson, R. V. P. Hutter and M. H. Myers. Philadelphia: J. B. by CGH. This case illustrates the potential risk of normalizLippincott Co., chapt. 33, pp. 199-201, 1988. ing the densitometric tracing of RFLP to the “noninvolved” J. C., Jr., Reuter, V. E., Galan, T., Fair, W. R. and allele. Such a practice, which has been commonly employed 15. Resti, Cordon-Cardo, C.: Molecular genetic alterations in superficial in the past, may lead to false interpretations of RFLP data. and locally advanced human bladder cancer. Cancer Res., 51: This method will introduce error if the “nondeleted” allele is 5405,1991. in fact present in additional copies. Normalization of the 16. Latif, F.,Tory, K., Gnarra, J., Yao, M., Duh, F. M., Orcutt, M. L.,
RCC GENETIC ANALYSIS Stackhouse, T., Kuzmin, I., Modi, W., Geil, L. et al.: Identification of the von Hippel-Lindau disease tumor suppressor gene. Science, 260 1317, 1993. 17. Shuin, T., Kondo, K., Torigoe, S., Kishida, T., Kubota, Y., Hosaka, M., Nagashima, Y., Kitamura, H., Latif, F., Zbar, B. et al.: 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, 1994. 18. Anglard, P., Trahan, E., Liu, S., Latif, F., Merino, M. J., Lerman, M. I., Zbar, B. and Linehan, W. M.: Molecular and cellular characterization of human renal cell carcinoma cell lines. Cancer Res., 52: 348, 1992.
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19. Tsai, Y. C., Nichols, P. W., Hiti, A. L., Williams, Z., Skinner, D. G. and Jones, P. A.: Allelic loss of chromosomes 9, 11, and 17 in human bladder cancer. Cancer Res., 5 0 44, 1990. 20. Huang, D. P., Lo, K. W., van Hasselt, C. A,, Woo, J . K., Choi, P. H., Leung, S. F., Cheung, S. T., Cairns, P., Sidransky, D. and Lee, J . C.: A region of homozygous deletion on chromosome 9p21-22 in primary nasopharyngeal carcinoma. Cancer Res., 54: 4003, 1994. 21. Kallioniemi, A., Kallioniemi, 0.P., Piper, J., Tanner, M., Stokke, T., Chen, L., Smith, H. S., Pinkel, D., Gray, J. W. and Waldman, F. M.: Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc. Natl. Acad. Sci. U.S.A., 91: 2156, 1994.
THEJOURNAL OF UROLOGY
Vol. 156, 281-285, July 1996 Printed in U.S.A.
Copyright 0 1996 by AMERICAN UROL~GICAL ASSOCIATION, INC.
RENAL CELL CARCINOMA GENETIC ANALYSIS BY COMPARATIVE GENOMIC HYBRIDIZATION AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS JOSEPH C. PRESTI, JR.," HOLGER MOCH, VICTOR E. REUTER, CARLOS CORDON-CARD0 FREDERIC M. WALDMAN
AND
From the Department of Urology, Division of Molecular Cytometry and the Department of Laboratory Medicine, University of California, San Francisco and the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York
ABSTRACT
Purpose: To compare comparative genomic hybridization (CGH) with restriction fragment length polymorphism (RFLP) analysis in renal cell carcinoma (RCC). Materials and Methods: Fifteen RCC specimens were analyzed by both CGH and RFLP analysis a t 18 loci. Results: Restriction fragment length polymorphism analysis was informative on 90 chromosomal arms. Allelic imbalance was identified on 27 chromosomal arms by RFLP and on 26 arms by CGH. Data from CGH and RFLP demonstrated a high degree of concordance (p <0.001). Comparative genomic hybridization identified previously documented areas of interest in RCC as well as potential new areas of interest including loss of genetic material on chromosome 2 and gains of genetic material on chromosome 16p. Conclusions: Comparative genomic hybridization can successfully be performed in RCC specimens. As it surveys the entire genome simultaneously, it may be more efficient than conventional cytogenetics or RFLP analysis in analyzing RCC. KEY WORDS:kidney neoplasms, genetics Numerous reports have identified genetic alterations in the pathogenesis of renal cell carcinoma (RCC).1-12 These studies have used conventional cytogenetics based upon short-term cultures or molecular genetic techniques, including Southern or polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) analysis on primary tumor specimens. Conventional cytogenetic techniques are labor intensive, require the establishment of short-term cultures for each sample, and often result in unsatisfactory metaphase spreads for interpretation. Molecular genetic techniques may be successfully applied to the analysis of primary tumors, yet are limited in scope as specific probes to presumptive areas of interest must be used. In addition, while genetic alterations may be more readily identified, the differentiation between allelic deletions or amplifications is often difficult. The technique of comparative genomic hybridization (CGH) has recently been developed.13 In CGH, total tumor genomic DNA and normal human genomic DNA (detected in different colors) are simultaneously hybridized to normal metaphase spreads. The ratio of the colors along the normal chromosomes provides a quantitative map of the relative copy number of DNA sequences in the tumor and normal genomes. Thus the entire tumor genome can be surveyed in a single step without the need to select which genetic loci to test. In addition the distinction between amplification and deletion is more readily apparent. The objectives of the present study were: 1) to validate CGH results with RFLP results in a group of 15 RCC specimens; 2) to identify potentially new chromosomal regions that may be of importance in the pathogenesis of RCC.
MATERIALS A N D METHODS
Tissue and histopathological diagnosis. Tumor specimens were obtained from radical nephrectomy specimens from 15 patients. Normal kidney tissue from an area away from the tumor or peripheral blood from each patient served as normal controls for RFLP analysis. Adjacent sections were processed for histopathological evaluation. Diagnostic specimens were formalin fixed and paraffin embedded. Six-micron thick sections were stained with hematoxylin and eosin. Each tumor was characterized for cell type, histological growth pattern, pathological grade using the Fuhrmann System and pathological stage according to the tumor-node-metastasis classification system without prior knowledge of genetic analysis results.14 Deoxyribonucleic acid isolation, Southern blotting, hybridizations and autoradiography. High molecular weight DNA was isolated from tumor and normal tissue by the nonorganic method developed by Oncop (Gaithersburg, Maryland). Restriction digests of 7.5-pg. aliquots of paired tumor and normal samples were performed in parallel, fractionated on 0.7% agarose gels and transferred to nylon membranes (Sure blot; Oncor? as previously d e ~ c r i b e dMembranes .~ were washed, prehybridized and hybridized with 32P-dCTPlabeled probes as previously described.9 Washes were performed under stringent conditions (final wash a t 65C). Densitometry. Autoradiographs were analyzed with an u1trascan XL Laser Densitometer (Pharmacia LKB Biotechnology, Piscataway, New Jersey) as previously described.15 If normal tissue demonstrated heterozygosity for a particular probe, then the ratio of alleles in both tumor and normal samples was calculated with densitometry. Normalization of Accepted for publication January 19, 1996. * Requests for reprints: Department of Urology, University of Cal- the densitometric tracing was done against the remaining ifornia, San Francisco, 533 Parnassus, San Francisco, California nondeleted allele. This method could introduce error in the 94143-0738. event of amplification of the nondeleted allele. A decrease in Dr. Presti is a recipient of an American Cancer Society Clinical signal intensity in 1allele of a t least 25%in tumor tissue was Oncology Career Development Award (#93-62). 281
282
RCC GENETIC ANALYSIS
considered loss of heterozygosity (LOH). All RFLP scoring ances were scored by CGH only if both hybridizations (CGH and inverse CGH) demonstrated imbalances. All CGH scorwas performed without knowledge of CGH results. Probes. Restriction fragment length polymorphism probes ing was performed without knowledge of RFLP results. Statistical analysis. For each specimen, allelic imbalance to chromosomal arms frequently reported to be altered in several RCC studies were used. The following probes with for each chromosomal arm as detected by RFLP analysis was chromosome map position, locus and restriction enzyme were scored and compared with CGH results. Only tumors inforobtained from the American Type Culture Collection (Rock- mative a t a particular chromosomal arm by RFLP were inville, Maryland) for use in this study: p627 (3~24-25;c N 1 ; cluded in each analysis. Chi square analysis and the kappa Taq I), pH3H2 i3p21; DNF15S2; Hind 1111, pHF12-32 ( 3 ~ 1 4 - statistic were used to investigate the relationship between 21; D3S2; Msp I), pEFD134.7 (3q26.2-qter; D3S45; Msp I), RFLP and CGH results. pCfms-MLA (5q23-q31; IL3; Bgl 11), pTP5E (5q23-q31; D6S44; Taq I), pJCZ30 D5S70; Taq I), pYNZ132 (6~21-qterm; RESULTS (6; D6S37; Hind III), pJCZ67 (7q; D7S396; Msp I), pEFD175 Histopathologic characteristics of all specimens are shown (10q26; D10S25; Taq I), pbc-N1 (111115.5; H W 1 ; Taq I), pCMM65 i16p; D16S84; Msp I), pYNH37.3 (17~13.3; in table 1. Comparative genomic hybridization was perD17S28; Taq I), pYNZ22 (171313.3; D17S5; Taq I), php53B formed on 15 primary RCC specimens, and the results are (17~13.1; TP53; Bgl II), pEW301(17~11.1-2;D17S58; Taq I), shown in table 2. Losses on chromosomes 1,2,3p, 6,8p, 9,113, pTHH59 (17q23-q25.3; D17S4; Taq I), and 0s-4 (18q21.3- 13q, 17 and gains on chromosomes 5q, 7 and 16p were freqter; D18S5; Taq I). The results of 8 of these probes have been quently observed in our study. The average chromosomal arm loss was 5.4 per specimen; average gain was 2.1. No high published. 10 Comparative genomic hybridization analysis. Comparative level amplification was seen in any specimen. A total of 18 RFLP probes directed at 12 chromosomal genomic hybridization was done essentially as desc15bed.l~ Briefly, DNAs from tumor and a sex-matched normal control arms was used in this study. A comparison between CGH and were labeled by nick translation with biotin-14-dATP (tumor) RFLP on all specimens is shown in table 3. For comparison, or digoxigenin-11-dUTP (normal). They were mixed with un- each chromosomal arm was scored as being informative or labeled Cot-1 DNA to block binding to repetitive sequences, not on the basis of RFLP analysis. Of a potential 180 chrodenatured and hybridized to a normal lymphocyte meta- mosomal arms, 90 were informative by RFLP analysis and phase spread for 2 to 3 days a t 37C. After washing to remove constitute the basis of comparison with CGH analysis. Allelic unbound probe, DNA was stained with avidin-FITC (green) imbalance by RFLP was detected on 27 chromosomal arms to detect biotinylated tumor DNA, and with antidigoxigenin while no imbalance was observed on 63 chromosomal arms. rhodamine (red) to detect normal DNA. The relative binding Comparative genomic hybridization detected allelic imbalof tumor (green) and normal (red) DNAs along each meta- ance on 26 chromosomal arms while no imbalance was obphase chromosome reflects the relative abundance of DNA served on 64 chromosomal arms. sequences in the tumor. Thus, DNA sequences that were The two modalities agreed on 73 chromosomal arms and overrepresented in the tumor show relatively increased disagreed on 17 chromosomal arms (p <0.001; table 4). The green fluorescence, and those regions that were under- discordance between RFLP and CGH for each informative represented in the tumor appear with relatively decreased chromosomal arm was 4 of 10 on 3p, 1of 8 on 3q, 0 of 3 on 5q, green fluorescence. The labeled normal DNA served as a 1 of 7 on 6p, 0 of 5 on 6q, 4 of 10 on 7q, 0 of 8 on lOq, 1of 7 control for regional variations in the ability to hybridize to on l l p , 2 of 6 on 16p, 3 of 10 on 17p, 1of 9 on 17q and 0 of 7 the target chromosomes. This DNA does not have to come on 18q. Nine chromosomal arms demonstrated allelic imbalfrom the same person as the tumor DNA. Inverse labeling ance by RFLP yet were not scored as having imbalance by CGH was performed on all samples (DNA was stained with CGH. In 5 of these 9, CGH demonstrated an imbalance in 1 antidigoxigenin rhodamine (red) to detect biotinylated tumor hybridization t h a t could not be confirmed by the inverse DNA and with avidin-FITC (green) to detect normal DNA) hybridization. Eight chromosomal arms demonstrated imbalfor confirmation of all alterations. Chromosomes in the nor- ance by CGH yet were not scored as imbalance by RFLP. In mal metaphase spread were counterstained with DAPI, all of these cases, the imbalance observed by CGH was a gain which also produces a banding pattern used for chromosome of genetic material. One case demonstrated imbalance by identification. RFLP, which was identified as a gain of genetic material by Digital image analysis. A quantitative image processing CGH (specimen 1, chromosome 16p). The figure demonsystem (QUIPS) was used for the acquisition of sequential three-color images using filters for green (FITC), red (rhodamine), and blue (DAPI) fluorescence. QUIPS is based on a Zeiss Axioplan microscope equipped with multiband pass TABLE1. Histopathological data filters, excitation filter wheel, motorized stage and a PhotoSpecimen # Grade* P-stage? Cell type: Histological Patterns metrics cooled CCD camera interfaced to a SUN workstation. 1 3 3b clear/granular acinar Software programs based on the SCILimage software pack2 3 3a clear acinar age were used to display and overlay multicolor images in 3 3 3a clear/granular acinar pseudocolors and to contrast-stretch the images to enhance 4 2 3a granular acinar 5 3 3b clear acinadpapillary green and red color differences and the blue DAPI banding. 6 1 3a clear acinar Chromosome boundaries were defined by thresholding after I 2 3a clear/granular acinar background subtraction, and the medial axis of each chromo8 2 3a cleadgranular acinar some was defined on the basis of the DAPI image. The inten9 4 3a cleadgranular acinar 1 3a 10 clear acinar sity profiles of green and red fluorescence were then calcu11 3 3a granular/clear acinar lated by integrating fluorescence values across the 2 3b 12 gramladclear acinar chromosome width along the medial axis. Green-to-red ratios 13 3 3a granular/clear acinar of each chromosome were then plotted as a function of dis14 2 3b clear acinar 15 1 3b clear acinar tance from the p to the q terminus (left to right). Generally, * Fuhrmann grade. 4 ratio profiles were averaged for each chromosome (2 sepat N and M categories were zero in all specimens. rate metaphases) to reduce noise. Green-to-red ratios >1.20 1: Cell types listed in order of most prevalent to least prevalent in tumor. were considered as gains of genetic material while ratios B Histologic patterns listed in order of most prevalent to least prevalent in c0.8 were considered as loss& of genetic material. Imbal- tumor.
283
RCC GENETIC ANALYSIS TABLE2 . Comparative genomic hybridization results in I5 primary renal cell carcinomas -
Soccimen #
~-
CGH Losses
CGH Gains
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
TABLE3. RFLP and CGH Results Specimen # 1
2 3 4 5 6 7
3P 01. 01. c/o 01. 010 -I0 010 01. 01. -I0 -I0 -1. 01.
3q
5q
-1. -1. 01.
-1.
01.
1 -.
01.
-10
01.
-1.
.I
Chromosome Arm 7q 1oq
6P
6q
-1. -1. -I. 010 01. -I.
-1. -1.
01. -I.
-1. -I0 -1. -I. 010 -I0
01. 1 -.. -1. 01. -1. 01. -I. 01.
1lP
16P
17p
17q
~ l m -1. -1. -1.
-1. 01. -1. -10
01 .. -1. mlo
01.
01. -10
01. 01. mlo -1. -1. 01. -I0
01. -Im -1. -Im -1. 01.
010
-I0
-1. -1. 01. clm 010 01. 01.
.I 01.
-10 01. -1.0 010 *I* -im 1 -. -I0 -I0 -..I 9 -10 -1. -I. *I* -1. 10 01. -h .I ./. 01. 01. 11 -1. 1-. -1. .I *I. *I. 01. 12 .I -I. 010 010 01. -10 01. 13 mlo 010 010 010 01. -I0 01. 14 -10 -I. -1. 01. -h 01. -1. - /m 15 010 01. 01. -I. -1. 01 .. 01. .I Data presented for each specimen as RFLPICGH results RFLP key: 0 , informative without allelic imbalance; 0, informative with allelic imbalance: -, noninformative CGH key: 0 , no imbalance: 0 , relative loss: H, relative gain 8
-1.
-I. -10
TABLE4. Correlation between RFLP and CGH results on all chromosomal arms*
1-. -I. -1. -10
-I. *I. 1-. mlm 01. -h
mlo -1.
18q
010
mlm 010 010
01. -/m
Chromosome 3
Allelic Imbalance by RFLP yes no 18 8f Allelic Imbalance yes by CGH no 9: 55 Chi-square = 26.8, p <0.001 Kappa statistic = 0.55 ( 9 5 6 confidence interval; 0.34-0.75) * 90 chromosomal arms informative by RFLP used for comparison t All 8 demonstrated gains by CGH $ 5 of 9 demonstrated loss by CGH in 1 of 2 hybridizations yet was not confirmed in the second hybridization.
1
I
Chromosome 17 strates representative CGH profiles and RFLP blots from chromosomes 3 and 17. DISCUSSION
We report a combined application of CGH and RFLP analysis in the evaluation of primary renal cell carcinoma tumors. Comparative genomic hybridization is a relatively new technique that offers tremendous advantages over conventional cytogenetic and other molecular genetic techniques. It does not require short-term cultures nor interpretation of complex karyotypes as does cytogenetics. In addition, CGH screens the entire genome simultaneously, unlike RFLP techniques which require locus-specificscreening. Great enthusiasm has developed over the past few years for the identification of genetic events associated with tumor initiation and progression. Comparative genomic hybridization may prove to be the most efficient means of screening for chromosomal areas of interest in the analysis of solid tumors. Our CGH results confirm alterations on several chromosomal regions of interest which have previously been re-
Comparative genomic hybridization profile of chromosome 3 demonstrating loss of genetic material (vertical arrows) from chromosome 3p (top left) and loss of heterozygosity (horizontal arrow) by probe pHF12-32 (3~14-213(top right). Comparative genomic hybridization profile of chromosome 17 demonstrating loss of entire chromosome (vertical arrows)(bottom left) and loss of heterozygosity (horizontal arrow) by probe pYNZ22 (171313.3)(bottom right).
ported in RCC, including losses on chromosomes 1 , 3 , 6 , 8 , 9 , 10, 13, 17 and gains on chromosomes 5 and 7. Our observed frequency of these alterations was lower than that commonly reported in the literature. The von Hippel-Lindau gene on 3p has recently been cloned and appears to be involved in the pathogenesis of most sporadic RCC.16.*7 In the present study, CGH identified loss on 3p in only 8 of 15 cases. Of the 7 cases which did not demonstrate loss on 3p by CGH, 1 demon-
284
RCC GENETIC ANALYSIS
strated a gain on 3p (specimen 4), and 3 demonstrated loss of densitometric tracing in this fashion will erroneously result 3p by one hybridization which could not be confirmed by in the reporting of LOH when in fact no loss of genetic inverse hybridization (1,2,13). If these 4 specimens had been material has occurred but rather a gain of the allele used for scored as a 3p alteration by CGH, then 3p alterations were normalization. I t is for this reason that allelic imbalance observed in 12 of 15 (80%) specimens. This percentage is rather than LOH may be the more proper term when reportlower than that reported by Anglard and associates (89%) ing RFLP data. An alternative means to correct for differen. and Kovacs and Frisch (95%)who analyzed cell line^.^.'^ tial DNA loading among the lanes of a Southern blot has been Contamination of tumor DNA by normal DNA is a well- to use constant bands from other probe hybridizations using recognized problem in the analysis of primary tumor tissue the same membranes. This also may be problematic as one and will decrease the sensitivity of detection of genetic ab- cannot be sure that the locus of the “constant” band is indeed normalities by any modality. The DNA of lymphocytes and unaltered in the tumor tissue. Comparative genomic hybridstromal cells found in primary tumors results in the dilution ization provides more reliable information with respect to of tumor DNA. Of the remaining 3 cases, 1 did not demon- allelic imbalances and more readily distinguishes between strate allelic imbalance by RFLP (8), 1 was noninformative gains and losses. Comparative genomic hybridization can successfully be apfor all RFLP probes at 3p (12) and 1 demonstrated allelic imbalance by RFLP which was not observed by CGH (9). I t plied to the evaluation of RCC specimens. As it surveys the should be noted that this specimen (9) did not demonstrate entire genome simultaneously, it may be more efficient than conventional cytogenetics or RFLP analysis in understandany abnormalities on any chromosomes by CGH. The gain of chromosome 5q has been reported to occur in as ing the genetic events involved in the initiation and progresmany as 48% of RCC cell lines analyzed by cytogenetic anal- sion of RCC. y s k 3 We observed this abnormality in only 4 of 15 specimens (27%). This observation may be a result of the small sample REFERENCES size; however, normal cell DNA contamination may also be a contributing factor. 1. 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