Genetic Changes in Primary and Recurrent Prostate Cancer by Comparative Genomic Hybridization'

[CANCERRESEARCH55,342-347,January15,19951 Genetic Changes in Primary and Recurrent Prostate Cancer by Comparative Genomic Hybridization'

Tapio Visakorpi,2 Anne H. Kallioniemi, Ann-Christine SyvÃ¤nen,Eija R. Hyytinen, Ritva Karhu, Teuvo Tammela, Jorma J- Isola, and Offi-P. Kalliomemi

Laboratory of Cancer Genetics, Department of Clinical Chemistry fT. V., A. H. K., E. R. H., R. K., 0-P. K.J, and Division of Urology, Department of Surgery IT. T.J, Tampere University Hospital, P. 0. Box 2000. FIN-33521 Tampere; Department of Human Molecular Genetics, National Public Health Institute, Helsinki (A-C. S.); and Department of Biomedical Sciences, University of Tampere. Tampere If. J. I.]. Finland

ABSTRACT of the known TSGs and oncogenes in prostate cancer development are also poorly known. For example, ras oncogene, which is commonly Genetic changes leading to the development of prostate cancer and affected in human malignancies, is involved relatively infrequently in factors that underlie the clinical progression of the disease are poorly prostate cancer (8, 9). Furthermore, factors that determine the prog characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes nosis of patients with prostate cancer are poorly known, and genetic in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim markers of tumor progression are urgently required (10). of the study was to identify those chromosome regions that contain genes CGH is a newly developed molecular cytogenetic method that important for the development of prostate cancer and to identify genetic makes it possible to survey the entire genome for gains and losses of markers of tumor progression. CGH analysis indicated that 74% of DNA sequences (11â€”17).CGH is based on the simultaneous hybrid primary prostate carcinomas showed DNA sequence copy number ization of differentially labeled tumor and normal DNA to normal changes. Losses were 5 times more common than gains and most often metaphase chromosomes. In such a hybridization, differences be involved Sp (32%), 13q (32%), 6q (22%), 16q (19%), lSq (19%), and 9p tween the binding of the labeled DNA sequences, as evidenced by (16%). Allelic loss studies with 5 polymorphic microsatellite markers for fluorescence intensity ratio measurements along all chromosomes, 4 different chromosomes were done from 13 samples and showed a 76% indicate those regions of the genome that were either over- or under concordance with CGH results. In local recurrencesthat developed during endocrine therapy, there were significantly more gains (P < 0.001) and represented in the tumor genome. The utility of CGH is based on the losses (P < 0.05) of DNA sequences than in primary tumors, with gains of concept that regions with increased copy number reveal chromosome Sq (found in 89% of recurrences versus 6% of primary tumors), X (56% sites that may contain dominant oncogenes, whereas regions with versus 0%), and 7 (56% versus 10%), as well as loss of Sp (78% versus decreased copy number may be putative TSG loci (11). Thus, in a 32%), being particularly often involved. In conclusion, our CGH results single hybridization, CGH allows screening of all chromosomal sites indicate that losses of several chromosomal regions are common genetic that are likely to contain genes with an important role in tumor changes in primary tumors, suggesting that deletional inactivation of development. putative tumor suppressor genes in these chromosomal sites is likely to We took advantage of the potential of CGH to screen for losses and underlie development of prostate cancer. Furthermore, the pattern of gains of DNA sequences in 31 primary uncultured prostate carcino genetic changes seen in recurrent tumors with the frequent gains of 7, Sq, mas. The aim was to identify those chromosomal regions that are and X suggests that the progression of prostate cancer and development of often involved in copy number aberrations and may thus contain hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of genes implicated in the development of prostate cancer. In the second prostate cancer progression. part of the study, genetic aberrations in the primary tumors were compared with those detected in 9 recurrent prostate tumors. The aim INTRODUCTION of this part of the study was to identify genetic changes that underlie clinical tumor progression. Genetic changes underlying the development and progression of prostate cancer are poorly known. Classical cytogenetic studies are MATERIALS AND METHODS very difficult to carry out in prostate cancer because of the preferential growth of nonmalignant cells. In many cases only a normal karyotype The material consisted of 31 primary and 9 recurrent uncultured prostate has been found (1). The aberrations that have been reported most often carcinomas. Six benign prostate hyperplasia samples were also evaluated. The include deletions of 7q, lOq, and 8 as well as gains of chromosome 7. ThM stagedistribution(18)of theprimaryprostatecarcinomaswas:T1N@M@, @ Occasionally, double minute chromosomes have been reported (1). 1; T2N@JM@J,14;T2N@M@,2;T3N0MIJ,3;T3N@M@,3;T4NXM@J,2; 1; LOH3 studies, which are thought to highlight chromosomal sites T2N1M,J,1;T2NXM1,1; T3N@M1,2;and unknown, 1. The histological grade distribution (19) ofprimary tumors was: grade I, 10; grade II, 19; and grade III, harboring mutated tumor suppressor genes, have implicated 8p, lOq, 2. That of recurrent tumors was: grade II, 4; and grade Ill, 5. The primary 13q, 16q, and 18q (2â€”7). However, LOH studies are typically limited tumor specimens were prostatectomy specimens (20 cases), transurethral re to extensive analyses of a single chromosome or analysis of all section specimens (7 cases), or Tm-Cut needle biopsy specimens (4 cases), all chromosomes with only 1â€”3probes/chromosome arm, thus leaving taken prior to the administration of any hormonal therapy, whereas recurrent the vast majority of the genome unexamined. The specific roles of any prostate carcinomas were all transurethral resection specimens taken from patients who received only endocrine therapy (orchiectomy 6 cases, luteinizing Received 8/12/94; accepted 11/10/94. hormone-releasing hormone agonist 2 cases, estrogen 1 case). The recurrent The costs of publication of this article were defrayed in part by the payment of page tumors came from patients with symptoms of urethral obstruction, indicating charges. This article must therefore be hereby marked advertisement in accordance with local progression of the disease despite ongoing therapy. 18 U.S.C. Section 1734 solely to indicate this fact. Five-@xmsectionswere cut from freshly frozen tumor blocks embedded in I Supported by the Reino Lahtikari Foundation, Paulo's Foundation, the Sigrid Juselius Foundation, The Academy of Finland, the Finnish Cancer Society, and the Finnish Cancer Tissue-Tek (Miles, Inc., Diagnostic Division, Elkhart, IN) and stained with Institute. hematoxylin and eosin to ensure the histological representativeness of the 2 To whom requests for reprints should be addressed, at Laboratory of Cancer Genet samples. Some specimens were trimmed by cutting normal tissue away with a irs, National Center for Human Genome Research, NIH, 9000 Rockville Pike, Building scalpel. High-molecular-weight tumor DNA was isolated either from homog 49, Room 4C20, Bethesda, MD 20892. 3 The abbreviations used are: LOH, loss of heterozygosity; TSG, tumor suppressor enized tumor specimens or from 200-sam frozen sections of the tumors using gene; CGH, comparative genomic hybridization; ThM, tumor-nodes-metastasis. standard protocols. DNA also was isolated from the peripheral blood of 13 342

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. GENETIC CHANGES IN PROSTATIC CARCINOMA patients (for LOH studies) and from normal male donors (reference DNA for Analysis and Interpretation of the LOH Studies. Aliquots (3 j@l)of the CGH). fluorescent PCR products were denatured in 50% formamide containing blue dextran and analyzed on 6% denaturing polyacrylamide gels using an auto Comparative Genomic Hybridization matic DNA sequencer (ALF; Pharmacia Biotech AB). The relative quantities of the PCR products were determined with the aid of ALF DNA Fragment Hybridization. CGH was performed using directly fluorochrome-conju gated DNAS, as described previously (1 1, 20). Briefly, DNA samples from the Manager program V1.1 (Pharmacia Biotech AB). When the ratio between the tumors were labeled with FITC-dUTP (DuPont, Boston, MA), and normal two alleles amplified from a tumor sample differed significantly (2â€”5-fold) male DNA was labeled with Texas red-dUTP (DuPont) using nick translation. from that obtained in the leukocyte sample, it was interpreted as a sign of LOH. Labeled tumor and normal DNAS (400 ng each) together with 10 g.@gof unlabeled Cot-i DNA (Gibco BRL, Gaithersburg, MD) in 10 @lofhybridiza Statistical Analysis tion mixture [50% formamide, 10% dextran sulfate, 2X SSC (1X SSC is 0.15

M NaCl-0.015 M sodium citrate, pH 7)] were denatured at 70Â°C for 5 mm and The statistical significance between the primary and recurrent tumors in the applied on normal lymphocyte metaphase preparations. Prior to hybridization, total number of genetic aberrations and the frequencies of selected changes the metaphase preparations were denatured at 72â€”74Â°Cfor3 mm in a form was calculated with the nonparametric Kruskal-Wallis test and 2-tailed amide solution (70% formamide, 2X SSC, pH 7) and dehydrated in a series of Fisher's exact test, respectively. The ic statistic was used to analyze the level 70, 85, and 100% ethanols; this was followed by proteinase K (0.1 mg/ml in of agreement between CGH and LOH results. 20 m@iTris-HCI, 2 mr@iCaC12,pH 7.5) treatment at room temperature and dehydration once again as described above. The hybridization was done at 37Â°Cfor 48 h. After hybridization, the slides were washed three times in 50% RESULTS formamide/2X SSC (pH 7), twice in 2X SSC, and once in 0.1 X SSC at 45Â°C followed by 2X SSC and 0.1 MNaH2PO4-0.1MNa2HPO4-0.1%NP4O(jH 8), Overview of Genetic Changes. None of the benign prostate hy and distilled water at room temperature for 10 mm each. After air drying, the perplasias showed any gains or losses of DNA sequences by CGH. Six slides were counterstained with 4',6-diamidino-2-phenylindole, 0.1 @xWml,in (19%) of the primary prostate cancers showed relative DNA sequence an antifade solution. gains, and 23 (74%) showed losses at 1 or more chromosomal sites Digital Image Analysis. Three single-color images (matching 4',6-dia midino-2-phenylindole, FITC, and Texas red fluorescence) were collected (Fig. 1). Eight tumors (26%) had no copy number alterations. On from each metaphase spread using a Nikon SA epifluorescence microscope average, there were 2.9 (range, 0â€”12)aberrations per primary tumor: (Nikon Corp., Tokyo, Japan) and a Xillix charge-coupled-devicecamera 0.5 gain (range, 0â€”4)and 2.4 deletions (range, 0â€”9).Fig. 2 shows an (Xilli.x Technologies Corp., Vancouver, BC, Canada) interfaced to a Sun LX example of green:red fluorescence ratio profiles of a primary prostate workstation (Sun Microsystems Computer Corp., Mountain View, CA). Four carcinoma analyzed by CGH. to six three-color digital images were collected from each hybridization. All recurrent prostate cancers showed both relative gains and losses Relative DNA sequence copy number changes were detected by analyzing the of DNA sequences (Fig. 3). The total numbers of aberrations per hybridization intensities of tumor and normal DNAS along the length of all chromosomes in the metaphase spreads, as described earlier (20). The absolute tumor (mean, 7.8; range, 4â€”15),as well as gains (mean, 2.2; range, fluorescence intensities were normalized so that the average green:red ratio of 1â€”4)and losses (mean, 5.6; range, 3â€”12)were significantly higher in all chromosome objects in each metaphase was 1.0. The fmal results were the recurrences than in primary tumors (Fig. 4A). Significance values plotted as a series of green:red ratio profiles and corresponding SDs for each for these differences were P < 0.01 for all aberrations, P < 0.001 for human chromosome from pter to qter. gains, and P < 0.05 for losses. Interpretation of CGH results followed previously described protocols (20). Losses and Gains. Chromosome arms that were lost most fre Hybridizations of FITC-labeled normal female DNA with Texas red-labeled quently in primary prostate cancers were 8p (32% of the cases), 13q normal male DNA were used as negative controls. The mean green:red ratio and the corresponding SD for all autosomes remained between 0.9 and 1.1 in (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). The minimal these control hybridizations. Chromosomal regions where the mean ratio and overlapping regions of loss in each chromosome were 8p12â€”pter, the corresponding SD were less than 0.85 were therefore considered lost, and 13q21â€”31,Ã³cenâ€”q21,l6cenâ€”q23,18q22â€”qter,and 9p23â€”pter.Gain regions where the mean and the corresponding SD were greater than 1.15 of the entire long arm of chromosome 8 was found in two (6%) cases. gained in the tumor genome. The entire Y chromosome was excluded from Chromosome arms that were lost most frequently in recurrent analysis. Hybridizations of DNA from the MCF-7 breast cancer cell line prostate cancers were 8p (78%), 13q (56%), 16q (56%), 6q (44%), and against normal female DNA were used as additional positive controls in each 5q (44%), and the minimal regions 8p21â€”pter, l3cenâ€”q21, 16q22â€” hybridization batch. qter, 6q13â€”q21,and 5q14â€”q23. Gain of 8q was seen in 8 of 9 (89%) Loss of Heterozygosity recurrent prostate cancers and usually affected the entire arm. Gains of chromosome 7 (minimal common region 7pl3) and X (Xpllâ€”q13 and Oligonucleotides. Primers for amplification of the microsatellite loci Xq23â€”qter) were both seen in 56% of cases. Fig. 4, B and C, D6S283 (6q16.3â€”q21), D8S265 (8p23.l), D8S282 (8p22), D13S153 (13q14â€” illustrates the main differences in the frequencies of genetic changes q22), and D16S413 (16q) were synthesized according to the sequences given in primary and recurrent prostate cancer. in the Genethon catalogue. The 5' ends of the upstream (AC strand) D6S283, D13S153, and D16S422 primers and the downstream (OT strand)D8S265 and Taking primary and recurrent tumors together, 10 cases had a gain D8S282 primerswere fluorescencelabeledduring the synthesis using the at 8q. In one tumor, gain was limited to 8q24, while nine were gains FluorePrime reagent (Pharmacia Biotech AB, Uppsala, Sweden). of the entire long arm. Seven of these tumors also had loss of 8p. PCR. Twenty ng of DNA isolated from patients' leukocytes or tumor Comparison between CGH and LOH Results. Detection of samples were amplified separately with each set of primers. The PCR mixtures losses by CGH was compared with data from LOH studies using five contained 50 pmol of both primers, the four deoxynucleotide triphosphates at polymorphic microsatellite markers (1)65283, D8S265, D8S282, 0.2 mr@tconcentration, and 1.25 units of DynaZyme DNA polymerase D13S153, and D16S413) for four different chromosome arms. In (Finnzymes Oy, Espoo, Finland) in 50 @tlofbuffer supplied with the enzyme. total, 37 comparisons were done in 5 loci. A 76% concordance was The PCR was initiated by heating the samples at 95Â°Cfor3 mm, followed by addition of enzyme at 80Â°C.Twenty-fivePCR cycles of 1 min at 95Â°C,1mm found between CGH and allelic loss results (Table 1). The ic coeffi at 58Â°C(markers D6S283, D8S282, and D16S422) or at 54Â°C(markers cient was 0.507. In 13% of cases, LOH was found without loss by D8S265 and D13S153), and 1 min at 72Â°Cwere carried out in a programmable CGH; in another 11% of cases, CGH showed loss of DNA sequences, heat block (FTC 100; Mi Research, Inc., Watertown, MA). but no LOH was detected. 343

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DISCUSSION be common mechanisms of LOH (27). Furthermore, CGH can detect only physical losses affecting regions larger than 10 mega-base pairs. This study represents a genome-wide survey of DNA sequence In four cases (11%), a loss was clearly detected by CGH, but no LOH copy number changes in prostate cancer using CGH. We found that was found. The reason for this inconsistency remains unknown, but it 74% of the primary carcinomas showed gains and/or losses of DNA is possible that in these cases the microsatellite markers, which have sequences, which is a significantly higher number than seen by been only genetically mapped, were localized outside the region of cytogenetic studies. This reflects the power of CGH in revealing loss. aberrations across the genome in uncultured cells. In contrast, none of The majority of prostate cancer patients have disease that is no the benign prostatic hyperplasias showed any genetic alterations by longer curable at the time of diagnosis. However, approximately 70% CGH. In primary tumors, losses predominated over gains with a ratio of these patients will respond to androgen ablation therapy. Although of 5:1. The complete absence of high-level amplification and the low endocrine treatment is initially effective, the cancer cells later become overall frequency of gains in primary prostate cancer are striking as androgen independent and the disease progresses despite the therapy compared to the extensive amplifications seen, for example, in breast (28). There are very few data on the genetic events that determine the cancer (13). This suggests that inactivation of putative recessive TSGs in several chromosomal sites is especially important in prostate cancer malignant potential of prostate cancer and its response to endocrine development. therapy. Knowledge of the mechanisms underlying hormone-indepen The most commonly lost chromosomal regions in primary prostate dent growth are important because at present, there are no effective cancer were 8p, 13q, 6q, 16q, 18q, and 9p. Several studies have shown therapies for hormone-resistant prostate cancers. We thought that the loss of heterozygosities at 8p, 13q, 16q, and 18q in prostate cancer analysis of genetic changes in local recurrences that arise during (2â€”7),but 6q and 9p losses have not been reported previously in hormonal therapy would highlight those aberrations causing prostate cancer. In one previous study, possible LOH at 6q and 9p was aggressive clinical behavior. studied but not found. However, only a single probe per chromosome The nine cases of androgen-resistant recurrent prostate carcinomas arm was used (7). According to CGH, the critical region at 6q was came from patients who had originally received endocrine therapy but 6cenâ€”q21,indicating that this region may harbor a TSG important in developed clinical signs oflocal tumor progression. We found that the the development of prostate cancer. Previously, LOH of the same total number of genetic changes per tumor was almost 3 times higher region was reported in melanoma and in ovarian and breast cancers in recurrences than in primary tumors. Whereas gains and amplifica (21â€”23).Furthermore, transfection of a normal human chromosome 6 tions were uncommon in primary tumors, all recurrences showed suppresses the tumorigenicity of both breast and melanoma cancer gains of at least one chromosomal region. In particular, gain of 8q, cell lines (24, 25). Taken together, these results support the presence either alone or in association with 8p loss, was found 8 of 9 recurrent of a TSG in 6q that may be involved in several tumor types, including prostate cancers. This suggests that the long arm of chromosome 8 prostate cancer. The putative TSG at 9p also remains unknown. may harbor a gene(s) involved in the progression of prostate cancer Recently, a new TSG, MTS1, was identified at 9p21 (26). Whether and its evolution toward hormone independence. The myc oncogene is this gene is also involved in prostate cancer remains to be determined. located at 8q24 and has been shown to be overexpressed in poorly According to CGH, the minimal deleted region was 9p23â€”pter,which differentiated prostate cancers (29, 30). Because the entire long arm of suggests that the target TSG for 9p loss in prostate cancer may reside chromosome 8 was usually present at an increased copy number, it is distal to MTSJ. likely that other genes instead of or in addition to myc are involved. The overall concordance between CGH and LOH results was 76%. Gains of chromosomes 7 and X also were found in more than In five cases (13%), LOH was detected but there was no loss by CGH. one-half of the recurrent prostate carcinomas but very infrequently in CGH is sensitive only to physical losses of DNA sequences and not to primary tumors. In two tumors, the entire chromosome 7 was gained, losses of specific alleles. Mitotic recombinations and losses followed while three tumors showed partial gains with the minimal overlapping by duplication of the remaining allele cannot be seen by CGH but may region at â€˜7pl3.Fluorescence in situ hybridization has shown that 345

trisomy 7 is common in clinically high-stage prostate cancer as well differentchromosomeTable I Comparison oflosses found by CGH with WH measurement of 4 @ as in progression specimens (3 1). Recently, aneusomy of chromosome arms D65283;D16S422)in 8q. D85265, D8S282; 13q, D135153; 16q, cancersLOHNo13 prostate 7 was shown to be associated with poor prognosis in prostate cancer (32).Chromosome7containsmanycandidategenes,suchasEGFR, PA!], RAF, and MDRJ, that may participate in the progression of TotalNoloss loss Loss prostate cancer. Gains of chromosome X were also variable. It is 21Loss 16 5 interesting that two recurrent tumors showed high-level amplification, 16Total 4 12 20 17 37 one at Xpl lâ€”q13and another at Xq23â€”qter. According to the Ge nome Data Base, the Xpl lâ€”q13region contains many possible target A genes such as AR, ARAFI, ELKJ, IL2RG, PGKJ, PGKIPJ, PHKAJ, 10 TFE3, TIMPJ, ZNF2J, and ZNF8J. Gains involving the same region have been found previously in about 35% of osteosarcomas by CGH,4 ** suggesting that this chromosomal region contains a dominantly acting 0 8 E oncogene involved in several tumor types. The other amplified region at X23â€”qter contains genes such as HPRT, LJCAM, MCF2, and I#1 * 0 6 MPPJ as a possible target gene. Further studies with specific probes C to these two regions and individual candidate genes are in progress. 0 .c Losses in the recurrent tumors in general involved the same regions as U 4 in primary tumors, but their overall frequency was higher. However, 0 the frequency of Sq losses was over 7 times higher in recurrent tumors z0 2 than in primary tumors. Adenomatous polyposis coli TSG is localized to 5q21. LOH of the adenomatous polyposis coli region has been found in 20-30% of advanced prostate carcinomas (5, 6). These results indicate that an increased overall number of genetic changes and, specif Total Gains Losses ically, gains and amplifications of certain chromosomes and chromo somal regions may underlie the progressionof prostate cancer. B In conclusion, these CGH results highlight several chromosomal 100 regions that may harbor important genes for prostate cancer tumori genesis and progression. Losses found by CGH in primary tumors U) 80 involving 6q (minimal overlapping region, 6cenâ€”q21)and 9p (9p23â€” 0 E pter) suggest two new regions that may contain prostate cancer TSGs in addition to the previously reported TSG loci 8p, 13q, 16q, and l8q. 60 ** ** 0 Gains of DNA sequences at 7 (7p13), 8q (8q24â€”qter),and X (Xpl 1â€” 0 q13 and Xq23â€”qter)appear important for prostate cancer progression. DI Further studies with specific probes are required to narrow down the 0 40 C critical regions in each chromosome and to identify the genes 0 U involved. 0 a. 20 ACKNOWLEDGMENTS 0 We thank Sari Pennanen and Ritva Timonen for their technical assistance. 7p 8q X

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Tapio Visakorpi, Anne H. Kallioniemi, Ann-Christine Syvänen, et al.

Cancer Res 1995;55:342-347.

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