Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[CANCERRESEARCH 55, 342-347,January15,19951 Genetic Changes in Primary and Recurrent Prostate Cancer by Comparative Genomic Hybridization' Tapio Visakorpi,2 Anne H. Kallioniemi, Ann-Christine Syvänen,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 factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinomas showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved Sp (32%), 13q (32%), 6q (22%), 16q (19%), lSq (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P < 0.001) and losses (P < 0.05) of DNA sequences than in primary tumors, with gains of Sq (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of Sp (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor underlie development genetic suppressor changes genes in these chromosomal of prostate cancer. seen in recurrent tumors sites is likely to the pattern of Furthermore, with the frequent gains of 7, Sq, and X suggests that the progression of prostate cancer and development of hormone-independent chromosome prostate growth aberrations may may have have a distinct diagnostic genetic utility basis. These as markers of Genetic changes underlying the development and progression of prostate cancer are poorly known. Classical cytogenetic studies are to carry out in prostate cancer because 13q, 16q, and 18q (2—7). However, LOH studies to extensive analyses of a single chromosome with only the vast majority it possible to survey the entire genome 1—3probes/chromosome of the genome unexamined. are typically fluorescence intensity ratio measurements along all chromosomes, indicate those regions of the genome that were either over- or under represented in the tumor genome. The utility of CGH is based on the concept that regions with increased copy number reveal chromosome sites that may contain dominant oncogenes, whereas regions with decreased copy number may be putative TSG loci (11). Thus, in a single hybridization, CGH allows screening of all chromosomal sites that are likely to contain genes with an important role in tumor development. We took advantage of the potential of CGH to screen for losses and gains of DNA sequences in 31 primary uncultured prostate carcino mas. The aim was to identify those chromosomal regions that are often involved in copy number aberrations and may thus contain genes implicated in the development of prostate cancer. In the second genetic aberrations in the primary tumors were AND METHODS 2. That of recurrent tumors was: grade II, 4; and grade Ill, 5. The primary tumor specimens were prostatectomy specimens (20 cases), transurethral re limited or analysis of all section specimens (7 cases), or Tm-Cut needle biopsy specimens (4 cases), all arm, thus leaving The specific taken prior to the administration of any hormonal therapy, whereas recurrent prostate carcinomas were all transurethral resection specimens taken from roles of any 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 Institute. for reprints should be addressed, at Laboratory of Cancer Genet irs, National Center for Human Genome Research, NIH, 9000 Rockville Pike, Building 49, Room 4C20, Bethesda, MD 20892. used are: LOH, loss of heterozygosity; TSG, tumor therapy (orchiectomy 6 cases, luteinizing hormone-releasing hormone agonist 2 cases, estrogen 1 case). The recurrent tumors came from patients with symptoms of urethral obstruction, indicating 18 U.S.C. Section 1734 solely to indicate this fact. abbreviations be by distribution (19) ofprimary tumors was: grade I, 10; grade II, 19; and grade III, The costs of publication of this article were defrayed in part by the payment of page 3 The differences as evidenced The material consisted of 31 primary and 9 recurrent uncultured prostate carcinomas. Six benign prostate hyperplasia samples were also evaluated. The ThM stagedistribution(18) of theprimaryprostatecarcinomaswas:T1N@M@, 1; T2N@JM@J, 14; T2N@M@, 2; T3N0MIJ,3; T3N@M@, 3; T4NXM@J, 2; 1; T2N1M,J,1; T2NXM1,1; T3N@M1,2; and unknown, 1. The histological grade charges. This article must therefore be hereby marked advertisement in accordance with requests in for gains and losses of metaphase chromosomes. In such a hybridization, tween the binding of the labeled DNA sequences, patients who received only endocrine whom infrequently DNA sequences (11—17).CGH is based on the simultaneous hybrid ization of differentially labeled tumor and normal DNA to normal MATERIALS Received 8/12/94; accepted 11/10/94. 2 To relatively of the preferential growth of nonmalignant cells. In many cases only a normal karyotype has been found (1). The aberrations that have been reported most often include deletions of 7q, lOq, and 8 as well as gains of chromosome 7. Occasionally, double minute chromosomes have been reported (1). LOH3 studies, which are thought to highlight chromosomal sites harboring mutated tumor suppressor genes, have implicated 8p, lOq, chromosomes makes is involved compared with those detected in 9 recurrent prostate tumors. The aim of this part of the study was to identify genetic changes that underlie clinical tumor progression. INTRODUCTION @ in human malignancies, cancer (8, 9). Furthermore, factors that determine the prog patients with prostate cancer are poorly known, and genetic of tumor progression are urgently required (10). is a newly developed molecular cytogenetic method that part of the study, cancer progression. very difficult affected prostate nosis of markers CGH local progression of the disease despite ongoing therapy. Five-@xmsections were cut from freshly frozen tumor blocks embedded in Tissue-Tek (Miles, Inc., Diagnostic Division, Elkhart, IN) and stained with hematoxylin and eosin to ensure the histological representativeness of the samples. Some specimens were trimmed by cutting normal tissue away with a scalpel. High-molecular-weight tumor DNA was isolated either from homog enized tumor specimens or from 200-sam frozen sections of the tumors using suppressor standard protocols. DNA also was isolated from the peripheral blood of 13 gene; CGH, comparative genomic hybridization; ThM, tumor-nodes-metastasis. 342 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research. GENETIC CHANGES IN PROSTATIC CARCINOMA patients (for LOH studies) and from normal male donors (reference DNA for CGH). Analysis and Interpretation of the LOH Studies. Aliquots (3 j@l)of the fluorescent PCR products were denatured in 50% formamide containing blue gated DNAS, as described previously (1 1, 20). Briefly, DNA samples from the dextran and analyzed on 6% denaturing polyacrylamide gels using an auto matic DNA sequencer (ALF; Pharmacia Biotech AB). The relative quantities of the PCR products were determined with the aid of ALF DNA Fragment 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 Comparative Genomic Hybridization Hybridization. CGH was performed using directly fluorochrome-conju (2—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.@g of unlabeled Cot-i DNA (Gibco BRL, Gaithersburg, MD) in 10 @l of hybridiza tion mixture [50% formamide, 10% dextran sulfate, 2X SSC (1X SSC is 0.15 Statistical M NaCl-0.015 M sodium citrate, pH 7)] were denatured at 70°C for 5 mm and applied on normal lymphocyte metaphase preparations. Prior to hybridization, Analysis The statistical significance between the primary and recurrent tumors in the total number of genetic aberrations and the frequencies of selected changes amide solution (70% formamide, 2X SSC, pH 7) and dehydrated in a series of was calculated with the nonparametric Kruskal-Wallis test and 2-tailed Fisher's exact test, respectively. The ic statistic was used to analyze the level 70, 85, and 100% ethanols; of agreement between CGH and LOH results. the metaphase preparations were denatured at 72—74°C for 3 mm in a form this was followed by proteinase K (0.1 mg/ml in 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 37°Cfor 48 h. After hybridization, the slides were washed three times in 50% formamide/2X SSC (pH 7), twice in 2X SSC, and once in 0.1 X SSC at 45°C followed by 2X SSC and 0.1 MNaH2PO4-0.1MNa2HPO4-0.1%NP4O(jH 8), and distilled water at room temperature for 10 mm each. After air drying, the slides were counterstained with 4',6-diamidino-2-phenylindole, 0.1 @xWml,in an antifade solution. Digital Image Analysis. Three single-color images (matching 4',6-dia midino-2-phenylindole, FITC, and Texas red fluorescence) were collected from each metaphase spread using a Nikon SA epifluorescence microscope (Nikon Corp., Tokyo, Japan) and a Xillix charge-coupled-devicecamera (Xilli.x Technologies workstation Corp., Vancouver, BC, Canada) interfaced to a Sun LX (Sun Microsystems to six three-color Computer Corp., Mountain digital images were collected View, CA). Four from each hybridization. Relative DNA sequence copy number changes were detected by analyzing the hybridization intensities of tumor and normal DNAS along the length of all chromosomes in the metaphase spreads, as described earlier (20). The absolute fluorescence intensities were normalized so that the average green:red ratio of all chromosome objects in each metaphase was 1.0. The fmal results were plotted as a series of green:red ratio profiles and corresponding SDs for each human chromosome from pter to qter. Interpretation of CGH results followed previously described protocols (20). Hybridizations of FITC-labeled normal female DNA with Texas red-labeled 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 these control hybridizations. Chromosomal regions where the mean ratio and the corresponding SD were less than 0.85 were therefore considered lost, and regions where the mean and the corresponding SD were greater than 1.15 gained in the tumor genome. The entire Y chromosome was excluded from analysis. Hybridizations of DNA from the MCF-7 breast cancer cell line against normal female DNA were used as additional positive controls in each hybridization batch. Loss of Heterozygosity Oligonucleotides. Primers for amplification of the microsatellite loci D6S283 (6q16.3—q21), q22), and D16S413 D8S265 (8p23.l), (16q) were synthesized D8S282 (8p22), according D13S153 (13q14— to the sequences given in the Genethon catalogue. The 5' ends of the upstream (AC strand) D6S283, D13S153, and D16S422 primers and the downstream (OT strand) D8S265 and D8S282 primerswere fluorescencelabeledduring the synthesis using the FluorePrime reagent (Pharmacia Biotech AB, Uppsala, Sweden). PCR. Twenty ng of DNA isolated from patients' leukocytes or tumor samples were amplified separately with each set of primers. The PCR mixtures contained 50 pmol of both primers, the four deoxynucleotide triphosphates at 0.2 mr@tconcentration, and 1.25 units of DynaZyme DNA polymerase (Finnzymes Oy, Espoo, Finland) in 50 @tlof buffer supplied with the enzyme. RESULTS Overview perplasias of Genetic Changes. showed None of the benign prostate hy any gains or losses of DNA sequences by CGH. Six (19%) of the primary prostate cancers showed relative DNA sequence gains, and 23 (74%) showed losses at 1 or more chromosomal sites (Fig. 1). Eight tumors (26%) had no copy number alterations. On average, there were 2.9 (range, 0—12)aberrations per primary tumor: 0.5 gain (range, 0—4)and 2.4 deletions (range, 0—9).Fig. 2 shows an example of green:red fluorescence ratio profiles of a primary prostate carcinoma analyzed by CGH. All recurrent prostate cancers showed both relative gains and losses of DNA sequences (Fig. 3). The total numbers of aberrations per tumor (mean, 7.8; range, 4—15),as well as gains (mean, 2.2; range, 1—4)and losses (mean, 5.6; range, 3—12)were significantly higher in the recurrences than in primary tumors (Fig. 4A). Significance values for these differences were P < 0.01 for all aberrations, P < 0.001 for gains, and P < 0.05 for losses. Losses and Gains. Chromosome arms that were lost most fre quently in primary prostate cancers were 8p (32% of the cases), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). The minimal overlapping regions of loss in each chromosome were 8p12—pter, 13q21—31,ócen—q21,l6cen—q23, 18q22—qter, and 9p23—pter.Gain of the entire long arm of chromosome 8 was found in two (6%) cases. Chromosome arms that were lost most frequently in recurrent prostate cancers were 8p (78%), 13q (56%), 16q (56%), 6q (44%), and 5q (44%), and the minimal regions 8p21—pter, l3cen—q21, 16q22— qter, 6q13—q21,and 5q14—q23. Gain of 8q was seen in 8 of 9 (89%) recurrent prostate cancers and usually affected the entire arm. Gains of chromosome 7 (minimal common region 7pl3) and X (Xpll—q13 and Xq23—qter) were both seen in 56% of cases. Fig. 4, B and C, illustrates the main differences in the frequencies of genetic changes in primary and recurrent prostate cancer. Taking primary and recurrent tumors together, 10 cases had a gain at 8q. In one tumor, gain was limited to 8q24, while nine were gains of the entire long arm. Seven of these tumors also had loss of 8p. Comparison between CGH and LOH Results. Detection of losses by CGH was compared with data from LOH studies using five polymorphic microsatellite markers (1)65283, D8S265, D8S282, D13S153, and D16S413) for four different chromosome arms. In total, 37 comparisons were done in 5 loci. A 76% concordance was found between CGH and allelic loss results (Table 1). The ic coeffi cient was 0.507. In 13% of cases, LOH was found without loss by CGH; in another 11% of cases, CGH showed loss of DNA sequences, but no LOH was detected. 343 The PCR was initiated by heating the samples at 95°Cfor 3 mm, followed by addition of enzyme at 80°C.Twenty-five PCR cycles of 1 min at 95°C,1 mm at 58°C(markers D6S283, D8S282, and D16S422) or at 54°C(markers D8S265 and D13S153), and 1 min at 72°Cwere carried out in a programmable heat block (FTC 100; Mi Research, Inc., Watertown, MA). Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research. @ I @R. __ GENETIC CHANGES IN PROSTATIC CARCINOMA I @ i I I i@ Fig.1. Summaryof all gainsand lossesof @ i@k 1@ I P I@I I DNA sequences observed in 31 primary pros tate carcinomas by CGH. Gains are shown on 8 6 I 9 theleftsideof thechromosomeideogramsand losses on the right. Chromosome Y was cx cluded from analysis. 4 16 17 19 @t 20 18 1 14 I@I1 • [email protected]. . I@@1 11 21 @I 22 Y 15 i@_ .iiiiii I. UI1@I@ 12 5 @lI I II Ii @iII 13 @ 11 2 @ @ 10 . @l I 11 . @.. t@• 4@:.:::;:..::.:::::::::.:.::.@ 5@:.@...:;;..:::;:::::.::::@:::::::: @ L@I@@IJ I I 1 1 I@I@ N I @@JI @ 6b@ @:@: .;..:: 7fT::T..!....:@r::.:::I.r.. . :@ . Et@ I 1 @I@II 1 1 iUi II I •I@ 1 ii @ 9HH ‘ @I10@H. ii -@. @.. 12@@ @ L @I @1 II @i@i@ii Ill_I_Il_I •i iii Fig. 2. Mean grcen:red ratio profiles for all chromosomes (except Y) from pter to qtcr oh tamed from CGH analysis of a prhnary pros tate cancer. ----, the baseline value (1.0) representing the mean grecn:red ratio for the entire sample; ratios 03 and 1.5. changes in the grecn:redratioprofileindicatelossesat 6cen—q22,8p, 11q14—q23, 16q, and 22q; and gains at Sp, 7p, 7q32—qter, and 19p. Bars, SD. @ 13H•iI.iI ]5@i@J@9 @TIi•@@•i @I__uI_I1 ioH—@ 17L @ @ @ t-I_--@II_I1 @I•I I 18@ LUE .1 ••@@I tI.ft I 19 I I@ I . 21@'@22@ I 20 ___ . ________ V __ •@FIJ @i i @i. i _________ 344 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research. OENFI1C cHANOE5 IN PROSTATIC CARCINOMA @ @II @II ‘1@k uli ‘i― !@‘ I@ Fig. 3. Summary of all gains and losses of Il DNAsequencesobservedin 9 recurrentpros tate carcinomas by CGH. Gains arc shown on theleftsideof thechromosomeideogramsand losses on the right. Chromosome Y was cx cluded from analysis @ ho' I Ioii@ Ii 1@ II 19 16 13 14 17 18 20 21 @I 22 Y iO@ x 15 be common mechanisms of LOH (27). Furthermore, CGH can detect only physical losses affecting regions larger than 10 mega-base pairs. In four cases (11%), a loss was clearly detected by CGH, but no LOH was found. The reason for this inconsistency remains unknown, but it is possible that in these cases the microsatellite markers, which have been only genetically mapped, were localized outside the region of loss. The majority of prostate cancer patients have disease that is no longer curable at the time of diagnosis. However, approximately 70% of these patients will respond to androgen ablation therapy. Although endocrine treatment is initially effective, the cancer cells later become androgen independent and the disease progresses despite the therapy (28). There are very few data on the genetic events that determine the malignant potential of prostate cancer and its response to endocrine therapy. Knowledge of the mechanisms underlying hormone-indepen dent growth are important because at present, there are no effective therapies for hormone-resistant prostate cancers. We thought that the analysis of genetic changes in local recurrences that arise during hormonal therapy would highlight those aberrations causing aggressive clinical behavior. The nine cases of androgen-resistant recurrent prostate carcinomas came from patients who had originally received endocrine therapy but developed clinical signs oflocal tumor progression. We found that the total number of genetic changes per tumor was almost 3 times higher in recurrences than in primary tumors. Whereas gains and amplifica tions were uncommon in primary tumors, all recurrences showed gains of at least one chromosomal region. In particular, gain of 8q, either alone or in association with 8p loss, was found 8 of 9 recurrent prostate cancers. This suggests that the long arm of chromosome 8 may harbor a gene(s) involved in the progression of prostate cancer and its evolution toward hormone independence. The myc oncogene is located at 8q24 and has been shown to be overexpressed in poorly differentiated prostate cancers (29, 30). Because the entire long arm of chromosome 8 was usually present at an increased copy number, it is likely that other genes instead of or in addition to myc are involved. Gains of chromosomes 7 and X also were found in more than one-half of the recurrent prostate carcinomas but very infrequently in primary tumors. In two tumors, the entire chromosome 7 was gained, while three tumors showed partial gains with the minimal overlapping region at ‘7pl3.Fluorescence in situ hybridization has shown that DISCUSSION This study represents a genome-wide survey of DNA sequence copy number changes in prostate cancer using CGH. We found that 74% of the primary carcinomas showed gains and/or losses of DNA sequences, which is a significantly higher number than seen by cytogenetic studies. This reflects the power of CGH in revealing aberrations across the genome in uncultured cells. In contrast, none of the benign prostatic hyperplasias showed any genetic alterations by CGH. In primary tumors, losses predominated over gains with a ratio of 5:1. The complete absence of high-level amplification and the low overall frequency of gains in primary prostate cancer are striking as compared to the extensive amplifications seen, for example, in breast cancer (13). This suggests that inactivation of putative recessive TSGs in several chromosomal sites is especially important in prostate cancer development. The most commonly lost chromosomal regions in primary prostate cancer were 8p, 13q, 6q, 16q, 18q, and 9p. Several studies have shown loss of heterozygosities at 8p, 13q, 16q, and 18q in prostate cancer (2—7),but 6q and 9p losses have not been reported previously in prostate cancer. In one previous study, possible LOH at 6q and 9p was studied but not found. However, only a single probe per chromosome arm was used (7). According to CGH, the critical region at 6q was 6cen—q21,indicating that this region may harbor a TSG important in the development of prostate cancer. Previously, LOH of the same region was reported in melanoma and in ovarian and breast cancers (21—23).Furthermore, transfection of a normal human chromosome 6 suppresses the tumorigenicity of both breast and melanoma cancer cell lines (24, 25). Taken together, these results support the presence of a TSG in 6q that may be involved in several tumor types, including prostate cancer. The putative TSG at 9p also remains unknown. Recently, a new TSG, MTS1, was identified at 9p21 (26). Whether this gene is also involved in prostate cancer remains to be determined. According to CGH, the minimal deleted region was 9p23—pter, which suggests that the target TSG for 9p loss in prostate cancer may reside distal to MTSJ. The overall concordance between CGH and LOH results was 76%. In five cases (13%), LOH was detected but there was no loss by CGH. CGH is sensitive only to physical losses of DNA sequences and not to losses of specific alleles. Mitotic recombinations and losses followed by duplication of the remaining allele cannot be seen by CGH but may 345 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research. GENETIC CHANGES IN PROSTATIC CARCINOMA @ trisomy 7 is common in clinically high-stage prostate cancer as well as in progression specimens (3 1). Recently, aneusomy of chromosome 7 was shown to be associated with poor prognosis in prostate differentchromosome Table I Comparison oflosses found by CGH with WH measurement of 4 arms D65283; D16S422)in 8q. D85265, D8S282; 13q, D135153; 16q, cancersLOHNo 13 prostate cancer (32).Chromosome 7 containsmanycandidate genes,suchasEGFR, TotalNoloss PA!], RAF, and MDRJ, that may participate in the progression of prostate cancer. Gains of chromosome X were also variable. It is interesting that two recurrent tumors showed high-level amplification, one at Xpl l—q13and another at Xq23—qter. According to the Ge nome Data Base, the Xpl l—q13region contains many possible target A 0 E 21Loss 16Total ** I#1 6 0 * C 0 .c U 4 0 0 z 2 Total B U) 0 Gains Losses 100 80 ** 60 ** 40 C 0 U 0 37 regions that may harbor important genes for prostate cancer tumori genesis and progression. Losses found by CGH in primary tumors involving 6q (minimal overlapping region, 6cen—q21)and 9p (9p23— pter) suggest two new regions that may contain prostate cancer TSGs in addition to the previously reported TSG loci 8p, 13q, 16q, and l8q. Gains of DNA sequences at 7 (7p13), 8q (8q24—qter),and X (Xpl 1— q13 and Xq23—qter)appear important for prostate cancer progression. Further studies with specific probes are required to narrow down the critical regions in each chromosome and to identify the genes 0 a. 5 12 17 somal regions may underlie the progression of prostate cancer. In conclusion, these CGH results highlight several chromosomal E 0 DI 0 16 4 20 Loss genes such as AR, ARAFI, ELKJ, IL2RG, PGKJ, PGKIPJ, PHKAJ, 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 oncogene involved in several tumor types. The other amplified region at X23—qter contains genes such as HPRT, LJCAM, MCF2, and MPPJ as a possible target gene. Further studies with specific probes to these two regions and individual candidate genes are in progress. Losses in the recurrent tumors in general involved the same regions as in primary tumors, but their overall frequency was higher. However, the frequency of Sq losses was over 7 times higher in recurrent tumors 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 ically, gains and amplifications of certain chromosomes and chromo 10 8 loss involved. 20 ACKNOWLEDGMENTS 0 7p 8q We thank Sari Pennanen and Ritva Timonen for their technical assistance. X REFERENCES C 1. Sandberg, A. A. Chromosomal abnormalities and related events in prostate cancer. Hum. Pathol., 23: 368—380,1992. 100 2. Carter, B. S., Ewing, C. M., Ward, W. S., Treiger, B. F., Aalders, T. W., Schalken, 3. A., Epstein. J. I., and Isaacs, W. B. Alldic loss of chromosomes 16q and l0q in U) 0 * 80 human prostate cancer. Proc. Nail. Aced. Sci. USA, 87: 8751—8755,1990. 3. Bergerheim, U. S. R., Kunimi, K., Collins, V. P., and Ekman, P. Deletion mapping of E chromosomes 8, 10, and 16 in human prostatic carcinoma. Genes Chromosomes & 60 Cancer, 3: 215—220,1991. 4. Bova, 0. S., Carter, B. S., Bussemakers, M. J. G., Emi, M., Fujiwara, Y., Kyprianou, * 0 0 DI 0 C 0 U 4) a. N., Jacobs, S. C., Robinson, J. C., Epstein, J. I., Walsh, P. C., and lasses, W. B. Homozygous deletion and frequent allelic loss of chromosome 8p22 loci in human prostate cancer. Cancer Res., 53: 3869—3873,1993. 40 5. Phillips, S. M. A., Morton, D. 0., Lee, S. .1., Wallace, D. M. A., and Neoptelemos, J. P. Loss of heterozygosityof the retinoblastomaand adenomatouspolyposis susceptibility gene loci and in chromosomes lOp, lOq and 16q in human prostate 20@ cancer. Br. J. Urol., 73: 390—395,1994. 6. Brewster, S. F., Browne, S., and Brown, K. W. Somatic allelic loss at the DCC, APC. nm23-HI and p53 tumor suppressor gene loci in human prostatic carcinoma. J. Urol., 151: 1073—1077,1994. 5q 6q 8p 13q 7. Kunimi, K., Bergerheim, U. S. P., Larsson, I-L., Ekman, P.. and Collins, V. P. Allelotyping of human prostatic adenocarcinoma. Genomics, II: 530—536,1991. 16q Fig. 4. Comparison of genetic aberrations between primary (0) and recurrent () prostate cancer, showing mean number of genetic changes per tumor (A) as well as frequency of gains (B) and losses (C) at selected chromosome arms. °,P < 0.05; **, P < 0.01; °°°, P < 0.001; Fisher's exact test. 8. Carter, B. S., Epstein, J. I., and Isaacs, W. B. Ras gene mutations in human prostate cancer. Cancer Res., 50: 6830—6832, 1990. 4M.Tarkkanen, personal communication. 346 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research. GENETICCHANGESIN PROSTATICCARCINOMA 9. Gummerlock, P. H., Poonamallee, U. R., Meyers, F. J., and de Vere White, R. W. Activated ras alleles in human carcinoma of prostate are rare. Cancer Res., 51: 1632—1637, 1991. 10. Visakorpi, T., Kallioniemi, 0-P., Koivula, T., and Isola, J. Review of new prognostic factors in prostatic carcinoma. Eur. Urol., 24: 438—449,1993. 11. Kallioniemi, A., Kallioniemi, 0-P., Sudar, D., Rutovitz, D., Gray, J. W., Waldman, F., and Pinkel, D. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science (Washington DC), 258: 818—821,1992. 12. du Manoir, S., Speicher, M. R., Joos, S., SchrOck, E., Lichter, P., and Cremer, T. Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum. Genet., 90: 590—610, 1993. 13. 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. Nail. Acad. Sci. USA, 91: 2156—2160, 1994. 14. SchrOck, E., Thiel, 0., Lozanova, T., du Manoir, S., Meffert, M-C., Jauch, A., 18. 19. 20. and Croce, C. M. Suppression of tumorigenicity of breast cancer cells by microcell mediated chromosome transfer: studies on chromosomes 6 and 11. Cancer Res., 54: 1331—1336, 1994. 25. Trent, J. M., Stanbridge, E. J., McBride, H. L., Meese, E. U., Casey, 0., Araujo, D. E., 568—571, 1990. 26. Kamb, A., Weaver-Feldhaus, lignant gliomas reveals multiple amplification sites and nonrandom chromosomal gains and losses. Am. J. Pathol., 144: 1203—1218,1994. 15. Ried, T., Petersen, I., Holtgreve-Grez, H., Speicher, M. R., SchrOck, E., du Manoir, 17. melanoma. Cancer Res., 51: 5449—5453, 1991. 24. Negrini, M., Sabbioni, S., Possati, L., Rattan, S., Corallini, A., Barbanti-Brodano, 0., Witkowski, C. M., and Nagle, R. B. Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6. Science (Washington DC), 247: Speicher, M. R., Ntlrnberg, P., Vogel, S., JSnisch, W., Doms-Keller, H., Ried, T., Witkowski, R., and Cremer, T. Comparative genomic hybridization of human ma 16. 21. Devilee, P., van Vliet, M., van Sloun, P., Dijkshoorn, K., Hermans, J., Pearson, P. L., and Cornelisse, C. J. Allelotype of human breast carcinoma: a second major site of loss of heterozygosity is on chromosome 6q. Oncogene. 6: 1705—1711. 1991. 22. Foulkes, W. D., Ragoussis, J., Stamp, G. W. H., Allan, G. J., and Trowsdale, J. Frequent loss of heterozygosity on chromosome 6 in human ovarian carcinoma. Br. J. Cancer, 67: 551—559,1993. 23. Millikin, D., Meese, E., Vogelstein, B., Witkowski, C., and Trent, J. Loss of heterozygosity for loci on the long arm of chromosome 6 in human malignant J., Liu, Q., Harshman, K., Stockert, E., Day. III., R. S., Johnson, B. E., and Skolnick, M. H. A cell cycle regulator potentially involved in genesis of many tumor types. Science (Washington DC), 264: 436—440,1994. 27. Cavanee, W. K. Accumulation of genetic defects during astrocytoma progression. S., and Cremer, T. Mapping of multiple DNA gains and losses in primary small cell Cancer (Phila.), 70: 1788—1793,1992. lung carcinomas by comparative genomic hybridization. Cancer Res., 54: 1801—1806, 28. Stearns, M. E., and McGarvey, T. Biology of disease. Prostate cancer: therapeutics, 1994. diagnostic, and basic studies. Lab. Invest., 67: 540—552,1992. Joos, S., Scherthan, H., Speicher, M. R., Schiegel, J., Cremer, T., and Lichter, P. 29. Bunyan, R., Sawczuk, I. S., Benson, M. C., Siegal, J. D., and Olsson, C. A. Enhanced Detection of amplified DNA sequences by reverse chromosome painting using expression of the c-myc proto-oncogene in high-grade human prostate cancers. genomic tumor DNA as probe. Hum. Genet., 90: 584—589,1993. Prostate, 11: 327—337,1987. Speicher, M. R., Prescher, G., du Manoir, S., Jauch, A., Horsthemke, B., Bornfeld, N., 30. Hemming, W. H., Hamel, A., MacDonald, R., Ramsey, E., Peuigrew, N. M., Becher, R., and Cremer, T. Chromosomal gains and losses in uveal melanomas Johnston, B., Dodd, J. 0., and Matusik, R. J. Expression of the c-myc proto-oncogene detected by comparative genomic hybridization. Cancer Res., 54: 3817—3823, in human prostatic carcinoma and benign hyperplasia. Cancer Res., 46: 1535—1538, 1994. 1986. Union Intemationale Contre Ic Cancer TNM Classification of Malignant Tumours, 31. Bandyk, M. 0., Thao, L., Troncoso, P., Pisters, L. L., Palmer, J. L., von Eschenbach, Ed. 4. Berlin: Springer-Verlag, 1987. A. C., Chung, L. W. K., and Liang, J. C. Trisomy 7: a potential marker of human Mostofi, F. K. Histological Typing of Prostate Turnouts. Geneva: World Health prostate cancer progression. Genes Chromosomes & Cancer, 9: 19—27,1994. Organization, 1980. 32. Alcaraz, A., Takahashi, S., Brown, J. A., Herath, J. F., Bergsralh, E. J., Larso-Keller, Kallioniemi, 0-P., Kallioniemi, A., Piper, J., Isola, J., Waldman, F. M., Gray, J. W., and Pinkel, D. Optimizing comparative genomic hybridization for analysis of DNA J. J., Lieber, M. M., and Jenkins, R. B. Aneuploidy and aneusomy of chromosome 7 detected by fluorescence in situ hybridization are markers of poor prognosis in sequence copy number changes in solid tumors. Genes Chrom. Cancer, 10: 231—243, prostate cancer. Cancer Rex., 54: 3998—4002, 1994. 1994. 347 Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research. Genetic Changes in Primary and Recurrent Prostate Cancer by Comparative Genomic Hybridization Tapio Visakorpi, Anne H. Kallioniemi, Ann-Christine Syvänen, et al. Cancer Res 1995;55:342-347. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/2/342 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on April 28, 2017. © 1995 American Association for Cancer Research.