Allelic Imbalance and Microsatellite Instability in Prostatic Adenocarcinoma1
Julie M. Cunningham,2 Ailin Shan,2 Myra J. Wick, Shannon K. McDonnell, Daniel J. Schaid, David J. Tester,Junqi Qian, Satoru Takahashi, Robert B. Jenkins, David G. Bostwick, and Stephen N. Thibodeau3
Department of Lubnratory Medicine anil Pathology. Mayo Clinic. Rochester. Minnesota 55905
ABSTRACT
Although prostate cancer is one of the most common malignancies ofmales in Western countries, relatively little is known about the molecularmechanisms involved in tumor initiation and progression. Allelic lossstudies have suggested the involvement of multiple tumor suppressorgenes (TSGs), but few detailed studies of all chromosomes have beenperformed. In an effort to localize and identify candidate TSGs, weperformed allelic imbalance (AI) studies on 55 prostate cancers, using 135polymorphic microsatellite markers. For the entire chromosome, AIranged from a low of 0% on chromosomes 14 and 20 to a high of 71 % onchromosome 8. Chromosomal regions demonstrating at least twice thebackground frequency of AI (ranging from 20 to 69%) included 5q, 6q,7q, X|>, 13, I(H|. 18q, and 21. In addition, AI was examined for associationwith a number of clinicopathological parameters. AI on chromosomes 7and 16 were each associated with greater age at diagnosis (P = 0.009 and
0.001. respectively), and AI on chromosomes 10. 16, and 18 was associatedwith aneuploidy/tetraploidy (P = 0.037, 0.013, and 0.054, respectively).
Furthermore, AI on chromosome 5 was associated with a higher pathological stage (P = 0.021) and on chromosome 8 and 16 with a higherGleason score (P = 0.027 and 0.041, respectively). No tumor exhibited a
phenotype of widespread microsatellite instability. These results indicatethat there likely exist multiple sites harboring candidate TSG in prostatecancer, some of which may have important clinical implications, andwhich argue against widespread microsatellite instability.
INTRODUCTION
Prostate cancer is one of the most common malignancies of malesin Western countries, accounting for41% of all male cancers and 14%of male cancer-related deaths ( 1). with an estimated lifetime incidencein the United States of about 19% (1-5). Despite the high level of
morbidity and mortality associated with this disease, very little isknown about the molecular mechanisms involved in tumor initiationand progression. In recent years, substantial progress has been madein predicting outcome of patients with early prostate cancer. Currently, the most important prognostic factors appear to be stage, grade,ploidy, and tumor volume (6). In spite of these advances, however, thepredictive ability of these factors for an individual patient remainslimited, and new prognostic markers are needed to more preciselyidentify patients at risk for tumor recurrence and death.
The biology of prostate cancer is still poorly understood, but recentadvances in our understanding of the molecular genetics of othercommon malignancies offer new insights that can be applied to thestudy of prostate cancer. Current evidence suggests that the process oftumorigenesis involves: (a) activation of dominantly acting oncogenesthat promote cell proliferation (7); (b) the loss of TSG4 functions that
normally regulate cell proliferation (8): and (c) mutator gene defects.
Received 4/10/96: accepted 7/26/96.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Prostate Spore. NIH P20 CA 58225.2 J. M. C. and A. S. contributed equally to this study.'To whom requests for reprints should he addressed. Phone: (507) 284-4696: Fax:
such as the recently described alterations in several DNA mismatchrepair genes (9-11).
The role of TSGs in prostate cancer has been explored by AI anddirect examination of known TSGs. Although there is a great deal ofvariability between studies, the cumulative data demonstrate frequentAI on chromosomes 7. 8. 10, 13, 16, 17, and 18 (12-41). Only onestudy has described a complete "allelotype" (20). with the highest
frequency of alÃeleloss occurring for chromosomes 8. 10, 16. and 18.Allelic loss at other chromosomal loci occurred at a lower frequencyof 5-20%. The major limitations of that study, however, were that a
limited number of DNA markers were available for each of thechromosomes examined, and the number of tumors examined wassmall. Two recent studies, using comparative genomic hybridization,reported loss on chromosome arms 8p. 13q. 6q, 16q. 18q. and 9p (27,28).
Several chromosomes have exhibited the presence of AI, but mostfine mapping studies have focused primarily on chromosome arm 8p(which has consistently shown the highest frequency of AI), 7q, andchromosomes 10 and 16. For chromosome 8. at least two regions havebeen implicated: 8p22 and 8p 12-21 (29-36), and Macoska et al. (36)
suggest an additional site. Allelic loss has been identified on botharms of chromosome 10 (19, 21, 25), and a recent report found a highrate of loss at 10q23-25 (37). A potential TSG mapped to 10q25,
Mxil, seems not to be a gene important for the pathogenesis ofprostate cancer (37). One region on chromosome 16 (16q22.1-24) is
lost frequently (21). and the common region of deletion for chromosome 7 is 7q31.1-q31.2 (38-40).
The demonstration of AI for certain chromosomes has implicatedpreviously identified TSGs. including APC on 5q, Rb on 13q, p53 on17p, DCC on 18q. and BRCA1 on 17q. Current data, however, suggestthat p53 and Rb do not appear to play a role in a significant numberof prostate tumors, but rather are restricted to a subset of moreadvanced cases (12-14). Loss on chromosome 5q included the APC
locus, but APC gene mutations are not always present (15). Similarly,the losses reported for the chromosome 17q and 18q arms include theregions to which BRCAÌ(16, 17) and DCC (18) map, respectively.However, their role in prostate cancer remains unclear.
In an effort to delineate further the role of TSGs in prostate cancer,we performed AI studies on 55 prostate cancers using 135 markers,with approximately 6 markers for each chromosome (3 markers onacrocentric chromosomes). The data were analyzed to determinecommon regions of loss for some chromosomes and for any association with routine clinicopathological parameters.
MATERIALS AND METHODS
Tissue Samples. Paired prosiate cancer and adjacent normal prostate tissues were obtained from patients undergoing radical prostatectomy at theMayo Clinic between 1992 and 1994. Those portions not required for diagnostic purposes were immediately frozen at —¿�7()°Ctor future studies. The
location, size. Gleason score, and pathological stage of each cancer wererecorded. As pan of the routine clinical practice. DNA ploidy results from flowcvtometric evaluation of paraffin-embedded sections were available (42).
DNA Extraction. Tissue processing and DNA extraction were performedas described previously (43). Briefly, using H&E-stained cryostat sections as
reference, normal tissue was trimmed by microdissection of the specimen and
DNA extracted from multiple 10-/^m sections containing >70% tumor cells.
Because of the nature of prostate cancer, it was not possible to entirelyeliminate contaminating normal cells. DNA was also extracted from pairednoncancerous tissue.
Microsatellite Analysis. We used 135 polymorphic microsatellite markersto examine chromosomes 1-22, with an average of 6 markers for eachchromosome and 3 on the acrocentric chromosomes. PCR and gel electro-phoresis were performed as described by Thibodeau er ill. (11). Autoradio-
graphs of normal/tumor pairs were analyzed by densitometry using NIH Image1.47 software. AI was considered to be present when the relative intensities ofthe two alÃelesin tumor DNA differed from that present in the normal DNAlane by a factor of at least 1.5 (29). Although not all experiments wererepeated, select samples (those with high frequencies of Al) were subjected toverification, and these showed good reproducibility. In general, normal DNAgave reproducible band intensities from run to run. although some variabilitywas encountered. In a separate series of experiments, in which replicate PCRreactions were evaluated, replicate values for the ratio of allelic bands variedtypically by a difference of approximately 0-25%. For this reason, the differ
ence between intensities had to exceed 1.5 to score a normal/tumor pair ashaving AI. Complete loss of an alÃelewas relatively rare, because most prostatecancer DNA preparations contain DNA from contaminating nonmalignantcells.
Microsatellite instability at a given locus was defined by the presence ofnovel fragments after PCR amplification of tumor DNA that were not presentin the PCR product generated by normal DNA (11).
Statistical Analysis. AI at each of the loci was assessed for associationswith each of the following clinical and pathological parameters: patient age atdiagnosis, preoperative PSA concentrations, pathological stage, Gleasonscores, and DNA ploidy status. )f tests were used to assess associationbetween AI for each of the markers with preoperative PSA levels and DNAploidy status. Fisher's exact test was used for Gleason scores and when the
sample sizes were insufficient for \~ analyses. The Mantel-Haenszel ^ test for
linear trend was used to determine whether there was a trend in the proportionsof AI over the levels of stage (44). The distribution of age at diagnosis wascompared using the Wilcoxon rank sum test. All analyses were performed
using the SAS software. Because of the difficulty in assessing the dependencyof various parameters, none of the reported P values are corrected for multipletesting.
53 24 23 1
NT NT NT NT NT
D8S258 D8S258 LPL LPL D8S261
D13S325 D13S325 D18S851 D21S156 D21S156
Fig. 1. Representative examples showing AI. Sample numbers are noted ahtive. N.DNA from normal tissue: T. DNA from corresponding lumor specimen. Tnji nw, AI alD8S25X. Liptiproiein Upase gene, and DXS261. Boittim rim. Al al D13S325, DIXSX5I.and 1)21SI 56.
RESULTS
Patient Population. Matched normal and malignant tissues wereobtained from 55 patients with prostate cancer. The clinicopatholog-
ical parameters of these patients and their tumors are shown inTable 1.
AI. Using 135 microsatellite markers, chromosomal AI was examined for all autosomes in 55 prostate cancers. Examples of AI at
Table I I'uiietii characteristics
CharacteristicsAge
atdiagnosisTumorDNA
ploidyDiploidTetraploidAneuploidGleason
seore3678')Tumor
stageT,N,,M(1T,N„M„TXN
+M„PSA(preoperative,ng/ml)•CIÒ==10n
(%)Median.
65yrRange.49-78yr21
(38.2)27(49.1)7(12.7)5
(9. 1)7(12.7)20(36.4)10(18.2)13(23.6)26
(47.3)21(38.2)8(14.5)31
(56.4)24(43.6)
selected markers are shown in Fig. 1. The markers were chosen fromthe telomeric, central, and centromeric regions of each chromosomalarm. Those selected for study and their chromosomal location areshown in Fig. 2. The order shown on the chromosomal ideogramrepresents the genetic order for each marker and. if known, its physical location. These data were obtained from current published genetic-
maps, and at this time, three markers (Fig. 2, *) cannot be placed at
specific regions on the designated chromosome. Fig. 2 also shows thefrequency of AI for each individual locus, and Fig. 3 shows thecumulative frequency of AI for each chromosome and chromosomalarm. The data for chromosome 7 were reported previously (38).
All but five tumors (91%) exhibited AI on at least one chromosome.AI on three or more chromosomes was noted in 35 (62.6%) tumors,although no specific combinations of chromosomal imbalance couldbe discerned. Chromosome 8 demonstrated the highest frequency ofAI (71%). and the chromosome or chromosomal arm exhibiting themost frequent AI was at 8p (69%). In addition to chromosome 8,significant AI (S20%) was found for chromosomes I. 4, 5. 6. 7, 9, 10,13, 16, 17, 18, and 21 (Fig. 3). When each of the markers wasevaluated individually, however. AI on chromosome I. 4, and 9 didnot appear to preferentially involve any particular region (Fig. 2).Those areas suggesting particular regions of interest for AI include3p25-26 (includes von Hippel Lindau gene), 5ql2-q23 (includes¿PC), 6q, 7q31.1,8p, 10q23-25. 13. 16q, 17p (includessi), 18, and21q22.2-q22.3.
Fig. 4 contains the data for those chromosomes exhibiting thehighest frequencies of AI, namely, chromosomes 8, 16, and 18. For
Fig. 2. Ideograms showing locations of microsatellilc markers and frequency of AI for chromosomes 1-22. The order shown represents the genetic order lor each marker. ßfUi'.v.regions demonstrating high rates of AI. *. markers lhat cannot he placed at specific regions on the chromosome.
chromosome 8. all but one of the tumors with AI on 8p exhibited AI locus. Of the tumors not showing AI on chromosome 8 (n = 16). all
at 8p22 loci. The highest frequency was at the Lipoprotein Upase gene but three were uninformative at one or more 8p22 loci. The twolocus (Figs. 2 and 4). although six tumors did not show AI at this tumors without AI at 8p loci exhibited AI at DKS567. which maps
UHI
9(1
Fig. 3. The frequency of AI on the p arm (D), qarm (^1 and whole chromosome (•)lor each chromosome examined.
Fig. 4. Pattern of Al on chromosomes 8. 16. and 18 for inJiMilu.il prostate tumors. Only cases demonstrating AI are shown. C. no allelic loss: •¿�.AI: Mimk A/KÕIÕ-V.marker notinformative. Sample numhers are noted above.
distal to D8S87 and proximal to D8SÌ64,and at DKS272. which mapsto the telomcric region of 8q (8q24.2-q24.3).
For chromosome 18, D18SX51 (18q21.1) exhibited the highestfrequency of AI. AI at D18S64 (18q21.3) was also frequent, hut DCC.which lies close to D18S85Õ at 18q21.1, was not often found toexhibit AI. One tumor exhibited imbalance at only D18SX5I: the othertumors with AI at DÕKSK5Õwere either uninformative or had AI atDÌKS64and/or DCC. For chromosome 16, AI was noted most frequently at DI6S5.W ( 16q24); only one tumor with AI on chromosome16 did not show AI at this marker.
Microsatcllitc Instability. None of the cancers demonstratedwidespread microsatellite instability. Eighteen tumors had alterations
at one or two loci. Of the 5803 genotypes, microsatellite instabilitywas detected at only 22. Changes were observed in both dinucleotide(14 of 22) and tetranucleotide (8 of 22) repeats. Compared to othercancers (such as colorectal carcinoma), the overall mutation rateobserved at these loci in prostate cancer was extremely low.
Correlation of AI with Clinicopathological Parameters. Forthose chromosomes demonstrating significant rates of AI (chromosomes 5. 6, 7. 8, 10, 13, 16. 17. 18. and 21 ). the relationships betweenAI on a given chromosome or chromosomal arm and tumor DNAploidy. pathological stage, and preoperative PSA concentration (Table2). and patient age at diagnosis were examined. For these analyses, thechromosome or chromosomal arm was scored as having AI if any one
Table 2 Correlation of clinicopathologicul parameters with AI
Percentage of Al within clinicopathological subgroups
Chromosomenumber"55q77q
88p111lOq
1616q18IgqDiploiü
(21)"14.314.3
191961.961.9
4.8014.320
23.814.3DNA
ploidyTetraploidy/
Ancuploidv(34)29.429.4
34.431.376.573.5
29.426.5
47.148.4
5032.4fUS'NSNSNS
NSNS
0.037''0.009''
0.0130.064
0.0540.205T2N0
(26)15.415.432.032.0
65.465.4
11.57.7
42.343.5
38.523TumorT3N0
(21)1919
151571.466.7
28.623.8
1922.2
38.123.8stageTXN+
(8)62.562.5
5037.5
87.587.5
2525
5080
5037.5Gleason
score/>'0.0210.021
NSNSNSNSNSNSNSNS
NSNS<7
(12)8.38.3
8.38.341.741.7
8.38.3
8.312.?
58.341.7a7
(43)27.927.9
34.231.7
79.176.7
23.318.6
41.944.7
34.920.9PNSNSNSNS
0.0270.033NSNS
0.0410.124
NSNS<10
(31)16.116.1
2020
61.361.3
16.19.7
2932
41.929PSA
ng/mlaio(24)33.333.3
39.134.8
83.379.2
2525
41.747.6
37.520.8rNSNS
NSNS0.0740.155
NSNS
NSNSNSNS
"Chromosomes 6. 13. 17. and 21 showed no significant correlations with any parameter tested.'' Number of patients within each subgroup.' P values are reponed when <0.1 for either the whole chromosome or a chromosomal arm. All reported P values are from \~ tests unless otherwise noted.'' Mantel-Hucnszel test.' Fisher's exact test.' NS. not significant.
of the markers demonstrated AI on that chromosome or chromosomalarm. Survival analyses were not performed, because the follow-up
times were limited and none of the patients had died. AI on chromosomes 10. 16q. and 18 (hut not 18q alone) was associated with tumorDNA aneuploidy/tetraploidy (P = 0.037, 0.013, and 0.054 respec
tively: Table 2). There was no tendency for the seven aneuploidtumors to show AI on entire chromosomes. With the exception ofchromosome 5. there was no association between AI and pathologicalstage for any of the chromosomes or chromosomal arms examined,either by examining for trends across all stages (Table 2) or bycomparing node-positive versus node-negative cases (data not
shown). Tumors showing AI on chromosome S were of higher stagethan those without AI (P = 0.021: Table 2). with cancers fromnode-positive patients having more frequent AI. compared to thosethat were node-negative (P = 0.013. Fisher's exact test). AI on
chromosomes 8 and 16 (hut not 16q alone) was associated with higherGleason scores (P = 0.03 for both 8 and 8p, 0.04 for 16, and 0.12 for
16q: Table 2). whereas preoperative PSA levels showed a trend withAI on chromosome 8, but not 8p alone (P = 0.074 and 0.155.
respectively: Table 2). Patient age at diagnosis was greater in patientswhose tumors had AI on chromosomes 7 and 16. with the median agesof 69 for each, compared to 61.5 years each for those without AI(P = 0.009 and 0.001. respectively). No other associations were
noted.We next examined whether AI at specific loci or regions was
associated with any of the above parameters. Although no associationswere found for either the entire chromosome or the q arm of 7. AI at7q31 was associated with higher Gleason scores as reported previously (P = 0.048, Fisher's exact test: Ref. 38). None of the loci on
chromosomes 6. 13. 16. 17, 18, or 21 showed association of AI withany of the parameters tested. For chromosome 5. however, AI atD5S806 was marginally significant for an association with pathological stage (P = 0.062). although this was not the case for D5S346(APCY. (P = 0.092). On chromosome 8. AI at D8S254 was associatedwith a higher Gleason score (P = 0.045). whereas DIOS254 onchromosome 10 was marginally associated with ploidy (P = 0.071).
In addition to evaluating AI on each chromosome, we examined thecumulative number of chromosomes demonstrating AI in a tumor.This cumulative loss was evaluated in two ways: (a) the total numberof chromosomes demonstrating any AI; and (hi the cumulative number of chromosomes demonstrating high frequency of AI (i.e..
>20%). There was no association between the cumulative changesand pathological staging or Gleason scores. Tumors that were aneuploid or tetraploid had AI on an average of 4.6 chromosomes, whereasdiploid tumors had AI on an average of 2.8 chromosomes(P = 0.0083). When only those chromosomes with high frequenciesof AI were considered, a similar trend was noted (P = 0.006).
Likewise, tumors from patients with preoperative PSA levels ^10ng/ml had AI on an average of four chromosomes compared withthree chromosomes for those with PSA levels < 10 ng/ml (P = 0.04).
This association, however, was no longer statistically significant whenonly the chromosomes with high frequencies of AI were considered(P = 0.114).
DISCUSSION
In this study, we performed a detailed allelotyping of 55 prostaticadenocarcinomas with 135 highly polymorphic microsatellite markers. Those regions demonstrating the highest frequency of AI included8p and 16q. followed by 18q, 6q, 7q, 5q, 21, lOq, 17p. 13, and 3p.Overall, these results are consistent with those published previously.As in other reports (29-36). chromosome 8 demonstrated the highest
frequency of AI. In our study, 71% of tumors were found to have AIon 8 and 69% on 8p. Frequencies of AI. comparable to those reportedpreviously, were detected for chromosomes 5q (25, 26), 6q (27), 7q(39. 40). 13q (27. 28). 16q (19, 20. 24. 25. 27). and 18q (18-20).
Compared to other studies, however, we observed relatively lowfrequencies of AI on chromosome 10 (20% overall versus previouslyreported 62% at 10q22-24: Ref. 37) and I7q (6% versus previously
reported 39% and 52%; Refs. 16 and 17). The region on 17q showingthe highest rate of AI in the study by Gao el al. (16) included BRCA1.A marker used in our study maps within this region (D17S589) but iscentramene to BRCAÌ.Of significance in the present report are thenovel findings of AI at 21q22.2-q22.3 in 23% of tumors and at3p25-26 in 20%. In the reports of genome-wide analysis (20, 27, 28),
none found AI on chromosome 3 or 21. One of these used Southernblotting (20), and the other two used comparative genomic hybridization (27. 28). Additional fine mapping studies on these chromosomes, however, will be required to verify these findings.
In our study, the marker demonstrating the most frequent AI on 18qwas at 18q 21.1, close to the DCC gene. Whereas other studies of AIon chromosome 18 in prostate cancer implicated either DCC (18, 23)
or a region distal to DCC (26), our results suggest that another TSGin the region of. but distinct from. DCC may be important in prostatecancer. A recently identified candidate gene localized to this region isDPC4 (45). It will be important to perform additional high densitymapping of this region and to examine DPC4 in more detail to exploreits potential involvement in prostate cancer.
Specific chromosomal regions exhibiting AI in the present studyare similar to those reported previously for 16q (16q2l-24) and 5q
(5q2l: Refs. 21 and 25). AI was detected with a marker proximal tothe Rh gene on chromosome 13; we did not examine Rb itself.Candidate TSGs that map proximal to Rb include KRCA2, DPCI, andDPC2 (46). For chromosome 8. AI was almost always observed at8p22. a region identified by others (29-31. 35. 36). Imbalance at other
8p loci that have been suggested as regions harboring TSG in prostatecancer (33. 34. 36). or on 8q. was seen in association with 8p22 AIwith only two exceptions. Although we did not use markers to identifyAI at 8p21 or 8p I 1. it is striking that 8p22 was invariably involved inthis cohort of prostate tumors.
Although our results are largely in agreement with those reportedpreviously, there are some differences. However, because of differences in analytical techniques and populations studied, a comparisonof results is difficult. For example, previously reported AI on 8p inprostate cancer ranged from 27 to 65% (21, 29-36). The lowerfrequencies were found in studies that used paraffin-embedded mate
rial (22. 24) or had small numbers of informative tumors ( 12). Withfew exceptions, only small numbers of tumors have been examined,and these have been of varied pathological stages, limiting clinical-
genetic correlations. Furthermore, the contribution of prostate tumorheterogeneity, which is very likely to exist (47), has rarely beenconsidered. The evaluation of tumor heterogeneity was not a focus ofthe present study, but deserves consideration in future studies. Variations in the state of the tumor tissue (autopsy, frozen, or paraffin-
embedded), the number of chromosomes examined, the number ofmarkers used, and the technology employed confound attempts tocompare studies. Additional differences among studies includewhether visualization or quantitative ¡mageanalysis were utili/ed, thecriteria established for assessment of imbalance or loss, and differingsensitivities of Southern blotting versus PCR or comparative genomichybridization studies.
The regions identified as possible sites for TSG relevant to prostatecancer (8p. 6q. I6q. and I8q distal and proximal to DCC} have alsobeen implicated in other cancers. Allelic loss on 8p occurs in hepa-toccllulur carcinoma (48), colorectal carcinoma (31. 48-50). head and
neck cancer (51). bladder cancer (52). collecting duct carcinoma ofthe kidney (53), and non-small cell carcinoma of the lung (54). Theregions commonly deleted include 8p23-21.3 (48). 8p21.3-22 (54),and 8p21-l 1.2 (51). AI on chromosome 16 has been found in breastcancer (55-57) and hepatocellular carcinoma (58-60), and the regions implicated were 16q22.1-qter (55-57), 16q22.1-23.2 (60). and16q24.l-qter (55-60). Loss telomeric to the DCC gene has been
reported for ovarian cancer (61). and losses at 7q3I.I have beenobserved in breast and lung cancer (reviewed in Ref. 38). Allelic losson 6q has been noted in colorectal carcinoma (62), gastric cancer (63),ovarian cancer (64). breast cancer (65), and melanoma (66). Conversely, chromosome 10 has been implicated in glioma (67. 68).endometrial cancers (69). and melanoma (70). Thus, many of theregions of AI identified in our study, as well as other investigations ofprostate adenocarcinoma, have been implicated as possible sites forTSGs in other cancers.
Instability at microsatellites was an infrequent finding in this study.Although these results are consistent with one report (71 ). two otherreports describe rates of tumor microsatellite instability of 43% (72)and 14.6% (73) in prostate cancer. However, these latter studies
scored tumors as having microsatellite instability when only singleloci demonstrated alterations. Several studies have now shown thatalterations at microsatellites occur at a higher frequency in cancercompared to benign tissue (7-9). However, it is important to distin
guish between tumors demonstrating a low frequency of alterationsand those having instability at multiple loci. In tumors with defectiveDNA mismatch repair, microsatellite instability is usually detected ingreater than 30% of loci (74). In our study, none of the tumorsexhibited this characteristic widespread instability. These data, therefore, suggest that defective mismatch repair does not play a significantrole in sporadic prostate cancer. Additionally, the frequency of instability at any locus (22 of 5803 loci) was extremely low. much lowerthan that described for other cancers (74).
The prognostic value of AI in prostate cancer is uncertain, possiblybecause of the small numbers of tumors examined in many studies andthe varied clinical and pathological stage of cancers examined. In ourstudy, the cumulative AI on a chromosome or chromosomal arm wasnot associated with any consistent clinicopathological parameter except for the following: tumors with AI on chromosome 5 or 5q wereof higher pathological stage than those without AI; those with AI onchromosomes 10, 16q. and 18q were more likely to be nondiploid;those with AI on 7 and 16q were of older age at diagnosis; and thosewith AI on chromosomes 8p and 16 were associated with higherGleason scores. A trend toward higher PSA concentrations was notedin patients whose cancers exhibited AI on chromosome 8. but not 8pspecifically. The association of AI al individual loci with these parameters, however, was limited, suggesting that further delineation ofAI is required to more fully investigate the role of specific sites asprognostic markers. Although some associations were found in thisdata set with a variety of clinicopathological parameters, it is important to note that these correlations were sometimes based on smallnumbers, and multiple statistical tests were performed, inflating thechance of false-positive findings. None of the reported P values are
corrected for multiple testing: rather, we caution that these correlations be only tentatively accepted until reproduced independently inother studies.
Although occurring at a consistently high rate. AI on chromosome8 has been found to be unrelated to the progression of cancer,suggesting that it occurs early in tumorigenesis (30. 36. 38). In ourstudy, chromosome 8 AI was associated with higher Gleason scoresbut not with any other prognostic parameter. In contrast, Takahashi eltil. have suggested that gain of 8 or 8q may be of prognostic relevancein stage T3 prostate cancer (41). The latter study also implicatedaneusomies of chromosome Y in prostate cancer progression. In asmall cohort. Brewster et cil. (26) found that allelic loss of DCC (or aTSG distal to DCC). APC. and />5.i genes was more frequent inadvanced rather than localized cancers. At present, however, nomolecular marker meets the criteria, established by the American JointCommittee on Cancer, of an important prognostic factor in prostatecancer (75).
In conclusion, we found high rates of AI on multiple chromosomesin prostate adenocarcinoma. These include sites described previously,as well new regions that may harbor TSGs relevant to the process ofprostate tumorigenesis. Additionally, it will be important to furthercharacterize known TSGs. such as DPC4 and BKCA2, and determinetheir role in prostate tumorigenesis. Although the number of patientsstudied is small, some associations were noted with several clinicopathological parameters. Additional studies, using multiple microsatellite markers clustered at chromosomal regions that frequently exhibit imbalance, are needed to more fully delineate relevant geneticchanges in prostate cancer and their correlations with clinicopathological parameters.
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