[CANCERRESEARCH54,3998-4002,August1, 19941

Advances in Brief

Aneuploidy and Aneusomy of Chromosome 7 Detected by Fluorescence in SituHybridization Are Markers of Poor Prognosis in Prostate Cancer'

Antonio Alcaraz, Satoru Takahashi, James A. Brown, John F. Herath, Erik J- Bergstralh, Jeffrey J. Larson-Keller,Michael M Lieber, and Robert B. Jenkins2Depart,nent of Urology [A. A., S. T., J. A. B., M. M. U, Laboratory Medicine and Pathology (J. F. H., R. B. fl, and Section of Biostatistics (E. J. B., J. J. L-JCJ, Mayo Clinicand Foundation@ Rochester, Minnesota 55905

Abstract

Fluorescence in situ hybridization is a new methodologj@which can beused to detect cytogenetic anomalies within interphase tumor cells. Weused this technique to identify nonrandom numeric chromosomal alterations in tumor specimens from the poorest prognosis patients with pathological stages T2N@M,Jand T3NOMOprostate carcinomas. Among 1368patients treated by radical prostatectomy, 25 study patients were ascertamed who died most quickly from progressive prostate carcinoma within3 years of diagnosis and surgery. Tumors from 25 control patients whosurvived for more than 5 years and who were matched for age, tumorhistological grade, and pathological stage also were evaluated. The tumorsfrom all 25 (100%) poor prognosis patients and from 11 of 25 (44%)control patients were found to be aneuploid by fluorescence in situ hybndization (P < 0.0001). Alterations ofchromosome 7 were observed in 24of the tumors (96%) from the poor prognosis patients versus 3 tumors(12%) from the control group (P < 0.0001). Moreover, a characteristicaneuploidy pattern with multiple abnormal chromosomes and a hypertetrasomic population was generally found in tumors from the poor prognosis patients. This preliminary study suggests that fluorescence in situhybridization studies of prostate cancer specimens may help to identifythose patients at highest risk for early cancer death.

Introduction

Prostate cancer is the most common cancer in United States men,with approximately 200,000 newly diagnosed cases and 38,000 deathsdue to prostate cancer per year (1). The distinction between thosecases of prostate cancer destined to progress rapidly to lethal metastatic disease and those with little likelihood of causing morbidity andmortality is a major goal of current prostate cancer research.

Factors such as clinical and pathological stage and histologicalgrade are conventionally used to help predict prognosis for patientswith localized prostate carcinoma (2). Among newer techniques,ploidyIDNA analysis using FCM3 may help refine prognostic risk forpatients with tumors of common histological grades and stages (3).However, it is widely recognized that FCM ploidy analysis cannotdetect small changes in DNA content or chromosome number. Routine cytogenetic studies of solid tumors are a more precise approach todetect numerical and/or structural defects. However, because metaphase cells are required for analysis, cytogenetic studies are hampered by the difficulties of specifically stimulating tumor cells todivide. It has been particularly difficult to perform routine cytogenetic

Received 4/6/94; accepted 6/14/94.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.

I Supported in part by a grant from Imagenetics Inc., Naperville, IL (to R. B. J.); GrantCA 58225 from NIH (to R. B. J., M. M. L., S. T.); and by Grant FIS 93/5326 from theHospital ainic de Barcelona and Fondo de Investigaciones Sanitarias (to A. A.).

2 To whom requests for reprints should be addressed, at Cytogenetics Laboratory,

Mayo Clinic, 200 First Street SW, Rochester, MN 55905.3 The abbreviations used are: FCM, flow cytometry; FISH, fluorescence in situ

hybridization; BPH, benign prostatic hyperplasia; SSC, standard sodium citrate; 2SD, twostandard deviations.

studies on prostate carcinoma samples. Interphase cytogenetic analysis using FISH to enumerate chromosomes has the potential to over

come many of the difficulties associated with traditional cytogeneticstudies. Previous studies from this institution have demonstrated thatFISH analysis with chromosome enumeration probes is more sensitivethan FCM for detecting aneuploid prostate cancers (4, 5, 7).

We designed a case-control study to test the hypothesis that specific, nonrandom cytogenetic changes are present in tumors removedfrom patients with prostate carcinomas with poorest prognoses . Weidentified 25 patients with clinically localized prostate carcinomatreated by radical prostatectomy who died from metastatic prostatecancer within 3 years after treatment. These patients were thenmatched for age, pathological stage, and tumor histological grade witha group of patients who survived for more than 5 years after surgery.FISH analysis was then carried out using chromosome enumerationprobes for 12 different chromosomes.

Patients and Methods

Study Design. Among 1386 patients with pathological stage T2NOM@JandT3N@M0prostate cancer who underwent a radical prostatectomy at the MayoClinic from 1966 to 1990, 31 died of prostate cancer between 6 months and 3years after surgery. These patients (30 stage T3N@MI@and 1 stage T2N@,M,@)were matched for age, pathological stage, and tumor grade with patientssurviving for more than 5 years after surgery. For those poor prognosis casesresected prior to 1988, the control cases were also matched for date of surgery.Six cases were excluded in both groups because the paraffin-embedded tissueblocks did not contain residual tumor, or the cells were poorly preserved. TenBPH surgical samples, from the same time period, were studied to obtainnormal value information.

In Situ Hybridization with Chromosome Enumeration Probes. Afterpathological confirmation, six adjacent 50-sm tumor tissue sections were usedfor FCM and FISH analyses. Tissue deparaffmization was performed. Sectionswere washed for 10 mm with 2 ml Histo-Clear (National Diagnostics, Atlanta,GA) three times. The tissue was then dehydratedin 100% ethanol (5 min,twice) and then rinsed in water (5 mm, twice). Tissues were digested in pepsinsolution (Sigma P.7012; Sigma Chemical Co., St Louis, MO; 4 mg/mI in 0.9%NaC1,pH 1.5) for 2.5 h at 37%. After filtering, isolated nuclei were washedtwice with phosphate-buffered saline and vortexed. The resultant nuclearsuspension was applied to Superfrost slides, and the slides were oven-dried at65°Cfor 10 min. FISH probes, labeled with SpectrumOrange or SpectrumGreen, specific for the centromere region of 11 chromosomes (4, 6—12,17, 18, and X) and for the mid-distal Yq (Yq12) region, were obtained fromImagenetics (Framingham, MA). Only single-probe hybridizations were per

formed. All of the currently available Imagenetics chromosome enumerationprobes were used in the study.

Slides containing isolated nuclei were dehydrated in an ethanol series (70,85, and 100%)for 2 mm each. DNA was denaturedby incubatingthe slides in70% formamide/2X SSC (300 mM sodium chloride and 30 mM sodium citrate)at 75°Cfor 4 mm, followed by dehydration in an ice-cold ethanol series.Simultaneously, the probe solution (7 @lHybridization Mix II; Imagenetics;2 @.tlprobe) was denatured for 6 min at 75°C and kept on ice until it was added

to the slides. After sealing, the slides were then incubated overnight at 37°C.Following hybridization, the slides were washed in 50% formamide/2X SSC

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FI5H MARKERS OF POOR PROGNOSIS IN PROSTATE CANCER

for 10 s, 2X SSC for 1 aria,and2X SSC/NonidetP-40 for 1 mm at 37°C.Thecounterstain 4,6-diamidino-2-phenylindole with antifade compound p-phenylenediaminewas addedpriorto analysis.

Analysis of Interphase in Situ Hybrkllzatlon. For each probe hybridization, signals from 300 nuclei were counted. For this number of enumerated

nuclei, the 95% confidence limits ofthe 0, 1, 2, 3, 4 and 5 signal proportions(p) were estimated as ±1.96V@j@00 using the binomial distribution andwere at most ±0.057at P 0.5.

Flow Cytometry. The method for DNA content analysis of paraffinembedded tissues has been described previously (4).

Statistical Analysis Comparisons of age and tumor characteristics between the poor prognosis (cases) and control groups were made using the

rank-sum and@ tests as appropriate. Matched analyses were not performed as

the primary goal of the matching was to obtain similar groups with respect totumor stage and grade. Age, which is not a risk factor for cause-specificsurvival, was used as a convenient way to select a specific control.

Within the case group, the generalized estimating equations of Liang andZeger (6) were used to model aneusomyfrequencyas a functionof chromosome, taking into account within patient correlations. All tests were two-sidedwith a = 0.05.

Results

Normal Value Studies. Mean and positive 2SD of the number ofcopies for each chromosome in the 10 cases of benign prostate tissuestudied is illustrated in Fig. 1. All of the chromosomes had an averagemonosomy (autosomes) or nullisomy (sex chromosomes) of less than5%, except for chromosome 17, which had an average monosomy rateof 7% (Fig. IA). We have previously shown that this higher rate is

most likely the result of homologous pairing (7). The mean + 2SD formonosomy (for autosomes) and nullisomy (sex chromosomes) waslower than 10% for all chromosomes. All of the autosomes had anaverage trisomy or tetrasomy level of less than 1.5% (Fig. 1B). Foreach of the autosomes, mean + 2SD for trisomy was less than 3% andfor tetrasomy less than 4%. The percentage nuclei with 4 signals foreach autosome was averaged to estimate the normal percentage oftetraploid cells with prostate tissue. The mean for all BPH specimenswas 2.58% with an SD of 0.62%.

Because of relative loss of chromosomes, inefficient hybridization,and/or homologous pairing, it was also necessary to establish thenormal proportion of trisomic to tetrasomic cells in apparently puretetraploid tumors. Several tumors in the control group and in ourprevious studies of two series of unselected prostate carcinomas (5, 7)contained a significant population of cells with 4 signals for allautosomes. In the control tumors from the current study, the tetrasomic populations averaged 13.3% of the cells enumerated (range, 7.5to 21%, eight tumors). These tumors contained no apparent aneusomicchromosomes and were thus apparently truly tetraploid (we cannotrule out aneusomies of any untested chromosome). We used theapparently true tetraploid tumors from the control group and established that, for autosomes, the mean trisomy:tetrasomy ratio was lessthan 0.51 for each chromosome. The mean + 2SD of the trisomy:tetrasomy ratio was less than 0.59 for each chromosome, except forchromosome 17, which was less than 0.82. Similar-sized tetrasomicpopulations and similar mean trisomy:tetrasomy ratios were alsoobserved in the apparently purely tetraploid tumors of our previousstudies (5, 7).

For the BPH specimens, the mean + 2SD of nuclei with 2 signalsfor chromosomes X and Y was less than 10% (Fig. 1B). There was noevidence of gain or loss of sex chromosomes in the BPH specimens.The population of cells with 2 signals for the sex chromosomesincludes both cells with the gain of one chromosome (aneusomy) andcells with twice the total chromosome content (tetraploidy). In orderto distinguish sex chromosomal aneusomy from tetraploidy, we analyzed the ratio of the sex chromosome 2-signal percentage to theaverage autosorne 4-signal percentage in the apparently true tetraploidtumors. The X and Y chromosomes had mean ratios of 1.23 and 1.30,respectively. The mean + 2SD ratios for chromosomes X and Y were1.95 and 2.08, respectively.

Abnormal Criteria. Based on the normal value studies describedabove and an inspection of the FISH data from the poor prognosis andcontrol groups, we established the following conservative cut-offcriteria: (a) a tumor was classified as tetraploid if the autosomalaverage of percentage nuclei with 4 signals (or the average autosomaltetrasomy) was 6%; (b) an abnormal monosomy (or nullisomy forsex chromosomes) required 12% of nuclei contain 1 (or 0) FISHsignals. Chromosomes that were disomic on a tetraploid backgroundwere also classified as lost; (c) an abnormal autosomal trisomy wasrequired to fulfill two criteria: (1) 7% nuclei contain 3 FISH signalsand, because of the incidence of tetraploidy, (2) the ratio of 3-signalnuclei to 4-signal nuclei be 1 for chromosome 17 and ½for theremaining autosomes; (d) sex chromosome gain or tetraploidy required 12% nuclei with 2 FISH signals; (e) to distinguish sexchromosornal gain from tetraploidy, the former required that the ratioof the percentage nuclei with 2 sex chromosome signals to the averageautosomal 4-signal percentage be 2.1; (f) abnormal hypertetrasomyrequired that the sum of the percentage nuclei with 5 signals for anyautosome or 3 signals for any sex chromosome be 6%; and (g)abnormal monosorny/nullisomy, trisomy, or hypertetrasomy defined atumor as aneuploid. Otherwise, the tumor was classified as apparentlydiploid or apparently pure tetraploid, based upon the overall averageautosomal tetrasomy.

A

10

8.50

a2

4

2

0—@ @@—@•—-——@ ______

4 6 7 8 9 10 11 12 17 18 X VChromosome

B12

100 Tnsomy

8 @TetraSOmy.50

a2

4 6 7 8 9 10 11 12 17 18 X VChromosome

Fig. 1. FISH signal distribution in BPH. Data are expressed as mean + 2SD for eachchromosomestudied.A, monosomyfor the autosomesandnullisomyfor the sex chromosomes. B, trisomy and tetrasomy for the autosomes. For the sex chromosomes, a singlecolumn illustrates the percentage of cells with two nuclear signals.

3999

0 @ionosom@'INulisomy

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Table 1 Centro,nerecopy number ofselected poorprognosis and controlcasesChromosomeCentromere

copynumber(%)°0123

4Control

22 (diploid)

FISH MARKERSOF POOR PROGNOSISIN PROSTATECANCER

No BPH specimens contained enumeration values which exceeded

these criteria.FCM and FISH Ploidy Evaluation. Table 1 shows typical results

from a FISH-diploid, FISH-tetraploid, and two FISH-aneuploid tumors. The FISH-diploid tumor (control tumor 22) had an averageautosomal tetrasomy of 3.1% and, based on the criteria listed above,contained no apparent chromosome centromere anomaly. The FISHtetraploid tumor (control tumor 17) had an average autosomal tetrasomy of 15.5% and, except for the increased tetrasomy for eachautosome and increased disomy for each sex chromosome, containedno apparent chromosome centromere anomaly. The first FISH aneuploid tumor (poor prognosis tumor 22) had an average autosomaltetrasomy of 4.3% and, based on the criteria listed above, was observed to have abnormal centromere signal distributions for chromosomes 4, 7, 18, and Y. The second FISH aneuploid tumor (poorprognosis tumor 5) had an average autosomal tetrasomy of 12.9% andwas observed to have abnormal centromere signal distributions forchromosomes 7, 9, and 10 relative to tetraploid.

Table 2 summarizes the clinical characteristics, the FCM ploidy

distribution, and the FISH ploidy distribution for the poor prognosisand control groups after the exclusion of the 6 patients from bothgroups (see “Patientsand Methods―).No significant differences werefound between the two groups for matched clinical variables: age,pathological stage, and Gleason grade. The patients from the poorprognosis group were more likely to receive adjuvant therapy(P = 0.047). There was no significant difference in the FCM DNAploidy distribution between two groups (P 0.14). By FISH analysis,all 25 tumors in the poor prognosis group were found to be aneuploid,while 11 tumors in the control group were found to be aneuploid, 8tetraploid, and 6 diploid. This difference in FISH ploidy distributionwas highly statistically significant (P < 0.0001).

Specific FISH Results. Table 3 summarizesthe aneusomic chromosomes from the poor prognosis and control groups. The aneusomies are listed as either a gain or loss relative to a reference ploidylevel inferred from the overall FISH results. Chromosomes withhypertetrasomic populations 6% were defined as a gain. Since thepoor prognosis cases were often observed to have a complex distribution of centromere enumeration counts, in some cases, the inferred

4.73.3634.01.32.02.02.72.02.30.00.0

13.715.013.716.011.719.020.015.014316.30.00.0

83234.04.75.74.04.063330.70.00.7

14.714.71439.0

14.08.7

16.014313.010.00.00.0

2.03.33.74.73.34.72.03.07.01.0

91.389.3

3.01.02.75.03.72.05.01.35.04.773.775.0

4.06.73.03-32.34.03.73.711.748.088.373.1

1.36.00.32.02.71.34.04.32.36.3

80.074.7

91.792.086.088.394.392.795.093.089.394.76.05.3

81.078.779.375.082.376.071.080.376.076.024.323.0

78.087.079.390.387.789.391.787.782.750.3

7.018.6

79.071.023.081.368.082.074.377.382.779.716.317.3

46789

1011121718xY

Control 17 (tetraploid) 46789

1011121718xY

Poor Prognosis 22 (aneuploid) 46789

1011121718xY

Poor Prognosis 5 (aneuploid) 46789

1011121718xY

a Percentageof 300 nuclei with indicated centromere copy number.

0.00.00.00.00.00.00.00.00.00.02.75.3

0.00.00.00.00.00.00.00.00.00.01.71.3

0.00.00.00.00.00.00.00.00.00.03.75.1

0.00.00.00.00.00.00.00.00.00.02.76.3

1.71.34.03.01.00.71.01.31.72.00.00.0

2.3534.34.02.32.74.03.34.73.0030.7

9.34.013.71.74.32.70.32.32.01.01.02.4

4.38.0

48.76.7

14.3735.74.02.04.01.01.7

0.00.00.00.00.00.00.00.00.00.00.00.0

0.00.00.00.00.0030.00.00.00.00.00.0

0.30.00.00.00.00.0030.00.30.00.00.0

0.70.3

13.61.01.00.70.00.00.00.00.00.0

4000

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Poor prognosispatients (n25)Control patients(n25)P°Age

atsurgery―0.67<604660—701515>7064Mean65.163.2Pathological

stage―0.99pT2NOMO10pT3NOMO2425Gleason

pathologicalgrade―0.162—4005—6377—102218Adjuvant

therapycؕ@47No39Yes2216FCM

ploidy0.14Diploid56Tetraploid710Aneuploid135Not

classifiable04FISH

ploidy<0.0001Diploid06Tetraploid08Aneuploid2511

Table 3 Summary of aneusomic chromosomesin the poor prognosis and controlgroupsPoor

prognosis groupControl groupInferredPresence

ofInferredPresenceofTumorAneusomies°stemlineploidy―hypertetrasomyTumorAneusomies°stemlineploidy@'hypertetrasomy2—6X3,—7,—8,—9,—10,—11,—12,—YX2T—1+11X2D—3—4,+6,—7,--8x3,+10,--18,—Yx2T+2T—4+4,—6X3,—7,—12,—YX2T+4T—5+7,—9,—10T+5—17D—6—11,—12,—17,—18,+XT+7T—7—7,—18T—8T—8—4,—7T—10—18D—10—4,—6,—7,—8,—9,—10,+11,—12,—17,—18,+X,+Y,—YT+11T—11—4,—6,—7,—11,+17,—18,+X,+YT+12+7D—13—7,—8X3,—12X3,—18X3T—13+6,+8,+9,+18,+YD—14—6,—7,+8,--17,—18T+14D—15+4,—6,+7,—10,—11,—YX2T+16D—16—7,+8,+11,—YX2T+17T—17—4X3,+6,+7,+8,+9,+17,+18,+X,+Y,--YX2T+18T-18—4,—6,—7,—8,—10,—11,—12,—18X3,—XX2,+YT—20—YX2T—20—4,—6,—7,—8X3,—9,—10X3,—11,--12,-—17X3,--18,+X,+YT—21—8D—21+4,+7D—22D—22+4,+7,—18,+YD—23+7,+12D—23+4,—6X3,+7,—8X3,—9,+10,—11,—12,+17,--18T+24D—25+7,-17D—25D—26—4,—6x3,--7,—8X3,—9X3,—10,—11,—12,—17,—18T—29—6X3T—27+4,—7,+11,+17,+18T+30D—29+7,+X,+YD—31—6T-30+7,—17,--18,+YD—32T—31—4,—6,—7,—9,—10,—11,—12,--17,—18,+YT—33—6,—7,—11,—12T—

FISH MARKERS OF POOR PROGNOSIS IN PROSTATE CANCER

reference ploidy level may be incorrect (e.g., diploid and not tetraploid or vice versa). However, this does not change the finalconclusion that a particular chromosome was abnormal (e.g., alters anapparent loss to an apparent gain or vice versa).

The tumors in the poor prognosis group generally had multiple aneusomies: 10 of the 25 cases containedseven or more numericchromosome

25-

! 20-

!r0@

gz

0

Table 2 Clinical characteristics and ploidy results ofpoor prognosis and control groups

4 I6 ‘7 ‘8 ‘9 ‘10‘11‘12‘17‘18‘X ‘VChromosome

Fig. 2. Distribution of aneusomic chromosomes in poor prognosis (study) and controlgroups. Ps have been calculated for each chromosome comparing the poor prognosis andcontrol groups. Twenty-four tumors were evaluated in the poor prognosis group forchromosomes 4, 10—12,and 17. For the remaining seven chromosomes in the poorprognosis group, all 25 tumors were evaluated. All 12 chromosomes were evaluated in all25 tumors of the control group.

alterations. Twenty ofthe 25 had a tetraploid-inferred stemline ploidy. No

control tumors were aneusomic for six or more chromosomes, and 8 ofthe 11 aneuploid tumors in the control group were found to have a singleaneusomic chromosome. Twelve of the control cases had a tetraploidinferred stemline ploidy. Hypertetraploidy was commonly found in tumors from the poor prognosis patients; 12 cases were found to havehypertetrasomy of one or more chromosomes, compared to zero cases inthe control group (P < 0.0001).

As can be seen in Fig. 2, for each of the 12 chromosomes, thefrequency of aneusomy in the tumors from the poor prognosis patientswas higher than in the tumors from the control patients. The largestdifferences in frequency were seen for chromosomes 7 (@= 35.5;P < 0.0001), 4 (x2= 22.5; P < 0.0001), and 18 (f 17.0;P < 0.0001).

Within the poor prognosis group, we found a significant differencein the frequency of aneusomy between chromosomes (x@ = 28.8 on11 degrees of freedom; P < 0.0025). To assess which chromosomes

a Comparing the distribution of each parameterfor the poor prognosis group with thatof the control group.

b Matching variables.

C Radiation or hormonal therapy within 3 months after surgery.

a Losses (—)and gains (+) of chromosomes are expressed relative to the inferred stemline ploidy level. For example, —6X3,—11, and +7 in poor prognosis tumor 23 refer tosignificant 1-, 3-, and 5-signal populations, respectively, relative to a tetraploid stemline ploidy.

b Ploidy level inferred on inspectionof the distributionof FISH signalsfor all twelve chromosomesanalyzed.D, diploid; T, tetraploid.

4001

I . o.oi<@ <0.05@ PoorPrognosis. 0.001<p<0.01@ •Control

@ 00.0001<p<tMlOl*. I * P<°.000l I ‘@

H@@@ @n

. 0

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FISHMARKERSOF POORPROGNOSISIN PROSTATECANCER

were producing this significant difference, each chromosome wastested using the generalized estimating equations model to determine

if it had a higher (or lower) rate of aneusomy than all the otherchromosomes combined. In view of the multiplicity of tests (8), theresulting Ps were multiplied by 12 (Bonferroni correction). The onlychromosome in the tumors from the poor prognosis group with asignificantly higher aneusomy rate than all the other combined chromosomes was chromosome 7 (adjusted P = 0.020). The independentaneusomy rates for chromosomes 4 and 18 were statistically similar tothe aneusomy rates of the other combined chromosomes (adjustedP > 0.20 for both chromosomes).

Discussion

To the best of our knowledge, this is the first study of matched poorprognosis and good prognosis patients to evaluate the association ofnumeric chromosome alterations ascertained by FISH with clinicalbehavior in prostate cancer or any solid tumor. We targeted high gradeT3NOMIJ prostate cancers treated by radical prostatectomy, tumorswhose rate of clinical progression is difficult to predict. Our results

indicate that an abnormal tumor FISH ploidy is significantly associated with a poor prognosis in patients with prostate cancer. Thequalitative analysis of the aneuploidy pattern is of particular importance. The typical aneuploid tumor found in the poor prognosis

patients was defined by aneusomies of multiple chromosomes relativeto a basic tetraploid population. Hypertetrasomic cells were frequent.In contrast, tumors in patients with a good prognosis are often diploidor tetraploid, and their aneusomies, when present, tended to be mono

somies of a single chromosome. These results suggest that tumor cells

in the poor prognosis group are cytogenetically unstable and mayrandomly lose and gain chromosomes, resulting in the different aneusomies. Such cells also had likely developed structural abnormalitiesin the retained chromosomes (9).

Except for loss of a Y chromosome and gain of chromosome 7,previous routine cytogenetic studies of metaphase cells have notclearly demonstrated nonrandom, whole chromosome loss or gain in

prostate cancer (10). In this study, we observed that all chromosomesare likely to be numerically altered in clinically aggressive prostatecancers. Because all chromosomes were more likely to be altered inthe tumors from the poor prognosis group and because a significantdifference in the frequency of chromosomal aneusomies was observedwithin the poor prognosis group, we performed detailed statisticaltests to determine which alteration(s) occurred nonrandomly in this

group. Only chromosome 7 aneusomy had a significantly higherincidence within the poor prognosis group when compared to theother chromosomes. This result, along with the almost constant presence (96%) of chromosome 7 aneusomy within the poor prognosisgroup, strongly suggests that aneusomy of chromosome 7 is a nonrandom alteration which correlates with clinically aggressive prostatecancer. Especially remarkable was the presence of trisomy 7 in thefive FCM diploid tumors in the poor prognosis group. This resultsuggests that trisomy 7 may be an early change that occurs prior to

other chromosomal aneusomies in poor prognosis prostate carcinoma.

Chromosome 7 aneusomies have been correlated withaggressiveness in melanoma (1 1) and bladder carcinoma (12).Recently, a significantly higher rate of trisomy 7 has been observedin advanced (T3NOMOand higher) but not in early (T2N0M@)stagesof prostate tumors (8), although neither survival information norclinical follow-up was reported. It is possible that aneusomies ofchromosome 7 are simply associated with poor prognosis tumors

and are not of etiological significance. However, there is evidencethat human chromosome 7 is necessary and sufficient for both theestablishment and maintenance of invasiveness and metastatic potential. For example, malignant mouse-human somatic cell hybrids retaintheir metastatic potential after losing all human chromosomes exceptchromosome 7 (13).

Several authors have criticized the significance of the presence oftrisomy 7 in human cancer cells (14). The most consistent criticism is

that the observed alterations in chromosome 7 number are found in theinfiltrating lymphocytes and not in the tumor cells (15). We haveperformed simultaneous FISH and immunohistochemical staining andhave observed trisomy 7 in prostate epithelial tumor cells but not ininfiltrating leukocytes (5).

In conclusion, we have found a characteristic FISH pattern ofaneuploidy defined by multiple aneusomies, hypertetrasomic cells,and the consistent presence of alterations of chromosome 7 to have astrong association with early death in patients with localized prostatecancer. These fmdings may be of important practical use, since wehave developed the methodology to perform rapid FISH pretreatmentanalysis on prostate biopsy core specimens that can subsequently beused for routine histopathological examination (5). If the results fromthe preliminary study reported here can be extended and confirmed,

the pretreatment detection of cytogenetic markers of poor prognosis

described above may be of help in selecting patients who wouldbenefit from a most aggressive therapeutic approach.

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G., and Smith,G. J. Consensusreviewof the clinicalutility of DNA contentcytometry in prostate cancer. Cytometry, 14: 497—500,1993.

4. Persons, D. L, Gibney, D. J., Katzmann, J. A., Lieber, M. M., Farrow, G. M., andJenkins, R. B. Use of fluorescent in situ hybridization for deoxyribonucleic acidploidy analysis of prostatic adenocarcinoma. J. Urol., 150: 120—125,1993.

5. Takahashi, S., Qian, J., Brown, J. A., Alcaraz, A., Bostwick, D. G., Lieber, M. M.,and Jenkins, R. B. Potential markers of prostate cancer aggressiveness detected byfluorescence in situ hybridization in needle biopsies. Cancer Res., 54: 3574—3579,1994.

6. Liang, K-Y., and Zeger, S. L Longitudinal data analysis using generalized linearmodels. Biometrika, 73: 13—22,1986.

7. Brown, J. A., Alcaraz, A., Takahashi, S., Persons, D. L, Lieber, M. M., Jenkins, R. B.Chromosomal aneusomies detected by fluorescent in situ hybridization (FISH) inclinically localized prostate carcinoma. J. Urol., 152, in press, October 1994.

8. Bandyk, M. 0., Thao, L C., Troncoso, P., Pisters, L L, Palmer, J. L, vonEschenbach, A. C., Chung, L W. K., and Liang, J. C. Trisomy 7: a potentialcytogenetic marker of human prostate cancer progression. Genes ChromosomesCancer, 9: 19—27,1994.

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1994;54:3998-4002. Cancer Res   Antonio Alcaraz, Satoru Takahashi, James A. Brown, et al.   Prognosis in Prostate Cancer

Hybridization Are Markers of Poorin SituFluorescence Aneuploidy and Aneusomy of Chromosome 7 Detected by

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