[CANCER RESEARCH 50, 3795-3803, June 15. 1990]

Frequency and Pattern of Karyotypic Abnormalities in Human Prostate Cancer1

Arthur R. Brothman,2 Donna M. Peehl, Ankita M. Patel, and John E. McNeal

Departments of Pediatrics [A. R. B.¡and Microbiology and Immunology [A. R. B., A. M. P.] Eastern Virginia Medical School, Norfolk, Virginia 23501, and Division ofUrology, Stanford University School of.Medicine, Stanford, California 94305 [D. M. P., J. E. M.]

ABSTRACT

The cytogenetic evaluation of 30 cultured primary prostatic cancerspecimens obtained during radical prostatectomies of patients with relatively early stage disease is reported. The majority of specimens examinedshowed a normal male karyotype, 46.XY. Nine samples contained clon-ally abnormal populations including five specimens which were hyperdip-loid (modal range, 65-92 chromosomes), one specimen containing doubleminute chromosomes, and three containing structural aberrations. Lossof the Y chromosome and a partial trisomy for chromosome 4 wasobserved in a sample from one patient. Another sample showed a trans-location between the long arms of chromosomes 5 and 7. The only tumorobtained from a previously irradiated patient contained no normal cells,a modal chromosome number of 45, loss of chromosomes 2 and Y, andmultiple structural rearrangements. The appearance of any clonal cytogenetic abnormality correlated in general with a poorly differentiatedstate of the cancer. A survey of all available previous cytogenetic data onhuman prostate adenocarcinoma indicated that the loss of chromosomes1, 2, 5, and Y, the gain of chromosomes 7, 14, 20, and 22, and rearrangements involving chromosome arms 2p, 7q, and lOq are the mostcommon changes observed. This suggests that, although the assignmentof a single chromosomal aberration as a marker for early stage prostaticcancer is unlikely, several consistent "hotspots" might be of significance

in the etiology of this disease.

INTRODUCTION

Advances in techniques of cell karyotyping in recent yearshave produced a wealth of cytogenetic information about manyhuman cancers (1, 2). In leukemias and lymphomas, a numberof specific chromosomal changes have been identified whichare clinically important for diagnosis, therapy, and prognosis(3). These changes may have great significance for understanding the pathogenesis of malignant transformation. By contrast,the cytogenetic changes in solid human tumors generally appearto be distinctively more complex, and for some common malignancies such as prostate cancer, correlation of chromosomeaberrations with disease has been hindered by insufficient data.

Difficulty in the culturing of prostatic epithelial cells is likelythe main reason that limited karyotypic information has beenobtained for prostate cancer. Only 11 primary prostatic tumorshave so far been karyotyped (4-8), two of which were leiomy-osarcomas. Two metastatic cancers of the prostate have alsobeen examined (9, 10). Three cell lines derived from primarytumors have been characterized (11-13), and five cell linesderived from metastatic prostatic tumors have been reported(14), four of which have been cytogenetically evaluated (15-18). To date no consistent chromosome changes have beenassociated with this malignancy.

We have overcome some of the technical problems of culturing prostate epithelial cells (19, 20) and have performed karyotypic analysis on 30 early stage prostate cancers obtained from

Received 11/21/89; revised 2/26/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by RO1-CA46269-02 from the National Cancer Institute (to A.

R. B. and in part by a grant from the R. M. Lucas Foundation, Mento Park, CA(D.M. P. andJ. E. M.).

2To whom requests for reprints should be addressed, at Department ofMicrobiology and Immunology. Eastern Virginia Medical School, P. O. Box1980, Norfolk, VA 23501.

radical prostatectomies. In addition, each cancer was subjectedto detailed morphological analysis so that the volume andhistological pattern of the tumor could be related to the cytogenetic findings.

MATERIALS AND METHODS

Cell Culture and Characterization. Radical prostatectomy specimenswere obtained from 29 patients referred to Stanford University MedicalCenter from 1986 to 1989. All patients except one (DP-30) receivedno previous radiation or chemotherapy. Two samples were derivedfrom a single patient (DP-12 and DP-19), while all other samplesrepresent independent patients. A wedge of tissue for cell culture wascut from each prostate by palpating the gland under sterile conditionsand incising into the center of the area of greatest firmness or IKulularity. Following collagenase digestion of the tissues, epithelial cell cultures were established in serum-free medium according to previouslydescribed methods (19, 21). Primary and secondary cultures weremaintained in medium PFMR-4A supplemented with 10 ng/ml ofcholera toxin (List Biologicals, Campbell, CA), 1 Mg/ml of hydrocorti-sone, 10 Mg/ml of bovine pituitary extract (Hammond Cell/Tech,Alameda, CA), 4 /jg/ml of insulin (Collaborative Research, Bedford,MA), 10 ng/ml of epidermal growth factor (Collaborative Research),0.1 mM phosphoethanolamine, 3 x IO"8 M selenous acid, 2.3 x IO"6 Ma-tocopherol, 3 x 10~" M retinoic acid, and 100 f¿g/mlof gentamicin.

Unless otherwise indicated, components of PMFR-4A and mediumsupplements were obtained from Sigma (St. Louis, MO). Dishes coatedwith type I collagen (Collagen Corp., Palo Alto, CA) were used routinely. This culture system was designed for the optimal growth ofprostatic epithelial cells (19, 20) and selects against the growth offibroblasta- cells.

The epithelial nature of cultures obtained by this method was verifiedroutinely by immunocytochemical staining for keratin, PAP,3 and PSA.

Fixed and permeabilized cells were labeled with antibodies specific forkeratin (EAB903; Enzo Biochemicals, New York, NY), PAP (Cambridge Research Laboratory, Cambridge, MA), and PSA (CambridgeResearch Laboratory). Labeling by primary antibodies was detectedwith biotinylated secondary antibodies followed by the ABC reagent(Vector Laboratories, Burlingame, CA). Diaminobenzidine was used asthe substrate.

Cytogenetics. Primary cultures were subcultured once followinggentle treatment with 0.2% trypsin/0.02% EDTA. First passage cultures were monitored carefully for mitotic activity, and colcemid wasadded (0.1 jig/ml) for 4 h. Cells were removed with trypsin/EDTA andcentrifuged at 200 x g. The cell pellet was washed with a buffered salinesolution (pH 7.6) and resuspended for hypotonie swelling in 0.075 MKC1 at 37°Cfor 20 min. The pellet was fixed in three parts metha-

nohone part glacial acetic acid, and slides were prepared for mitoticfigures by routine procedures. Metaphase cells were G banded followingbrief trypsin treatment and stained with Wright stain. Confirmation ofloss of the Y chromosome was done by routine staining with quinacrinemustard.

All cells were examined microscopically, and a minimum of two cellswas karyotyped for each cell population. Evaluation of 15 metaphasecells was sought from each sample; if fewer than 10 mitoses were found,that sample was not used in this study. Thirty-nine samples wereattempted in this series, of which 30 are reported here. Of the nine notdiscussed, one karyotypically normal specimen was shown to be obtained from normal tissue, and the remaining eight samples resulted inlow mitotic yields of poor morphology. While no gross chromosome

'The abbreviations used are: PAP. prostatic acid phosphatase; PSA. prostate-

specific antigen.

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CYTOGENETICS OF PROSTATE CANCER

abnormalities were observed in these specimens, we believed they wereuninformative to this study. Clonal abnormalities were defined as thosepresent in at least two cells from a particular specimen.

Determination of Cancer Volume and Histológica! Grade. After obtaining a wedge of tissue for culture, the prostate was fixed in undiluted(37%) formalin for 24 h at room temperature. The entire prostate glandwas sectioned at 0.3-cm intervals in transverse planes perpendicular tothe rectal surface as previously described (22). Levels of section werelabeled serially, and slides were cut at 5 /MUand stained with hematox-ylin and eosin. On each slide the outlines of prostate and the boundaryof the cancer were drawn with a fine point marking pen, and theseoutlines were transferred by tracing to a comprehensive map of theentire cancer and gland. The total area of cancer on the map wasdetermined by computer planimetry and multiplied by section thickness(0.3 cm) to obtain the cancer volume. Calculated volume was multipliedby a factor of 1.5 to correct for tissue shrinkage in processing. Aspreviously described (22), the factor of 1.5 was obtained by comparingthe areas of fresh and formalin-fixed tissue slices from several prostatesto the areas of the same slices after paraffin embedding, sectioning at5 (ini, and mounting on glass slides.

Quantitative histological grading using the Gleason system (23) wasapplied to the entire cancer, and the percentage of the tumor occupiedby each Gleason histological pattern was estimated. The cut boundaryof the tissue culture wedge was also graded separately, and the presenceof normal tissue at the boundary was quantitated. If greater than 90%of the cut boundary' was composed of cancer, then the corresponding

cell culture was classified as cancer. The corresponding culture wasclassified as "mixed" if the boundary of the wedge contained both

normal and cancer areas.

RESULTS

The immunological characterization of primary and secondary cultures derived from prostate tumor specimens showedthat 100% of the cells were epithelial, as demonstrated bypositive labeling with antikeratin (Fig. 1). Prostatic origin andretention of function was also indicated by the presence ofprostate-specific antigen and prostatic acid phosphatase, whichwere most prominent in dense areas as cultures became confluent (Fig. 1).

A compilation of cytogenetic data from the 30 specimens

PSA

Fig. 1. Immunocytochemical characterization of prostatic cell cultures forkeratin. PAP. and PSA. Staining was done using biotinylated secondary antibodies as described in the text. Left, control cultures for which the respective primaryantibodies were eliminated.

examined is shown in Table 1. Twenty-one of the specimensexamined showed only normal male karyotypes, 46,XY (Fig.2). Nine of the 30 samples, however, showed some clonalaberration. Five samples (DP-13, DP-25, DP-27, DP-37, andDP-39) contained hyperdiploid (tetraploid) populations (Fig.3). The presence of double minute chromosomes was observedin DP-17 (Fig. 4).

Chromosomal rearrangements were observed in three of thetotal specimens studied. DP-9 contained a clone missing the Y

il if

ff U II If il It U6 8 9 10 11 12

II II13 14 15

«I/l

il II <Ì16 17

il

18

I19 20 21 22 X X X Y

Fig. 2. Representative normal male karyotype from one of the primary prostate cultures, DP-19. This G-banded metaphase is 46.XY.

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CYTOGENETICS OF PROSTATE CANCER

Table 1 Cytogenetic and pathologic evaluation of 30 primar}- prostate tumors

caseDP

1DP-2DP-4DP-5DP-6DP

7DP-8DP-9DP-

10DP-11DP-

12mixDP-13DP-14DP-15mixDP-16

mixDP-17DP-19mixDP-22mixDP-23mixDP-24mixDP-25mixDP-27DP-29

mixDP-30DP-31DP-33DP-34

mixDP-37mixDP-38mixDP-39

mixTotal

cancervol6.0313.884.445.70.776.043.076.114.7225.108.961.542.082.200.564.108.964.855.37N

D4.294.602.602.083.543.665.807.943.60NDPathologyGleason

histologiepattern3/44/54/3/53/33/44/3/53/44/33/34/33/34/33/33/34/43/43/33/33/33/33/43/33/43/33/43/43/34/33/33/4CytogeneticsStage(24)BlAlB3B2B3B2B2B2B2B3B2B3BlB2BlB3B2B2B2BlBlB2BBlBBlBlCBlB3Total

cellsanalyzed11202012221915242019161820202017151615111617152117IS17IS1516Chromosome

no. and no. ofcells<4313111020000400000001000400000044110101430000010001110015000000454513421905052002301020292010014651118715168102012156181820151114139121412314151610IS134700002001010001000000000010000048000000010110000010000000000000Hyperdiploid

AbnormalCells'0000000

Randomloss045.X.-Y/46X.-Yder(4)t(4;?)(q27;?)|9]00030

46,XY,t(5;7)(ql4;q31)(5]000

46,XY,+dmin[2]01002300

(seebelow)'0

Random loss andgain00502

" Numbers in brackets, number of cells with abnormality observed.4 ND, not determined.cClonalkaryotypeofDP-30 = 45,X,-Y,-2,+der(IO)t(10;?)(q24;?), + 13,-14,t(l;6)(p32:q21).inv(3)(p21ql2).inv(4)(P12q33),der(7)t(7;12)(q32;ql5).der(8)t(8;?)-

(q24;?),del(8)(q22),der(9)t(9;?)(p 13;?),del( 10)(p 13)(q24).del( 12)(q22).der( 14)t( 14;?)(q24;?),del( 19)(p 13. 1).

Table 2 Cytogenetic findings associated with prostate adenocarcinoma

CelllinesPC-3TSU-PR1DU-

145LNCaPPC-82PPC-1VCRU-Pr-2Metastatic

strains12Cell

strains12345678910111213-161718-41Double

Homogenouslyminute staining

chromosomesregions+

———+-———-++———

———+

+++

—————_—_—+—+—

———-———+—-Marker

chromosomes Hyperdiploidy 46,XY15

+10+3+7"

+18"+18

+5"--6"

+++"++10"

++4

+8+6

+16+4

++5"2

++2--—

+10"++

+++Ref.161718191112141094444456778Table

18Table

1Table1Table

11Identifiable chromosomes from these were analyzed and used in Fig. 9 for breakpoint localization.

chromosome and another clone which also contained an extraderivative chromosome 4 (Fig. 5). DP-14 contained a clonewith a translocation between chromosomes 5 and 7 (Fig. 6).DP-30 contained no normal cells; the modal number was 45,and numerous aberrations were seen involving many chromo

somes (Fig. 7). DP-30 was the only specimen obtained from apatient who had received previous radiation therapy; the aberrations associated with this sample may thus not reflect thenatural history of prostate cancer.

From Table 1 it can be seen that total cancer volume, stage,3797

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CYTOGENETICS OF PROSTATE CANCER

s v Ur -

v»

Fig. 3. Representative metaphase cell from one of the hyperdiploid prostate populations (DP-27). This cell is 91,XXYY,-21.

Fig. 4. Representative metaphase cell from DP-17, showing double minute chromosomes (some shown by arrows).

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CYTOGENETICS OF PROSTATE CANCER

it fti

If Xf *t tt II8 9 10 11 12

O» it13 14 15 16 17 18

Fig. 5. Representative karyotypes from thetwo abnormal populations in DP-9. A,45.X,-Y; B, 45,X.-Y,-8.+der(4)t(4;?)(q37;?).This is the only cell missing chromosome 8.Arrows indicate aberrant chromosomes.

ÀI19 20 21 22 XX X Y

B n n «Ü

il ir i ti li li it8 10 11 12

•aft* 4413 14

A4 i*15 16 17 18

19 20

4'

21 22 X X X Y

or Gleason histológica! pattern of the sample did not appear tocorrelate with specimens containing cytogenetic changes. Aninteresting correlation was suggested, however, when the differentiation of the cancer was considered (Fig. 8). Using theGleason histológica! pattern as a measure of differentiation, wefound that the majority of specimens with cytogenetic aberrations were derived from prostates in which quantitative gradingof the entire tumor showed greater than 65% of Gleason patterns 4/5. Five of the 9 (56%) specimens in this category wereabnormal, whereas only 3 of the 19 (16%) specimens fromtumors with less than 65% of Gleason patterns 4/5 were aberrant. On the Gleason grading scale, patterns 4 and 5 representthe category of poorly differentiated carcinoma.

Specimens derived from tissues composed of greater than

90% cancer appeared to have a slightly higher frequency ofclonal abnormalities [6/17 (35%)] than did "mixed" specimens

derived from tissues containing normal as well as malignantglands [3/13 (23%) had clonal abnormalities]. Two additionalsamples, DP-8 and DP-31, lacked clonal changes but had anumber of random whole chromosome gains or losses; thesetwo samples were also derived from tissues composed of greaterthan 90% cancer.

DISCUSSION

The cases reported in this series increase the total number ofkaryotyped primary prostate cancers in the literature from 11to 41. While this number is still small, some preliminary

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H II-

Fig. 6. Representative karyotype from DP-14,46,XY,t(5;7)(ql4;q31). Arrows indicate breakpoints.

lì6

fi - Ilai18*•siQ*WW

•»At O13 14 15

A* »ft16 17 18

-- ir -r-19 20 21 22 X X X Y

ÕII) it HI«

Fig. 7. Karyotype from DP-30, with arrows indicating the many aberrant chromosomes detected. Thiscell is representative of the major abnormal clonalpopulation, 45, X,-Y,-2, +der(10)t(10;?) (q24;?),+ 13,-14,t(l;6) (p32;q21). inv(3) (p21qI2), inv(4)(pl2;q33), der(7) t(7;12) (q32;ql5), der(8)t (8;?)(q24;?), del(8)(q22). der(9)t(9;7) (pl3;?),del(10)(pl3)(q24),del(12) (q22),der(14) 1(14;?) (q24;?),

Tfe* »i15 i|* ^4 _^„ * «

11IM t

19 20

10 11

13 14 15

\

* •' - A~2\ 22

12

li tt li16 17 18

iXX X Y

10090

80

70

60

50

40

30

20

10

O1.0 2.0 3.0 4.0 5.0 6.0

TOTAL TUMOR VOLUME (cm3)

conclusions may be drawn. Cytogenetic change appears not tobe a consistent event in prostate cancer, since the majority ofour samples obtained from patients with clinical stages Athrough C tumors had a normal diploid karyotype. This findingcorroborates flow cytometry studies, in which the majority ofprostate tumors analyzed have been diploid (25-27). A higher

Fig. 8. Relationship of the karyotype of 28 cultured cancer strains to the totalvolume and differentiation (Gleason grade) of the tumors from which they werederived. Two specimens of the 30 which were karyotyped were eliminated fromthis graph. DP-30 was not plotted since it was derived from an irradiated tumor,and DP-12 and DP-19 were represented by a single point in the graph since theywere derived from different areas of the same tumor. •,specimens with a normalkaryotype; +, those with a karyotypic aberration.

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1-4ei

-i+292e~33

f1-1+40

1-5 +7e

e

20

6-

20

e

Fig. 9. Composite ideogram of chromosome changes in prostatic cancer. Points, breakpoints involved in translocations, deletions, or inversions. Numbers aboveeach chromosome followed with a plus or minus, independent observations of whole chromosome gains or losses, respectively. Arrows with a plus or minus, whole armduplications or deletions. Symbols not in circles, observations from primary cell strains: symbols in circles, observations from cell lines. All data were obtained fromreports referenced in Table 2.

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CYTOGENETICS OF PROSTATE CANCER

frequency of aneuploidy has been found in nodal métastases(28), but these may represent selected subpopulations of theprimary tumors.

A pattern potentially related to progression of prostate canceremerged, however, when karyotypic lesions were correlated withthe overall differentiated state of the cancer. The majority ofspecimens with karyotypic abnormalities were derived fromtumors with greater than 65% of Gleason patterns 4/5 (poorlydifferentiated). Evidence has previously been presented thatpoorly differentiated areas (Gleason patterns 4 and 5) in prostate cancer arise by evolution with time from initially well-differentiated cancers and represent biological progression to astate of more rapid tumor growth and enhanced metastaticpotential (22, 29). Other explanations are possible for theobserved correlation between high volume and poor differentiation. If, however, a strong correlation can be confirmed betweenloss of histological differentiation and acquisition of karyotypiclesions, it may be that cytogenetic abnormalities play an important role in the changing biological features of prostatecarcinoma with time rather than in tumor initiation.

It should be noted that cancers with Gleason patterns 1 and2 were not identified in our material. These two best differentiated of the Gleason patterns have recently been discoveredalmost exclusively in the anatomic region of the prostate knownas the transition zone (30, 31). This zone is the site of origin ofmodular hyperplasia and is located far anteriorly away fromthe rectal surface. Cancers in the transition zone are difficult topalpate, even in the excised prostate specimen. Our materialwas limited to cancers which could be palpated through therectal capsule; hence, transition zone tumors were not wellrepresented.

Since no consistent chromosomal changes were found in ourstudy, we thought that it would be informative to compare ourfindings with those from previous studies and to summarize thecytogenetic aberrations associated with prostate cancer (Fig. 9and Table 2).

Fig. 9 shows the number of additions, deletions, and specificbreakpoints observed in both cell lines and primary specimens.The most commonly lost chromosomes are numbers 1, 2, 5,and Y, while the most commonly gained chromosomes arenumbers 7, 14, 20, and 22. It appears that the most frequentlyobserved breakpoints are at 2pl3-p23, 7q22-q31, and 10q22-q24. The sites on the long arms of chromosomes 7 and 10 havepreviously been implicated in prostate adenocarcinoma (5) andwe concur that these sites are likely "hotspots" for re

arrangement in prostate cancer.Table 2 records other cytogenetic observations regarding

prostate cancer. In some cases marker chromosomes have beenobserved in which specific chromosomal breakpoints could notbe defined. These data are omitted in Fig. 9, and therefore,some sites of rearrangement may be underrepresented. Sincemany of the marker chromosomes observed cannot be betteridentified accurately, the possibility exists that some breakpoints may be common to prostatic cancer but have so far beenoverlooked. These markers are likely a result of multiple structural rearrangements.

Evidence for gene amplification characterized by the presenceof double minute chromosomes or homogenously staining regions has been observed in six primary tumors and three prostatic tumor-derived cell lines. This may be suggestive of someoncogene amplification in prostate cancer as has been observedin other solid tumors (32).

Hyperdiploidy has been seen both cytogenetically and by flowcytometric analysis of primary prostatic samples (25). It can beseen from Table 2 that 6 of the 7 cell lines studied and 10 of

the 43 cell strains studied exhibited some degree of hyperdiplo-idy. Whether hyperdiploidy or the general phenomenon ofchromosome instability is involved in prostate tumor progression remains to be seen.

In conclusion, the data presented indicate that no simplechromosomal change can yet be defined as a marker of prostaticcancer. The most common finding in the present study was anormal male karyotype, with the lack of clonal abnormalities.One implication of the results is that the earliest primary changein prostate cancer occurs at the submicroscopic or individualgene mutation level. A similar finding was recently reported inhuman breast cancer where clonal abnormalities were detectedin only 2 of 40 primary breast carcinomas (33). We hypothesizethat a similar mechanism may be involved in these two commoncancers. The involvement of tumor suppressor genes has beenimplicated in a growing number of human cancers (34). Molecular analyses, in conjunction with further cytogenetic studies tolocalize hotspots, should provide useful information regardingthe etiology of prostatic carcinoma.

ACKNOWLEDGMENTS

The authors wish to thank Perry C. Wilkins for his technical assistance and Drs. Kenneth D. Somers and Donald J. Merchant for theirhelpful discussions and critical reading of this manuscript. We alsowish to acknowledge, posthumously, Lori J. Lesho, for her inspirationand valuable technical help during the initiation of these studies.

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1990;50:3795-3803. Cancer Res   Arthur R. Brothman, Donna M. Peehl, Ankita M. Patel, et al.   Prostate CancerFrequency and Pattern of Karyotypic Abnormalities in Human

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