Analysis of Prostatic Tumor Cultures Using Fluorescence In-Situ Hybridization (FISH) Arthur R. Brothman, Ankita M. Patel, Donna M. Peehl, and Paul F. Schellhammer
ABSTRACT: A n a l y s i s of ten primary prostatic tumor cultures using fluorescence in-situ hybridization (FISH) with pericentromeric probes for chromosomes 7, 8, 10, 16, 17, and 18 revealed aneusomies in nine of these specimens. Classical cytogenetics by G-banding indicated that only four of those same ten specimens had any (but not consistent) clonal abnormalities. This preliminary study suggests that a n e u s o m y is a c o m m o n event in early-stage prostatic tumors, and also supports the notion that multiple chromosomes are involved. In combination with routine cytogenetic analysis, FISH is thus likely to be a powerful tool in the evaluation of prostatic cancer.
INTRODUCTION Cancer of the prostate is the leading malignancy in U.S. males [1] yet few advances in the understanding of the etiology of this disease have been made. Several reports describing cytogenetic analysis of primary human prostatic tumors have been published [2-9] but no consistent chromosome change has been associated with prostate cancer. While chromosome abnormalities have been observed, the majority of tumors studied (approximately 75%) have shown only 46,XY, normal diploid male karyotypes. The addition of fluorescence in-situ hybridization (FISH) techniques using chromosome-specific probes to the field of cancer cytogenetics has clearly shown that information can be gained from interphase nuclei [11-13]. This has allowed for the expansion of analyses to include nondividing cells. We demonstrate in this report the value of FISH analysis in the study of prostatic tumor specimens.
MATERIALS AND METHODS Procurement of Samples Fresh tumor material was obtained from radical prostatectomies as previously described [7, 8]. Briefly, a wedge of tissue from an area of firmness or nodularity in the prostate was
From the Departments of Pediatrics and Microbiology and Immunology (A. R. B., A. M. P.) and Department of Urolagy (P. F. S.) Eastern Virginia Medical School, Norfolk, Virginia and Department of Urology (D. M. P.), Stanford University School of Medicine, Stanford, California. Arthur R. Brothman' s current address is: Department of Pediatrics, Rm. 413 MREB, University of Utah School of Medicine, Salt Lake City, UT 84132. Address reprint requests to: Arthur R. Brothman, Department of Pediatrics, Rm. 413 MREB, University of Utah School of Medicine, Salt Lake City, UT 84132. Received March 5, 1992; accepted May 1, 1992.
180 Cancer Genet Cytogenet62:180-185(1992) 0165-4608/92/$05.00
dissected under aseptic conditions for the establishment of a primary culture. The samples were digested overnight with collagenase [14] and cultures were initiated in either PMFR4A medium [15] or PC-1 medium (Endotronics, Coon Rapids, MN). Histologic grading of the cancer was done using the Gleason system [16], and tumor samples were coded so histology could be correlated with the specimen used for cell culture. If adjacent sections of the initial specimen were not predominantly composed of tumor cells, outgrowth from those cultures was not used in this study. Briefly, the epithelial nature of cellular outgrowth was determined by morphologic examination or by immunocytochemical staining for keratin, prostatic acid phosphatase, and prostate-specific antigen as previously described [7]. Metaphase cells were prepared from primary culture within 7 to 10 days of initiation using routine procedures. Fixed cultured prostatic cells which had previously been used for cytogenetic analysis were used for this insitu hybridization study. The slides were prepared using standard procedures and slides that were not immediately used for in-situ hybridization were briefly rinsed in PBS, dehydrated in an ethanol series (70%, 95%, 100%), and stored at - 70°C.
Probes Biotinylated centromeric probes for chromosomes 8, 10, 17, and 18 (D8Z1, DIOZ1, D17Z1, and D18Z1, respectively) were purchased from Oncor (Gaithersburg, MD). Centromeric probes for chromosome 16 (pHuR195) and 7 (7C-181-29) were kindly provided by Dr. R. Stallings (Los Alamos National Laboratories) and Dr. E. Jabs (Johns Hopkins University), respectively. These probes were labeled by nicktranslation using Bio-11-dUTP (Enzo Biochem). Hybridization The technique used was a modification of that described by Pinkel et al. [17]. Chromosomal DNA on the slides was © 1992Elsevier SciencePublishingCo., Inc. 655 Avenueof the Americas,New York,NY 10010
FISH of Prostatic Tumors
denatured by i m m e r s i o n in 70% formamide/2 x SSC, pH 7.0, at 70°C for 2 minutes, followed by dehydration in an ethanol series (70%, 95%, 100%) at 4°C. Ten microliters of hybridization mixture containing form a m i d e (65% for Oncor probes, 45% for probe pHuR195, and 55% for probe 7C-18-1-29), 2 x SSC, 500/zg/ml salmon sperm DNA, 5% dextran sulfate, and 0.5 ~g/ml biotinylated probe was heated at 70°C for 5 min, rapidly cooled, and applied to the slide. The hybridization was performed at 37°C overnight in a moist chamber. Post-hybridization washes were done in 65%, 45%, and 55% formamide/2 x SSC for Oncor, pHuR195, 7C-18-1-29 probes, respectively, for 20 m i n u t e s at 43°C. The slides were then washed twice in 2 x SSC pH 7.0 for 5 minutes each at 37°C, and transferred to PN buffer (0.1 M NaH2PO 4 and 0.1 M Na2HPO 4 to pH 8.0, 0.1% NP-40) at room temperature. Detection of the biotinylated probes was carried out by applying FITC-labeled avidin (5/zg/ml in PNM buffer: PN buffer + 5% Carnation non-fat dry milk). Amplification of the signal was accomplished by applying biotinylated antiavidin (10/zg/ml in PNM buffer) followed by another layer of FITC-avidin. The nuclei were stained with p r o p i d i u m iodide (0.5 tzg/ml) in an antifade solution (100 mg/ml Pp h e n y l e n e d i a m i n e dihydrochloride pH 9.0 in 90% glycerol [18]). Cells were viewed with an O l y m p u s BH2 microscope fitted with a BH2-DMB dichroic mirror cube and a blue filter. Photomicrographs were taken using Kodak Ektachrome 400 Daylight film.
Statistics Nuclei were scored for fluorescence signals (spots) by two i n d e p e n d e n t observers and a m i n i m u m of 150 total cells were evaluated for each probe hybridized to each specimen. S i m u l t a n e o u s hybridization with each probe to chromosomally normal fibroblasts were used as control studies. Populations were determined to be significant for aneusomy using the following criteria. Loss of a chromosome was considered significant if greater than or equal to 15% of all cells showed 0 plus 1 spots in addition to less than or equal to 80% of cells showing 2 spots. Gain of a chromosome was considered significant if greater than or equal to 5% of cells showed 3 plus 4 spots.
RESULTS This initial study involved the use of m e t h a n o l - a c e t i c acid-fixed cell pellets from specimens that had previously been evaluated using classical cytogenetic methods. Representative interphase and metaphase cells hybridized with the respective probes (pericentromeric sequences for chromosomes 7, 8, 10, 16, 17, and 18) are shown in Figure 1. Data from this study are s h o w n for each prostate specim e n plus three control samples in Table 1. Significant gains or losses of particular chromosomes are also shown in Table 1. Nuclei in which signals were not clearly discernable are i n c l u d e d in the " u n s u r e " category. These values were
181
helpful in the ascertainment of the efficiency of each assay. These data are summarized in Table 2. Routine cytogenetic results following G-banding analysis in addition to the pathologic characterization of samples can also be seen from that table. All except samples CAP 239, CAP 253, CAP 267, and DP 53 showed normal karyotypes in our initial examination. By FISH analysis, only case 267 showed no aneusomies using our statistical criteria described above. All other specimens showed chromosomal gains or losses as indicated in Table 1.
Table 1
FISH of prostate tumor cells
CAP 239 Signal/nucleus
Chromosome (%) 10 16 17
7
8
Unsure
0 3 87 0 6 4
3 11 83 1 1 1
2 7 78 4 4 5
1 5 80 1 1 11
5 17 72 1 1 5
5 11 79 2 2 2
Total Change
197 G
493 --
650 G
370 --
534 L
335 L
7
8
Chromosome (%) 10 16 17
18
0 1 2 3 4 Unsure
2 4 82 2 4 6
4 15 74 3 3 1
4 1~ 73 3 4 5
8 15 72 2 1 2
6 10 74 1 5 4
1 7 84 1 1 6
Total Change
461 G
395 L/G
645 L/G
303 L
286 L/G
348 --
7
8
17
18
1 5 83 1
1 6 83 2
2 6 77 2
6 9 72 2
5 11 7-6 2
3 7 85 0
Unsure
4
2
5
7
3
2
Total Change
409 G
457 G
312 G
382 L/G
313 L/G
276 --
7
8
Unsure
1 2 96 0 1 2
3 11 84 1 1 2
2 5 86 1 3 3
5 8 77 2 1 10
6 18 72 1 0 3
4 19 70 1 1 5
Total Change
420 .
500 .
304 .
402
611 L
298 L
0 1 2 3 4
CAP 243 Signal/nucleus
CAP 246 Signal/nucleus 0 1 2 3
CAP 248 Signal/nucleus 0 1 2 3 4
.
Chromosome (%) 10 16
Chromosome (%) 10 16 17
18
18
182
Table 1
Continued
CAP 253 Signal/nucleus
7
8
0 I 2 3 4
1 3 89 0 0
2 1-7 73 1 2
2 1 83 3 -6
Unsure
7
5
Total Change
336 --
338 L
7
8
Unsure
0 1 96 0 3 0
2 12 82 1 2 1
Total Change
206 .
CAP 267 Signal~nucleus 0 1 2 3 4
DP-44 Signal/nucleus
C h r o m o s o m e (%) 10 16
18
DP-54 Signal~nucleus
7
8
5 17 76 1 2
4 1-9 72 1 2
9
0
2
10
67 1 2
2 3
68 2 5
5
5
2
4
Unsure
303 G
374 L
474 L
292 L
Total Change
17
18
1 4 92 1 0 2
0 4 93 0.3 0.5 2
380
397
C h r o m o s o m e (%) 10 16 4 10 77 4 0 5
211 .
17
.
313 .
67 0.5 0.5
71 1 1
69 1 1
67 1 2
74 3 1
13
7
5
5
5
3
453 G
232 L
475 L
861 L
411 L
510 L
N o r m a l Control Signal~nucleus
7
8
17
18
0 1
1 3
2
96
3 4
0 0 0
0.5 8 88 0 0 3.5
2 10 84 0.3 0.6 3.1
4 8 84 0 0 4
2 11 84 0.5 0.5 2
313
642
329
501
17
18
2 8 89 0 0 1
I 5 91 0 0 3
451
451
17
18
O.6 6 87 0 0 5
3 3 81 0 0 6
478
317
4
Unsure Total
214
Change
.
3 8 86 0 0
i 4 82 1 3
0 1 2 3 4
5
8
3
9
Unsure
344 L/G
311 G
295 --
231 --
Total Change
17
18
5 1-7 71 I 1 5
4 15 76 2 1 2
0.7 8 86 1 2 2.3
Unsure
399 L
412 L
411 --
Total Change
17
18
2 10 77 3 2 6
2 7 84 2 4 7
2 8 81 2 -5
456 G
467 G
530 G
Unsure
3
--1-
Total Change
178 L
318 G
7
8
Unsure
0 3 88 0.3 3 5.7
6 1-0 67 2 -7 -8
4 12 78 1 2 3
Total Change
342 --
443 L/G
776 L
7
8
Unsure
3 17 72 1 1 8
1 9 84 1 3 2
1 7 86 O.4 3 2.6
Total Change
247 L
490 --
475 --
0 1 2 3 4
5
2 7 75 3 -5
5 14 68 5 5
DP-53 Signal~nucleus
6
N o r m a l Control Signal~nucleus
1 2 79 6 17
C h r o m o s o m e (%) 10 16
C h r o m o s o m e (%) 10 16
18
4
18
7 17 75 1 3
0 1 2 3 4
.
17
5
17
8
DP-45 Signal~nucleus
471 .
C h r o m o s o m e (%) 10 16
7
0 1 2 3 4
1 3 80 2 2 12
C h r o m o s o m e (%) 10 16
N o r m a l Control Signal~nucleus 0 1 2 3 4
.
7
C h r o m o s o m e (%) 10 16
.
8
1 2 94 1 0 2
1 11 87 0.2 0 .8
424
461 .
7
8
0 5 84 1 0 10
1 7 86 0,5 0.5 5
150
369 .
2 5 85 1 0 7 344
.
.
.
C h r o m o s o m e (%) 10 16 2 4 90 0.3 0 3.7
3 7 89 0 0.2 .8
304
462 .
.
.
.
C h r o m o s o m e (%) 10 16 1 4 93 0 0 2
O.2 5 92 0.2 0 2.6
380 .
.
417 .
.
Observations of fluorescence signal (spots) per nucleus observed following hybridization with centromeric sequence for respective probes. Totals represent total number of nuclei scored while figures showing signal~nucleus are percentages. G and L indicate significant gain or loss of a chromosome, respectively, as described in the text. Data contributing to these significance figures are shown underlined in the tables.
183
b ~ i i I¸
d
e
9
f
8
~
1
I
Figure I
Representative prostatic t u m o r cells hybridized with respective pericentromeric probes for c h r o m o s o m e (a) 7, disomic m e t a p h a s e and interphase; (b) 7, disomic m e t a p h a s e and trisomic interphase; (c) 7, d i s o m i c and m o n o s o m i c interphase; (d) 8, disomic and m o n o s o m i c interphase; (e) 10, trisomic and disomic interphase; (f) 10, m o n o m o s i c and disomic interphase; (g) 10, m o n o s o m i c metaphase; (h) 16, m o n o m s o m i c and d i s o m i c interphase; (i) 17, m o n o s o m i c m e t a p h a s e and interphase; (j) 18, trisomic interphase; (k) 18, m o n o s o m i c interphases.
Table 2
Chromosome
Specimen CAP 239 CAP 243 CAP 246 CAP 248 CAP 253 CAP 267 DP 44 DP 45 DP 53 DP 54 Totals
(G = g a i n , L :
loss, "--"
-- n o c h a n g e )
7
8
10
16
17
18
Cytogenetics/Pathology
G G G . -. L -L G 20% L 40% G
-L/G G
G L/G G
-L L/G
L L/G L/G L L
L --L L
-L G L 70% L 30% G
--G L 40% L 10% G
4 6 , X Y / d m i n / m o d diff. 46,XY/GL 3 + 4 46,XY/well diff. 46,XY/poorly diff. 46,XY/dmin/GL 2 + 3 46,XY/1 cell del(lOQ) - / G L 2 + 3 46,XY/GL 3 + 3 46,XY/GL 3 ÷ 3 46,XY/dmin/GL 4 + 3 46,XY/GL 4 + 3
.
.
.
L .
G .
G L/G -L 40% L 40% G
L .
L/G L -L 40% L 50% G
. G L G L 50% L 30% G
.
Summary of observations following hybridizations and statistical comparison of alpha-satellite probes on cultures from ten primary prostatic tumors. Abbreviations: G = gain; L = loss; " - - " = no change as indicated from Table 1. Cytogenetic data represent a summary of G-banded analyses on these same samples. Pathologic characterization (well, moderate, or poorly differentiated), or Gleason grading (GL) is shown where it could be determined.
184 DISCUSSION Our findings suggest substantial differences in the interpretation of cytogenetic abnormalities in prostatic cancer. Using routine b a n d i n g methods, only 25% of the patients studied by us and others [8] showed any chromosomal change. Using FISH with the six probes indicated above, we have already determined that 90% (9 of 10) samples have clones with abnormal copy n u m b e r s of at least one of those six chromosomes. The rationale for using the six probes noted above came from previous cytogenetic data suggesting the involvement of chromosomes 7 and 10 in prostatic cancer [2], molecular data suggesting the i n v o l v e m e n t of chromosomes 7, 8, 10, and 16 [19, 20], and the presence of tumor suppressor genes on chromosomes 17 and 18 [21, 22]. Examination of other chromosomal loci is obviously necessary. The finding of gains of chromosomes (defining each cell with a single extra copy of the whole chromosome as trisomic) led to the suspicion that hyperdiploid cells were being examined. The absence of gains of all of the other chromosomes studied (e.g., chromosome 18 in CAP-246) could mean that there is some technical artifact (false negative results) or could imply that certain chromosomes are under-represented in polyploid cells and may indeed represent a true loss. The loss of chromosomes (defining each cell with less than two signals as m o n o s o m i c or nullisomic) suggests that specific chromosome loss may in fact be involved in prostatic cancers. This w o u l d i m p l y an u n u s u a l finding of whole chromosome loss in cancer of the prostate, and could be representative of a m e c h a n i s m of tumor suppression. If specific loss of more than one chromosome occurs, the order of that loss is not readily apparent. Likewise, there is no correlation of loss (or gain) at this time with Gleason stage or clinical course. While the frequency of loss in m a n y of the samples is significant by our statistical criterion described, there is still a large population of cells in those samples that do not show loss. This could be a result of the presence of normal prostate epithelial outgrowth (there is not yet available a well-defined surface marker for prostate tumor cells) or could simply represent tumor heterogeneity. It should be noted that the type of analysis described is a crude study which examines gain or loss of peri-centromeric sequences and thus implies total chromosome involvement. Cancers have been studied in w h i c h specific chromosomal loci are lost; if the centromeric sequences are not involved, specific loss of loci w o u l d not be detected by the methods described above. The difference in findings of chromosome i n v o l v e m e n t by FISH compared with classical cytogenetics suggests the need for further study using FISH analysis. It is possible that genetically abnormal cells in prostatic cancer do not respond to the artificial m e d i u m e n v i r o n m e n t in cell culture as well as genetically normal cells. This of course would yield a dichotomy of results if metaphase or interphase cells were studied. We have recently begun the examination of interphase nuclei from fresh prostatic tumor samples without cell culturing. This approach may elucidate better the specific
A.R. Brothman et al.
i n v o l v e m e n t of chromosome gain or loss in prostatic cancer. Without complete cytogenetic evaluation, however, obscure or subtle chromosomal changes would be missed using FISH alone. A c o m b i n e d evaluation, with aims to improve current cell culture methods, appears imperative.
Supported by a grant from the National Cancer Institute (#R01CA46269} and, in part, by a grant from Children's Health Systems of the Children's Hospital of The Kings Daughters, Norfolk, VA.
REFERENCES 1. Boring CC, Squires TS, Tong T (1991): Cancer Statistics in CaA Journal for Clinicians. American Cancer Society 41:1. 2. Atkin NB, Baker MC (1985}: Chromosome studies of five cancers of the prostate. Hum Genet 70:359-364. 3. Limon J, Dal Cin PD, Sandberg AA (1986): Cytogenetic findings in a primary leiomyosarcoma of the prostate. Cancer Genet Cytogenet 22:159-167. 4. Limon J, Lundgren R, Elfving P, Heim P, Kristoffersson U, Mandahl N, Mitelman F (1989}: Double minutes in two primary adenocarcinomas of the prostate. Cancer Genet Cytogenet 39:191-194. 5. Brothman AR, Lesho LJ, Somers KD, Schellhammer PF, Ladaga LL, Merchant DJ (1989}: Cytogenetic analysis of four primary prostatic cultures. Cancer Genet Cytogenet 37:241-248. 6. Babu VR, Miles BJ, Cerny JC, Weiss L, Van Dyke DL (1990}: Cytogenetic study of four cancers of the prostate. Cancer Genet Cytogenet 48:83-87. 7. Brothman AR, Peehl DM, Patel AM, McNeal JE (1990): Frequency and pattern of karyotypic abnormalities in human prostate cancer. Cancer Res 50:3795-3803. 8. Brothman AR, Peehl DM, Patel AM, MacDonald GR, McNeal JE, Ladaga LE, Schellhammer PF (1991): Cytogenetic evaluation of 20 cultured primary prostatic tumors. Cancer Genet Cytogenet 55:79-84. 9. Lundgren R, Mandahl N, Heim S, Limon S, Limon J, Henrickson H, Mitelman F (1992}: Cytogenetic analysis of 57 primary prostatic adenocarcinomas. Genes Chrom Cancer 4:16-24. 10. Devilee P, Thierry RF, Keivits T, Rukmini K, Hopman AHN, Willard HF, Pearson PL, Cornelisse CJ (1989}: Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes. Cancer Res 48:5825-5830. 11. Van Dekkan H, Pizzolo JG, Reuter VE, Melamed MR (1990): Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes. Cytogenet Cell Genet 54:103-107. 12. Waldman FM, Caroll PR, Kerschmann R, Cohen MB, Field FG, Mayall BH (1991}: Centromeric copy number of chromosome 7 is strongly correlated with tumor grade and labeling index in human bladder cancer. Cancer Res 51:3807-3813. 13. Hopman AHN, Moesker O, Smeets WGB, Pauwels RPE, Vooijs GP, Ramaekers FCS (1991}: Numerical chromosome 1, 7, 9, and 11 aberrations in bladder cancer detected by in situ hybridization. Cancer Res 51:644-651. 14. Peehl DM (1992): Culture of human prostatic epithelial cells. In: Culture of Epithelial Cells, Freshney RI, ed. Wiley-Liss, New York, pp. 159-180. 15. Peehl DM, Stamey TA (1986}: Serum-free growth of adult human prostatic epithelial cells. In Vitro Cell Devel Biol 22:82-90.
16. Gleason DF (1977): Histologic grading and clinical staging of prostatic carcinoma. In: Urologic Pathology: The Prostate.
F I S H of Prostatic T u m o r s
Tannenbaum M, ed. Lea and Febiger, Philadelphia, pp. 171-197. 17. Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938. 18. Johnson GD, Nogueira JGM (1981): A simple method for reducing the fading of immunofluorescence during microscopy. J Immunol Methods 47:349-350. 19. Carter BS, Ewing CM, Ward WS, Treiger BF, Aalders TW, Schalken JA, Epstein JI, Isaacs WB (1990): Allelic loss of chromosomes 16q and lOq in human prostatic cancer. Proc Natl Acad Sci (USA) 87:8751-8755.
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20. Bergenheim USR, Kazuto K, Collins VP and Ekman P (1991): Deletion mapping of chromosomes 8, 10 and 16 in human prostatic carcinoma. Genes Chrom Cancer 3:215-220. 21. Isaacs WB, Carter BS, Ewing CM (1991): Wild-type 53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. Cancer Res 51:4716-4720. 22. Fearon ER, Cho KR, Nigro JM, Kern SE, Jonathon WS, Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kinzler KW, Vogelstein B (1990): Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247:49-56.
ABSTRACT: A n a l y s i s of ten primary prostatic tumor cultures using fluorescence in-situ hybridization (FISH) with pericentromeric probes for chromosomes 7, 8, 10, 16, 17, and 18 revealed aneusomies in nine of these specimens. Classical cytogenetics by G-banding indicated that only four of those same ten specimens had any (but not consistent) clonal abnormalities. This preliminary study suggests that a n e u s o m y is a c o m m o n event in early-stage prostatic tumors, and also supports the notion that multiple chromosomes are involved. In combination with routine cytogenetic analysis, FISH is thus likely to be a powerful tool in the evaluation of prostatic cancer.
INTRODUCTION Cancer of the prostate is the leading malignancy in U.S. males [1] yet few advances in the understanding of the etiology of this disease have been made. Several reports describing cytogenetic analysis of primary human prostatic tumors have been published [2-9] but no consistent chromosome change has been associated with prostate cancer. While chromosome abnormalities have been observed, the majority of tumors studied (approximately 75%) have shown only 46,XY, normal diploid male karyotypes. The addition of fluorescence in-situ hybridization (FISH) techniques using chromosome-specific probes to the field of cancer cytogenetics has clearly shown that information can be gained from interphase nuclei [11-13]. This has allowed for the expansion of analyses to include nondividing cells. We demonstrate in this report the value of FISH analysis in the study of prostatic tumor specimens.
MATERIALS AND METHODS Procurement of Samples Fresh tumor material was obtained from radical prostatectomies as previously described [7, 8]. Briefly, a wedge of tissue from an area of firmness or nodularity in the prostate was
From the Departments of Pediatrics and Microbiology and Immunology (A. R. B., A. M. P.) and Department of Urolagy (P. F. S.) Eastern Virginia Medical School, Norfolk, Virginia and Department of Urology (D. M. P.), Stanford University School of Medicine, Stanford, California. Arthur R. Brothman' s current address is: Department of Pediatrics, Rm. 413 MREB, University of Utah School of Medicine, Salt Lake City, UT 84132. Address reprint requests to: Arthur R. Brothman, Department of Pediatrics, Rm. 413 MREB, University of Utah School of Medicine, Salt Lake City, UT 84132. Received March 5, 1992; accepted May 1, 1992.
180 Cancer Genet Cytogenet62:180-185(1992) 0165-4608/92/$05.00
dissected under aseptic conditions for the establishment of a primary culture. The samples were digested overnight with collagenase [14] and cultures were initiated in either PMFR4A medium [15] or PC-1 medium (Endotronics, Coon Rapids, MN). Histologic grading of the cancer was done using the Gleason system [16], and tumor samples were coded so histology could be correlated with the specimen used for cell culture. If adjacent sections of the initial specimen were not predominantly composed of tumor cells, outgrowth from those cultures was not used in this study. Briefly, the epithelial nature of cellular outgrowth was determined by morphologic examination or by immunocytochemical staining for keratin, prostatic acid phosphatase, and prostate-specific antigen as previously described [7]. Metaphase cells were prepared from primary culture within 7 to 10 days of initiation using routine procedures. Fixed cultured prostatic cells which had previously been used for cytogenetic analysis were used for this insitu hybridization study. The slides were prepared using standard procedures and slides that were not immediately used for in-situ hybridization were briefly rinsed in PBS, dehydrated in an ethanol series (70%, 95%, 100%), and stored at - 70°C.
Probes Biotinylated centromeric probes for chromosomes 8, 10, 17, and 18 (D8Z1, DIOZ1, D17Z1, and D18Z1, respectively) were purchased from Oncor (Gaithersburg, MD). Centromeric probes for chromosome 16 (pHuR195) and 7 (7C-181-29) were kindly provided by Dr. R. Stallings (Los Alamos National Laboratories) and Dr. E. Jabs (Johns Hopkins University), respectively. These probes were labeled by nicktranslation using Bio-11-dUTP (Enzo Biochem). Hybridization The technique used was a modification of that described by Pinkel et al. [17]. Chromosomal DNA on the slides was © 1992Elsevier SciencePublishingCo., Inc. 655 Avenueof the Americas,New York,NY 10010
FISH of Prostatic Tumors
denatured by i m m e r s i o n in 70% formamide/2 x SSC, pH 7.0, at 70°C for 2 minutes, followed by dehydration in an ethanol series (70%, 95%, 100%) at 4°C. Ten microliters of hybridization mixture containing form a m i d e (65% for Oncor probes, 45% for probe pHuR195, and 55% for probe 7C-18-1-29), 2 x SSC, 500/zg/ml salmon sperm DNA, 5% dextran sulfate, and 0.5 ~g/ml biotinylated probe was heated at 70°C for 5 min, rapidly cooled, and applied to the slide. The hybridization was performed at 37°C overnight in a moist chamber. Post-hybridization washes were done in 65%, 45%, and 55% formamide/2 x SSC for Oncor, pHuR195, 7C-18-1-29 probes, respectively, for 20 m i n u t e s at 43°C. The slides were then washed twice in 2 x SSC pH 7.0 for 5 minutes each at 37°C, and transferred to PN buffer (0.1 M NaH2PO 4 and 0.1 M Na2HPO 4 to pH 8.0, 0.1% NP-40) at room temperature. Detection of the biotinylated probes was carried out by applying FITC-labeled avidin (5/zg/ml in PNM buffer: PN buffer + 5% Carnation non-fat dry milk). Amplification of the signal was accomplished by applying biotinylated antiavidin (10/zg/ml in PNM buffer) followed by another layer of FITC-avidin. The nuclei were stained with p r o p i d i u m iodide (0.5 tzg/ml) in an antifade solution (100 mg/ml Pp h e n y l e n e d i a m i n e dihydrochloride pH 9.0 in 90% glycerol [18]). Cells were viewed with an O l y m p u s BH2 microscope fitted with a BH2-DMB dichroic mirror cube and a blue filter. Photomicrographs were taken using Kodak Ektachrome 400 Daylight film.
Statistics Nuclei were scored for fluorescence signals (spots) by two i n d e p e n d e n t observers and a m i n i m u m of 150 total cells were evaluated for each probe hybridized to each specimen. S i m u l t a n e o u s hybridization with each probe to chromosomally normal fibroblasts were used as control studies. Populations were determined to be significant for aneusomy using the following criteria. Loss of a chromosome was considered significant if greater than or equal to 15% of all cells showed 0 plus 1 spots in addition to less than or equal to 80% of cells showing 2 spots. Gain of a chromosome was considered significant if greater than or equal to 5% of cells showed 3 plus 4 spots.
RESULTS This initial study involved the use of m e t h a n o l - a c e t i c acid-fixed cell pellets from specimens that had previously been evaluated using classical cytogenetic methods. Representative interphase and metaphase cells hybridized with the respective probes (pericentromeric sequences for chromosomes 7, 8, 10, 16, 17, and 18) are shown in Figure 1. Data from this study are s h o w n for each prostate specim e n plus three control samples in Table 1. Significant gains or losses of particular chromosomes are also shown in Table 1. Nuclei in which signals were not clearly discernable are i n c l u d e d in the " u n s u r e " category. These values were
181
helpful in the ascertainment of the efficiency of each assay. These data are summarized in Table 2. Routine cytogenetic results following G-banding analysis in addition to the pathologic characterization of samples can also be seen from that table. All except samples CAP 239, CAP 253, CAP 267, and DP 53 showed normal karyotypes in our initial examination. By FISH analysis, only case 267 showed no aneusomies using our statistical criteria described above. All other specimens showed chromosomal gains or losses as indicated in Table 1.
Table 1
FISH of prostate tumor cells
CAP 239 Signal/nucleus
Chromosome (%) 10 16 17
7
8
Unsure
0 3 87 0 6 4
3 11 83 1 1 1
2 7 78 4 4 5
1 5 80 1 1 11
5 17 72 1 1 5
5 11 79 2 2 2
Total Change
197 G
493 --
650 G
370 --
534 L
335 L
7
8
Chromosome (%) 10 16 17
18
0 1 2 3 4 Unsure
2 4 82 2 4 6
4 15 74 3 3 1
4 1~ 73 3 4 5
8 15 72 2 1 2
6 10 74 1 5 4
1 7 84 1 1 6
Total Change
461 G
395 L/G
645 L/G
303 L
286 L/G
348 --
7
8
17
18
1 5 83 1
1 6 83 2
2 6 77 2
6 9 72 2
5 11 7-6 2
3 7 85 0
Unsure
4
2
5
7
3
2
Total Change
409 G
457 G
312 G
382 L/G
313 L/G
276 --
7
8
Unsure
1 2 96 0 1 2
3 11 84 1 1 2
2 5 86 1 3 3
5 8 77 2 1 10
6 18 72 1 0 3
4 19 70 1 1 5
Total Change
420 .
500 .
304 .
402
611 L
298 L
0 1 2 3 4
CAP 243 Signal/nucleus
CAP 246 Signal/nucleus 0 1 2 3
CAP 248 Signal/nucleus 0 1 2 3 4
.
Chromosome (%) 10 16
Chromosome (%) 10 16 17
18
18
182
Table 1
Continued
CAP 253 Signal/nucleus
7
8
0 I 2 3 4
1 3 89 0 0
2 1-7 73 1 2
2 1 83 3 -6
Unsure
7
5
Total Change
336 --
338 L
7
8
Unsure
0 1 96 0 3 0
2 12 82 1 2 1
Total Change
206 .
CAP 267 Signal~nucleus 0 1 2 3 4
DP-44 Signal/nucleus
C h r o m o s o m e (%) 10 16
18
DP-54 Signal~nucleus
7
8
5 17 76 1 2
4 1-9 72 1 2
9
0
2
10
67 1 2
2 3
68 2 5
5
5
2
4
Unsure
303 G
374 L
474 L
292 L
Total Change
17
18
1 4 92 1 0 2
0 4 93 0.3 0.5 2
380
397
C h r o m o s o m e (%) 10 16 4 10 77 4 0 5
211 .
17
.
313 .
67 0.5 0.5
71 1 1
69 1 1
67 1 2
74 3 1
13
7
5
5
5
3
453 G
232 L
475 L
861 L
411 L
510 L
N o r m a l Control Signal~nucleus
7
8
17
18
0 1
1 3
2
96
3 4
0 0 0
0.5 8 88 0 0 3.5
2 10 84 0.3 0.6 3.1
4 8 84 0 0 4
2 11 84 0.5 0.5 2
313
642
329
501
17
18
2 8 89 0 0 1
I 5 91 0 0 3
451
451
17
18
O.6 6 87 0 0 5
3 3 81 0 0 6
478
317
4
Unsure Total
214
Change
.
3 8 86 0 0
i 4 82 1 3
0 1 2 3 4
5
8
3
9
Unsure
344 L/G
311 G
295 --
231 --
Total Change
17
18
5 1-7 71 I 1 5
4 15 76 2 1 2
0.7 8 86 1 2 2.3
Unsure
399 L
412 L
411 --
Total Change
17
18
2 10 77 3 2 6
2 7 84 2 4 7
2 8 81 2 -5
456 G
467 G
530 G
Unsure
3
--1-
Total Change
178 L
318 G
7
8
Unsure
0 3 88 0.3 3 5.7
6 1-0 67 2 -7 -8
4 12 78 1 2 3
Total Change
342 --
443 L/G
776 L
7
8
Unsure
3 17 72 1 1 8
1 9 84 1 3 2
1 7 86 O.4 3 2.6
Total Change
247 L
490 --
475 --
0 1 2 3 4
5
2 7 75 3 -5
5 14 68 5 5
DP-53 Signal~nucleus
6
N o r m a l Control Signal~nucleus
1 2 79 6 17
C h r o m o s o m e (%) 10 16
C h r o m o s o m e (%) 10 16
18
4
18
7 17 75 1 3
0 1 2 3 4
.
17
5
17
8
DP-45 Signal~nucleus
471 .
C h r o m o s o m e (%) 10 16
7
0 1 2 3 4
1 3 80 2 2 12
C h r o m o s o m e (%) 10 16
N o r m a l Control Signal~nucleus 0 1 2 3 4
.
7
C h r o m o s o m e (%) 10 16
.
8
1 2 94 1 0 2
1 11 87 0.2 0 .8
424
461 .
7
8
0 5 84 1 0 10
1 7 86 0,5 0.5 5
150
369 .
2 5 85 1 0 7 344
.
.
.
C h r o m o s o m e (%) 10 16 2 4 90 0.3 0 3.7
3 7 89 0 0.2 .8
304
462 .
.
.
.
C h r o m o s o m e (%) 10 16 1 4 93 0 0 2
O.2 5 92 0.2 0 2.6
380 .
.
417 .
.
Observations of fluorescence signal (spots) per nucleus observed following hybridization with centromeric sequence for respective probes. Totals represent total number of nuclei scored while figures showing signal~nucleus are percentages. G and L indicate significant gain or loss of a chromosome, respectively, as described in the text. Data contributing to these significance figures are shown underlined in the tables.
183
b ~ i i I¸
d
e
9
f
8
~
1
I
Figure I
Representative prostatic t u m o r cells hybridized with respective pericentromeric probes for c h r o m o s o m e (a) 7, disomic m e t a p h a s e and interphase; (b) 7, disomic m e t a p h a s e and trisomic interphase; (c) 7, d i s o m i c and m o n o s o m i c interphase; (d) 8, disomic and m o n o s o m i c interphase; (e) 10, trisomic and disomic interphase; (f) 10, m o n o m o s i c and disomic interphase; (g) 10, m o n o s o m i c metaphase; (h) 16, m o n o m s o m i c and d i s o m i c interphase; (i) 17, m o n o s o m i c m e t a p h a s e and interphase; (j) 18, trisomic interphase; (k) 18, m o n o s o m i c interphases.
Table 2
Chromosome
Specimen CAP 239 CAP 243 CAP 246 CAP 248 CAP 253 CAP 267 DP 44 DP 45 DP 53 DP 54 Totals
(G = g a i n , L :
loss, "--"
-- n o c h a n g e )
7
8
10
16
17
18
Cytogenetics/Pathology
G G G . -. L -L G 20% L 40% G
-L/G G
G L/G G
-L L/G
L L/G L/G L L
L --L L
-L G L 70% L 30% G
--G L 40% L 10% G
4 6 , X Y / d m i n / m o d diff. 46,XY/GL 3 + 4 46,XY/well diff. 46,XY/poorly diff. 46,XY/dmin/GL 2 + 3 46,XY/1 cell del(lOQ) - / G L 2 + 3 46,XY/GL 3 + 3 46,XY/GL 3 ÷ 3 46,XY/dmin/GL 4 + 3 46,XY/GL 4 + 3
.
.
.
L .
G .
G L/G -L 40% L 40% G
L .
L/G L -L 40% L 50% G
. G L G L 50% L 30% G
.
Summary of observations following hybridizations and statistical comparison of alpha-satellite probes on cultures from ten primary prostatic tumors. Abbreviations: G = gain; L = loss; " - - " = no change as indicated from Table 1. Cytogenetic data represent a summary of G-banded analyses on these same samples. Pathologic characterization (well, moderate, or poorly differentiated), or Gleason grading (GL) is shown where it could be determined.
184 DISCUSSION Our findings suggest substantial differences in the interpretation of cytogenetic abnormalities in prostatic cancer. Using routine b a n d i n g methods, only 25% of the patients studied by us and others [8] showed any chromosomal change. Using FISH with the six probes indicated above, we have already determined that 90% (9 of 10) samples have clones with abnormal copy n u m b e r s of at least one of those six chromosomes. The rationale for using the six probes noted above came from previous cytogenetic data suggesting the involvement of chromosomes 7 and 10 in prostatic cancer [2], molecular data suggesting the i n v o l v e m e n t of chromosomes 7, 8, 10, and 16 [19, 20], and the presence of tumor suppressor genes on chromosomes 17 and 18 [21, 22]. Examination of other chromosomal loci is obviously necessary. The finding of gains of chromosomes (defining each cell with a single extra copy of the whole chromosome as trisomic) led to the suspicion that hyperdiploid cells were being examined. The absence of gains of all of the other chromosomes studied (e.g., chromosome 18 in CAP-246) could mean that there is some technical artifact (false negative results) or could imply that certain chromosomes are under-represented in polyploid cells and may indeed represent a true loss. The loss of chromosomes (defining each cell with less than two signals as m o n o s o m i c or nullisomic) suggests that specific chromosome loss may in fact be involved in prostatic cancers. This w o u l d i m p l y an u n u s u a l finding of whole chromosome loss in cancer of the prostate, and could be representative of a m e c h a n i s m of tumor suppression. If specific loss of more than one chromosome occurs, the order of that loss is not readily apparent. Likewise, there is no correlation of loss (or gain) at this time with Gleason stage or clinical course. While the frequency of loss in m a n y of the samples is significant by our statistical criterion described, there is still a large population of cells in those samples that do not show loss. This could be a result of the presence of normal prostate epithelial outgrowth (there is not yet available a well-defined surface marker for prostate tumor cells) or could simply represent tumor heterogeneity. It should be noted that the type of analysis described is a crude study which examines gain or loss of peri-centromeric sequences and thus implies total chromosome involvement. Cancers have been studied in w h i c h specific chromosomal loci are lost; if the centromeric sequences are not involved, specific loss of loci w o u l d not be detected by the methods described above. The difference in findings of chromosome i n v o l v e m e n t by FISH compared with classical cytogenetics suggests the need for further study using FISH analysis. It is possible that genetically abnormal cells in prostatic cancer do not respond to the artificial m e d i u m e n v i r o n m e n t in cell culture as well as genetically normal cells. This of course would yield a dichotomy of results if metaphase or interphase cells were studied. We have recently begun the examination of interphase nuclei from fresh prostatic tumor samples without cell culturing. This approach may elucidate better the specific
A.R. Brothman et al.
i n v o l v e m e n t of chromosome gain or loss in prostatic cancer. Without complete cytogenetic evaluation, however, obscure or subtle chromosomal changes would be missed using FISH alone. A c o m b i n e d evaluation, with aims to improve current cell culture methods, appears imperative.
Supported by a grant from the National Cancer Institute (#R01CA46269} and, in part, by a grant from Children's Health Systems of the Children's Hospital of The Kings Daughters, Norfolk, VA.
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