the location of several critical genes in prostate can- cer. For example, initial evidence of nonrandom prostate cancer chromosome aberrations included.
American Journal of Pathology, Vol. 149, No. 5, November 1996 Copyrnght © Amenican Societyfor Itnvestigative Pathology
Fluorescence in Situ Hybridization Evaluation of Chromosome Deletion Patterns in Prostate Cancer
Shiu-Feng Huang,* Sheng Xiao,* Andrew A. Renshaw,* Kevin R. Loughlin,t Thomas J. Hudson,t and Jonathan A. Fletcher*§ From the Departments of Pathology* and Surgey,t Brigham and Women's Hospital and Harvard Medical School, Boston, the Center for Genome Research,* Whitehead Institute for Biological Sciences/Massachusetts Institute of Technology, Cambridge, and the Dana-Farber Cancer Institute,5 Boston, Massachusetts
Various nonrandom chromosomal aberrations have been identified in prostate carcinoma. These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm. Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases. However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deletedfrequently in other common types of cancer. In this study, we performed dual-colorfluorescence in situ hybridization on intact nuclei from touch preparations of 16prostate cancers. Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chomosome (YAC) and Pl probes. Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer. Other YACs and Pls were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer. Hybridization efficiencies and signal intensities were exceUent for both repeat sequence (a-satellite) and singlecopy (YAC and P1) fluorescence in situ hybridization probes. Of 16 prostate cancers, 11 had clonal aberrations of I or more of the 13 chromosome regions evaluated, and 10 cases (62.5%)
had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted ceUs. Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and I case, respectively, and each of those cases had a similarly sized cell population with 8p deletion. These studies confirm 8p deletion in the majority of prostate carcinomas. 8p deletions appear to be early events in prostate tumortigenesis, often antedating aneuploidy. Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and efficient means for determiningfrequency and progression of oncogenetic events in prostate cancer. (Am J Pathol 1996, 149:1565-1573)
Cytogenetic studies have provided useful clues to the location of several critical genes in prostate cancer. For example, initial evidence of nonrandom prostate cancer chromosome aberrations included cytogenetic demonstration of deletions involving the chromosome 8 short arm (8p) and chromosome 10 long arm (10q).)-4 However, clonal chromosome aberrations were detected in only a minority of prostate cancers by cytogenetic analyses. It is now apparent that many prostate cancer cytogenetic analyses yielded negative (diploid) results because non-neoplastic epithelial and stromal cells overgrew cancer populations in the cytogenetic cultures.56 More recently, fluorescence in situ hybridization (FISH) and molecular studies have been performed using uncultured primary prostate cancer cells. Such approaches have demonstrated clonal chromosome aberrations in most primary prostate cancers.7'8 Accepted for publication June 17, 1996. Address reprint requests to Dr. Jonathan A. Fletcher, Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. S-F. Huang's present address is Department of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan.
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After nonrandom prostate cancer chromosome deletions were demonstrated cytogenetically, the deleted chromosome regions were evaluated further by Southern blotting and microsatellite polymorphism analyses. These molecular studies confirmed deletion of the 8p and 10q in 20 to 50% of primary prostate cancers.9 15 Several groups have also used comparative genomic hybridization to identify chromosomal deletions and amplifications in primary prostate cancers. Those studies demonstrated deletions at 8p, 1 0q, 16q, and 18q in 20 to 35% of cases but also revealed deletions of 6q and 13q in approximately 20% of cases. 16-18 Most Southern blotting, microsatellite polymorphism, and comparative genomic hybridization studies in prostate cancer have been performed using DNA isolated from uncultured tumor specimens. Accordingly, these studies were not confounded by in vitro selection as can be the case in cytogenetic analyses. However, the sensitivity of molecular approaches is influenced by the dilutional effect of wild-type DNA from reactive cells. This potential problem is a particular concern in prostate cancer because the predominant populations in many cases are non-neoplastic epithelial and stromal cells. Nonetheless, the potential confounding influence of non-neoplastic cell DNA has been minimized by selection of tissue sections predominantly composed of neoplastic cells and by microdissection of tissue sections. 10'15'19 An alternate approach that also minimizes the confounding effect of non-neoplastic populations is FISH. FISH analyses of fresh and archival tumor specimens permit evaluation of genetic aberrations on a cell-by-cell basis, and several groups have now used FISH approaches in demonstrating the extent of chromosome aneusomies8'20-22 and 8p deletions23,24 in prostate cancer. Notably, extra copy number of chromosome 7 was implicated in several FISH studies as a potential prognostic marker in localized prostate cancer. Clinicopathological correlations demonstrated increased risk of local and metastatic progression in prostate cancers with chromosome 7 aneusomy.25'26 FISH is also a particularly effective method for detection of homozygous chromosome deletions in primary tumor tissues.27 Demonstration of homozygous deletions has been a useful step in pinpointing tumor suppressor gene locations, but polymerase chain reaction (PCR) detection of homozygous deletions has been hampered by the masking effect of DNA from non-neoplastic cell populations in primary tumors. There have been several obstacles in applying FISH approaches to solid tumor specimens. FISH
approaches were developed and optimized originally for evaluation of metaphase cells, and initial methods were less reliable when applied to archival tissue specimens. Over the past 5 years, however, efficient protocols were established for FISH detection of chromosome aberrations in archival paraffinembedded tumor specimens,28 frozen tumor tisand tumor touch preparations.29 Another sues, obstacle has been the limited availability of FISH probes to chromosome regions of interest in tumors. Commercially available centromere-region FISH probes are very effective in demonstrating numerical chromosome aberrations, ie, losses or gains of whole chromosomes, and such probes have been used to define numerical chromosome aberrations in prostate cancer specimens.8'20-22,25,26 Centromeric and whole-chromosome FISH probes are of limited use, however, in gene mapping and gene identification. It is notable, therefore, that FISH gene mapping has been facilitated recently by widespread availability of several large-insert probe resources. One such resource is the Centre d'Etudes du Polymorphisme Humain collection of yeast artificial chromosomes (YACs), which generate intense FISH signals when hybridized to archival tumor specimens.29'30 The goals of the primary prostate cancer studies reported herein were to 1) evaluate a YAC and P1 FISH approach, 2) evaluate the frequency of chromosome 8 short arm deletions, 3) determine whether chromosome regions commonly deleted in other types of cancer might also be affected in prostate cancer, and 4) determine whether particular chromosome aberrations might be associated with genetic progression.
Materials and Methods Prostate Tissue Materials Sixteen prostate cancer specimens were obtained from radical prostatectomy specimens in patients with clinical stage B or stage C disease. Gleason grades were 6 (n = 2), 7 (n = 13), and 8 (n = 1; Table 1). None of the cases had lymph node metastases. All tissues were frozen at -80°C immediately after prostatectomy and were verified to contain >50% tumor cells by histological evaluation of adjacent tissue sections. Non-neoplastic prostate tissues were also obtained from radical prostatectomy specimens. These noncancerous tissues were used to establish expected FISH distributions and as controls during the cancer FISH analyses.
Chromosome Deletions in Prostate Cancer 1567 AJP November 1996, Vol. 149, No. 5
Table 1. Pathological Features of 16 Prostate Cancers Case
Grade
EPE
1 2 3 4 5 6 7 8 9 10 11 12
4+3 3+4 3+4 3+4 3+4 3+4 4+4 4+3 3+4 4+3 3+4 3+3 4+ 3 3+3 3+4 3+4
+ +
13 14 15
16
+ + + +
LVI
SVI
-
-
+
+
+
-
+ + +
+ + + +
+
+
-
-
+ + +
+ +
+
+
+
+
+
+ + + -
Chromosome aberrations Chromosome 8 deletion Other aberrations
-
-
+
EPE, extraprostatic extension; LVI, lymphovascular invasion; SVI, seminal vesicle invasion.
FISH Slide Preparation Touch preparations were made using small pieces (
