Allelic Loss on Chromosome Arm 8p: Analysis of Sporadic Epithelial Ovarian Tumors

Gynecologic Oncology 74, 98 –102 (1999) Article ID gyno.1999.5439, available online at http://www.idealibrary.com on

Allelic Loss on Chromosome Arm 8p: Analysis of Sporadic Epithelial Ovarian Tumors Monica R. Brown, M.D.,* ,1 Rodrigo Chuaqui, M.D.,† Cathy D. Vocke, Ph.D.,‡ Andrew Berchuck, M.D.,§ Lavinia P. Middleton, M.D.,* Michael R. Emmert-Buck, M.D., Ph.D.,† and Elise C. Kohn, M.D.* *Laboratory of Pathology, †Pathogenetics Unit, Laboratory of Pathology, and ‡Urologic Oncology Branch, National Cancer Institute, Bethesda, Maryland 20892; and §Division of Gynecologic Oncology, Duke University Medical Center, Durham, North Carolina 27710 Received December 21, 1998

genes have been shown to be important in human cancer development, and several have been linked to ovarian cancer. Mutations in BRCA1 and BRCA2, p53, and NM23 have been described in association with familial, advanced stage, and metastatic ovarian cancer, respectively [2– 6]. Identification of novel tumor suppressor genes has been facilitated by allelic deletion studies that have guided the localization of minimal gene intervals. This method of analysis has resulted in the identification of several chromosomal regions likely to harbor ovarian cancer tumor suppressor genes [7–10]. Studies of allelic loss on chromosome 8 in ovarian cancer are limited despite a high frequency of loss on this chromosome in other solid tumors [11–23]. Our group recently identified loss of heterozygosity (LOH) at 8p12–21 in 7/9 epithelial ovarian tumors from BRCA1 mutation-positive ovarian cancer patients [11]. These findings prompted us to investigate this specific region in sporadic primary epithelial ovarian cancers to determine if this pattern of loss is observed in both sporadic and familial forms of the disease.

Objective. Our objective was to determine the frequency of allelic loss at 8p21 in sporadic epithelial ovarian cancer. We recently described allelic loss at this locus in 7/9 ovarian cancers from patients with BRCA1 gene mutations. Methods. We anonymously obtained and examined 40 unselected invasive epithelial ovarian cancers and 5 low-malignantpotential (LMP) ovarian tumors for loss of heterozygosity (LOH) at 8p12–22. Pure epithelial and stromal cell populations were procured selectively by laser capture microdissection and extracted DNA was amplified with polymorphic microsatellite markers spanning the region of interest. Results. LOH was highest (50%) at marker D8S136 located at 8p21 with 15 of 30 informative cases exhibiting an allelic deletion. None of the LMP tumors evaluated showed LOH at 8p12–22. A trend toward more frequent LOH at 8p12–22 was identified with increasing disease aggressiveness from LMP to early stage invasive ovarian cancer to advanced stage invasive ovarian cancer (Lehman’s test, P 2 < 0.024). Conclusions. Fifty percent allelic loss at the distal portion of 8p21 has not been reported to date for sporadic epithelial ovarian carcinomas. The higher rate of loss in our cohort, in contrast to previous allelotyping studies, is due likely to analysis from homogenous cell populations. These results, in concert with our previous study of BRCA1 mutation-positive patients, suggest a tumor suppressor gene locus at 8p21 for epithelial ovarian cancer.

MATERIALS AND METHODS Tissue Acquisition Twenty-nine frozen and 11 paraffin-embedded, formalinfixed invasive primary ovarian cancer samples were obtained from the Cooperative Human Tissue Network, Duke University Division of Gynecologic Oncology, and the National Cancer Institute. Three paraffin-embedded, formalin-fixed, and 2 frozen low-malignant-potential (LMP) tumors were also analyzed. All identifying information was removed from each sample prior to its receipt for analysis. The age range of the patients was 31– 81 years, with a median age of 55 years.

INTRODUCTION Epithelial ovarian cancer is the fifth leading cause of death from cancer in women, with over 14,000 deaths each year [1]. The high mortality of ovarian cancer is due in part to the lack of reliable methods for detection of early stage disease and the resultant late stage of presentation. The majority of genetic events responsible for the development and progression of epithelial ovarian cancer remain unknown. Tumor suppressor

Microdissection Five-micrometer sections from paraffin-embedded blocks and 10-mm sections from frozen blocks were cut, mounted onto plain glass slides, and stained with hematoxylin and

1 To whom correspondence and reprint requests should be addressed at National Cancer Institute, Building 10, Room 2A33, 10 Center Drive, MDC 1500, Bethesda, MD 20892-1500. Fax: (301) 480-5142.

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eosin. Histologic diagnosis for each specimen was confirmed prior to microdissection (R.C. and L.M.). Laser capture microdissection was performed as described previously [24]. Briefly, tumor or normal stromal cells were visualized under microscopic guidance and selectively procured by activation of the laser. DNA Extraction and PCR and LOH Analysis Dissected cells were placed directly into 50 ml DNA extraction buffer (200 mg/ml proteinase K, 50 mM Tris– HCl, 1 mM EDTA, 0.5% Tween 20, pH 8.0) and incubated for 12 h at 43°C. The proteinase K was inactivated by heating at 95°C for 5 min prior to PCR. Two microliters of DNA from control and tumor samples was amplified with primers for the following microsatellite markers: D8S278, D8S1820, D8S137, NEFL, D8S136, and D8S254 (Research Genetics, Huntsville, AL). These markers span the region of 8p12–22 from centromere to telomere. A final 20-ml PCR reaction mixture contained 200 mmol of each dNTP, 13 reaction mix, 20 mmol of each primer, 2 U ampliTaq Gold polymerase (Perkin–Elmer, Foster City, CA), and 4 mCi of [ 32 P]dCTP (NEN Dupont, Boston, MA). Eleven cycles of touchdown PCR were performed consisting of a standard denaturation step for 20 s, a 1° decline in annealing temperature per cycle from 65 to 55°C each for 20 s, and a 20-s extension step at 72°C. Twenty-four cycles of 94, 55, and 72°C for 20 s each completed the reaction [25]. All reactions were carried out in a Perkin–Elmer 9600 thermocycler. PCR products were electorphoresed on a denaturing 6% polyacrylamide gel and visualized by autoradiography. Loss of heterozygosity was defined by complete loss of an allele in the tumor specimen as determined visually by four independent examiners. Each PCR reaction was run in duplicate and analyzed a minimum of two times at each marker. RESULTS Forty invasive epithelial ovarian cancers and 5 LMP tumors were evaluated for allelic loss using six microsatellite markers spanning the chromosomal region 8p12– 22. Representative autoradiographs of an invasive ovarian cancer case demonstrating allelic loss at 8p21 with retention at surrounding loci is shown in Fig. 1. The location of each microsatellite marker tested and a summary of LOH frequency for each case is shown in Fig. 2. Overall, 23 of 40 (58%) invasive ovarian tumors exhibited a deletion at one or more markers for 8p12–22. Of the 23 cases with deletions, 18 had LOH at one or more of the tested 8p21 markers. The highest rate of allelic loss, 15/30 informative cases (50%), was observed at marker D8S136 with lower rates at adjacent loci. There was minimal loss

FIG. 1. Autoradiographs of case 29. Normal (N) and tumor (T) DNA were amplified with the microsatellite markers described under Materials and Methods. Samples were loaded in duplicate, electrophoresed on a denaturing 6% polyacrylamide gel, and exposed to film. Loss of heterozygosity is seen at markers D8S136 and NEFL (arrow). Allelic retention is shown at markers D8S254 and D8S1820.

at the more centromeric loci evaluated and no allelic deletions at 8p12–22 in the 5 FIGO stage III LMP tumors examined. Clinical and histopathological parameters of the tumors were examined to determine if allelic loss at 8p12–22 correlated with these features (Table 1). There was no correlation between chromosome 8p LOH and patient age, tumor histologic subtype, or grade. However, there was a statistically significant trend for allelic loss on 8p with increasing disease aggressiveness from LMP to advanced stage invasive ovarian cancer (P 2 , 0.024, Lehman’s test for trend). DISCUSSION The present data identify chromosomal locus 8p21 as a putative tumor suppressor gene site in sporadic ovarian carcinoma. The highest frequency of loss in our study was 50% (15/30 informative cases) and was identified at marker D8S136. Allelic loss rates in excess of 35% have been proposed by Cliby et al. to be outside the 95% confidence interval for random chromosomal aberrations and are probable causal genetic events in tumorgenesis [13]. Thus, the 50% LOH found at marker D8S136 in our study likely represents a significant deletion event in sporadic invasive ovarian cancer. The majority of the cases with loss at D8S136 (13/15 cases) suggest that the 8p21 tumor suppressor gene locus is telomeric to marker NEFL and centromeric to marker D8S254. One case with loss at D8S136 is homozygous for the surrounding markers and one case contains a large deletion of the entire chromosomal arm including marker

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FIG. 2. Summary of 45 cases analyzed for LOH at 8p12–22. (A) Deletion map of 45 ovarian tumors evaluated for LOH on chromosome 8p. Cases 1– 40 are invasive ovarian tumors. Cases 41– 45 are low-malignant-potential tumors. Microsatellite marker location is shown at the top of the figure. Closed circles represent allelic loss in the tumor, open circles represent allelic retention, and a straight line represents homozygous cases. (B) Histogram showing the frequency of allelic loss at each of the microsatellite markers evaluated as well as their estimated location (in centimorgans) from the centromere of chromosome 8.

D8S136, making these two cases supportive but not definitive for an 8p21 tumor suppressor gene locus. Only two of the cases examined (cases 2 and 5) do not support 8p21 as a single tumor suppressor gene site. Case 2 shows two separate regions of loss (D8S1820, D8S254) with allelic retention between the two markers. Case 5 shows loss more centrimerically on 8p21 at marker D8S1820 (Fig. 2A). The patterns of loss observed in these two cases may represent inactivation of an additional 8p tumor suppressor gene or alternatively may reflect genomic instability which is known to occur at a low baseline level in advanced cancers. A recent investigation of allelic loss on the short arm of chromosome 8 identified three additional loci that may be important in ovarian cancers: two loci at 8p23 and one at 8p22 (Table 2) [15]. These authors, in contrast to our study, found a lower frequency of allelic loss at D8S136 (31%) and loss of the entire chromosomal arm in 60% of the cases examined. We attribute our higher rate of loss at this locus

to analysis of a pure population of epithelial cells obtained by LCM. One allelotyping study identified a region of chromosomal deletion more distal at 8pter–32 in ovarian carcinoma; however, this was only identified in 4 of 10 informative cases and not confirmed by subsequent studies [13]. The current analysis and our recent study of BRCA1 mutation-positive patients supports 8p21 as a putative tumor suppressor gene site. Allelic loss studies of 8p in other human cancers have suggested there may be more than one tumor suppressor gene located on this chromosomal arm. Prostate cancer has been shown to exhibit LOH in three distinct regions, 8p12, 8p21, and 8p22. Other cancers (breast, lung, and colorectal) have identified multiple independent regions of loss on 8p as well [16 –23]. Although the relationship between LMP tumors and invasive epithelial ovarian tumors remains controversial, we thought it important to investigate LOH at our locus of interest in this subset of ovarian neoplasms. None of the five

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advanced staged papillary serous LMP tumors were found to have loss at 8p12–22. This follows the overall low frequency of loss identified in LOH studies of LMP tumors to date [26]. Our findings of increasing loss on 8p with disease aggressiveness was supported by the findings of Wright et al. They found a statistically significant increasing trend for 8p LOH with progression to late stage ovarian cancer [15]. These results are in contrast to previous reports from our laboratory and others suggesting 8p LOH as an early event in other cancers. We reported a 60% frequency of LOH at 8p21 in prostatic intraepithelial neoplasia, the precursor lesion of invasive prostate cancer [16]. Yaremko et al. found common patterns of loss on 8p21–22 in primary and metastatic tumors from the same breast cancer patients and no difference in allelic loss frequency between large (.2 cm) and small (,2 cm) lesions, suggesting that LOH at 8p21–22 may occur in early stage breast cancer [19]. Identification, cloning, and mutation analyses of the putative 8p21 tumor suppressor gene(s) are necessary to determine if a single common gene is involved in prostate, breast, and ovarian tumorigenesis and, if so, the timing of gene inactivation relative to tumor progression. We combined the specificity of laser capture microdissection and the sensitivity of PCR-based microsatellite LOH analysis to identify a 50% frequency of allelic loss at 8p21 in sporadic invasive epithelial ovarian cancer. The findings of this study in concert with our previous findings in BRCA1 mutation-positive patients suggest the presence of a tumor suppressor gene in this region. Efforts to further refine the minimal interval and identify the gene are underway.

TABLE 1 Frequency of 8p LOH by Disease Aggressiveness, Histologic Grade, and Subtype 8p12–22 LOH/total (%) A. FIGO stage* LMP tumors (all stages) I II III IV B. Grade 1 2 3 C. Histologic type Papillary serous Endometroid Clear cell * P 2 , 0.024, Lehman’s test for trend.

0/5 1/4 2/4 16/26 2/3

(0) (25) (50) (62) (66)

5/7 (71) 12/17 (70) 12/16 (75) 18/25 (72) 8/11 (72) 2/3 (66)

TABLE 2 Summary of Studies Evaluating Allelic Loss on 8p in Sporadic Ovarian Carcinomas

Author

n

Design

Locus

% LOH

Brown et al. Wright et al. [15] Tapper et al. [14] Osborne et al. [12] Emi et al. [21] Cliby et al. [13]

45 53 20 37 24 25

PCR/LOH PCR/LOH CGH Allelotype RFLP/LOH Allelotype

8p12–22 8p23 8p22 8p11.1–23.3 8p11.2–23.3 8pter–p32

58 43–50 38 25–29 33 40

Note. PCR, polymerase chain reaction; LOH, loss of heterozygosity; CGH, comparative genomic hybridization; RFLP, restriction fragment length polymorphism.

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