Oncogene (1998) 16, 197 ± 202  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

A one centimorgan deletion unit on Xq12 is commonly lost in borderline and invasive epithelial ovarian tumors

MI Edelson1, CC Lau4, CV Colitti1, WR Welch2, DA Bell3, RS Berkowitz1 and SC Mok1

1Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive , and 2Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, 02115; 3Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114; 4Division of Hematology-Oncology, Texas Children's Hospital, Houston, Texas, 77030, USA

We have used polymerase chain reaction (PCR) have been unable to detect any signi®cant level of ampli®cation of tandem repeats to study the pattern of genetic alterations speci®c to borderline tumors allelic loss on chromosome X11.2-q12 in borderline and emphasizing the possible separate pathogenesis of invasive epithelial ovarian tumors. Using eight micro- these tumors (Rodabaugh et al., 1995b; Wertheim et satellite markers spanning Xq11.2-q12, 41 borderline and al., 1994; Tangir et al., 1996; Rodabaugh et al., 1995a; 65 invasive ovarian tumors, together with their corre- Dodson et al., 1993; Kim et al., 1994). Recently, Zheng sponding normal tissues, were analysed. The highest et al. have demonstrated that 50% of informative percentage of loss of heterozygosity (LOH) was observed borderline tumors (eight of 16 informative cases) and at the DXS1194 in borderline tumors (four of 16 approximately 44% of high grade IEOC displayed informative cases, 25%) and at the androgen receptor LOH at the androgen receptor (AR) locus in the (AR) locus in invasive epithelial ovarian tumors (18 of 47 proximal portion of chromosome Xq (Cheng et al., informative cases, 38%). activation 1996). These results suggest that LOH in this region studies performed in cases with LOH at the AR locus may be important for the development of borderline showed that the allelic loss at the AR locus is not tumors and high grade invasive epithelial ovarian con®ned to the inactive . A one centimorgan region carcinomas. including the AR locus and ¯anked by the primers In this study, we report the construction of a DXS1161 and PGK1P1 was identi®ed as the smallest detailed deletion map of chromosome Xq11.2-q12 common loss region in both borderline and invasive from 41 borderline tumors and 65 invasive epithelial epithelial ovarian tumors. ovarian carcinomas using eight microsatellite markers in an e€ort to more accurately localize a region(s) Keywords: ovary; cancer; borderline; loss of hetero- which may be involved in the development of zygosity; chromosome Xq borderline ovarian tumors and invasive EOC.

Results Introduction LOH in Xq11.2-Xq12 Borderline ovarian tumors, also known as ovarian epithelial tumors of low malignant potential, are a A set of eight microsatellite markers spanning the distinct entity within the classi®cation of ovarian region from Xq11.2 to Xq12 (Figure 1) was used to neoplasms. These tumors have a histological appear- perform the LOH study on 41 borderline ovarian ance and biologic behavior which are intermediate tumors and 65 invasive epithelial ovarian carcinomas between benign and frankly malignant ovarian with various stages and grades. All cases showed neoplasms (Scully, 1982). While these tumors are interstitial LOH for several of the microsatellite loci cytologically malignant with evidence of cellular tested. LOH a€ecting at least one locus was observed pleomorphism, they do not exhibit any stromal in 29% of the borderline tumors (12 of 41 informative invasion. A prolonged survival is expected with these cases) and 34% of the invasive epithelial carcinomas tumors even without the use of chemotherapy (22 of 65 informative cases). The LOH results derived (Bostwick et al., 1986; Barakat, 1994; Leake, 1992; from paran embedded tissue and frozen materials Leake et al., 1992; Casey et al., 1993). were not di€erent statistically, and thus were combined Genetic studies have been used to understand the in the analysis of both borderline ovarian tumors and biology of ovarian cancers. Loss of heterozygosity invasive epithelial ovarian carcinomas. Figure 2 shows (LOH) studies have been used to detect allelic losses of autoradiographs of ®ve borderline tumor cases speci®c in order to identify possible depicting LOH at the AR locus in the tumor lane tumor suppressor which may be relevant to the with the normal control in the adjacent lane. development of ovarian cancer. While many of these studies have focused on invasive epithelial ovarian LOH in borderline tumors carcinomas, only a few studies have examined genetic alterations of borderline ovarian tumors. These studies The percentage of tumors with allelic loss at each locus for borderline tumors is shown in Figure 1. The highest Correspondence: SC Mok percentage of LOH was observed at DXS1213 (four of Received 8 February 1997; revised 5 August 1997; accepted 6 August 16 informative cases, 25%) and at the AR locus (seven 1997 of 30 informative cases, 23%). All other loci showed a Deletion mapping of chromosome Xq in ovarian cancer MI Edelson et al 198

Figure 1 Summary of the loci, map position, and frequency of loss of heterozygosity at Xq11.2-q12 in borderline ovarian tumors and invasive epithelial ovarian carcinomas

mately one centimorgan, is located between the markers DXS1161 and PGK1P1. The identi®cation of B5 B43 333 466 470 this region was based on the study of cases 315, 317, T N T N T N T N T N 416 and 490 (Figure 4).

AR LOH with respect to grade, stage and histological type Clinicopathological characteristics of the tumors were analysed based on allelic loss at the AR locus (Table Figure 2 Autoradiographs of PCR ampli®cations of DNA from 1). No signi®cant di€erence was observed between paran blocks (patients B5 and B43) and frozen materials LOH rate at the androgen receptor locus and stage or (patients 333, 466, 470) for borderline ovarian tumors at the androgen receptor (AR) locus. T, tumor DNA; N, normal DNA; tumor type for borderline tumors. Similar analysis for Allelic loss is visualized in the tumor lanes invasive epithelial ovarian carcinomas did reveal a statistically signi®cant di€erence of allelic loss at the AR locus for Stage III/IV tumors (P=0.04) and serous much lower percentage of LOH. A one centimorgan tumors (P=0.02). Comparison of LOH rate at the AR region including the AR locus and ¯anked by the locus between borderline tumors (23%), low grade primers DXS1161 and PGK1P1 was identi®ed as the carcinomas (32%), and high grade carcinomas (53%) smallest common loss region using selected cases which did not reveal any statistical di€erence (P=0.10), showed restricted LOH patterns (Figure 3). Cases B5, though a trend towards a greater frequency of LOH 466, and 543 speci®cally show LOH at the AR locus at the AR locus with less di€erentiated tumors was and no LOH at the ¯anking primers DXS1161 and observed. PGK1P1. No selective loss of the inactive allele at the AR locus LOH in invasive epithelial ovarian carcinomas Selected paired normal and tumor DNA from cases Similar to the borderline tumors, the AR locus showed which showed LOH at the AR locus were subjected to the highest percentage of allelic loss (18 of 47 HpaII restriction enzyme digestion to evaluate whether informative cases, 38%, Figure 1). However, the the lost allele is active or inactive. After digestion, the ¯anking primers showed a higher percentage of LOH remaining allele of cases 324, 336, 490, 528, 545 and in the invasive epithelial ovarian carcinomas as 558 disappeared after digestion suggesting that in these compared to the borderline tumors. Five cases (ES-3, cases the inactive AR allele was lost (Figure 5a). In 351, 384, 528 and 545) showed allelic loss at almost all contrast, the remaining allele of cases 332, 384, and 386 the loci suggesting that these samples had LOH in the could still be observed, suggesting that the active AR whole Xq11.2-q12 region. Therefore, these cases were allele was lost in these cases (Figure 5b). When DNA not informative for the localization of the smallest from normal tissue from cases 324, 332, 386, 490, 528, region of allelic loss. The remaining 17 cases showed and 558 were digested with HpaII, a skewing pattern of restricted LOH patterns which allowed us to de®ne the the two AR was observed. This was particularly smallest common loss region. This region, approxi- evident in case 386 suggesting a relatively selective X- Deletion mapping of chromosome Xq in ovarian cancer MI Edelson et al 199

Figure 3 Deletion map of 12 borderline ovarian tumors. Shaded region indicates loss of region. Primers used in the PCR reaction are listed above. For map of location of the primers, see Figure 1. L, loss of heterozygosity. N, no loss. U, uninformative

Figure 4 Deletion map of 22 invasive epithelial ovarian carcinomas. Shaded region indicates loss of region. Primers used in the PCR reaction are listed above. For map of location of the primers, see Figure 1. L, loss of heterozygosity. N, no loss. U, uninformative

chromosome inactivation in the normal tissue of these Table 1 Relationship between allelic loss for androgen receptor loci cases. and clinicopathologic characteristics in 30 borderline ovarian tumors and 47 invasive epithelial ovarian carcinomas Invasive The AR allele was not totally deleted in a majority of epithelial ovarian cases that showed LOH Clinico- Borderline tumors carcinomas pathologic LOH No loss LOH No loss To evaluate whether loss of heterozygosity observed at characteristics (n=7) (n=23) P (n=18) (n=29) P the AR locus was due to total allelic deletion or Stage deletion with the replacement by the remaining allele, I/II 7 18 1 13 we performed multiplex PCR. Primers D7S523 and III/IV 0 5 0.30 17 26 0.02 D11S1334 did not exhibit LOH at the AR locus for Histology Serous 5 17 16 16 these selected cases, and thus were used as internal Non-Serous 2 6 0.89 2 13 0.02 standards. After normalization of the intensity of the Gradea % LOH P alleles generated by the two control primer sets, the Borderline 7/30 (23%) intensity of the retained AR allele in the tumor tissue Low 8/25 (32%) of cases 332, 324 and 558 was found to be comparable High 10/19 (53%) 0.10 to that of the corresponding normal tissue (Figure 6). a Clear cell cases are excluded from grade analysis such that n=44 for In contrast, the intensity of the retained AR allele in invasive epithelial ovarian carcinomas Deletion mapping of chromosome Xq in ovarian cancer MI Edelson et al 200 a a b 324 336 384 386 490 528 545 332 558 Control Hpall + Control Hpall + Control Hpall + T N T N T NT N T N T N T NT N T N T N T N T N T N T N T N AR AR

D7S523 D11S1334 Case 324 Case 336 Case 490

Figure 6 LOH study on IEOCs using multiplex PCR. Markers D7S523 (a) and D11S1334 (b) were used to normalize the intensity of the remaining AR allele of IEOCs. Cases 332, 324, and 558 showed deletion of one of the AR alleles, while the others showed a duplication of the remaining AR allele. T=tumor, Control Hpall + Control Hpall + Control Hpall + N=normal tissue T N T N T N T N T N T N

IEOC and high grade IEOC. This genetic alteration may represent an early event in ovarian tumorigenesis, suggesting a continuous spectrum of development similar to the colorectal carcinoma model (Fearon et Case 528 Case 545 Case 558 al., 1990). Whether ovarian carcinomas develop de novo or by a multistep clonal expansion from preexisting benign tumors is unknown. Evidence that borderline tumors and low grade IEOC appear phenotypically stable over time with no evidence of b progression towards a biologically more aggressive lesions support a de novo hypothesis. A recent study of 14 cases of incidentally discovered ovarian carcinomas Control Hpall + Control Hpall + Control Hpall + con®ned to one ovary supports this theory (Bell and T N T N T N T N T N T N Scully, 1994). Furthermore, studies have shown an absence of a common pattern of genetic alterations between borderline tumors and invasive epithelial ovarian carcinomas suggesting a separate pathogenesis Case 332 Case 384 Case 386 for these tumors (Wertheim et al., 1994; Tangir et al., 1996; Rodabaugh et al., 1995a,b). However, our data Figure 5 LOH in the AR locus involves both the inactive (a) and the active allele (b). DNA obtained from the normal (N) and shows similar genetic alterations in borderline tumors tumor tissue (T) of the same patient was incubated in bu€er in and invasive carcinomas with fewer abnormalities in absence (control) or presence (HpaII+) of methylation-sensitive borderline tumors. This suggests that a certain endonuclease HpaII. All samples were ampli®ed by PCR using percentage of high grade lesions may have progressed primers ¯anking the trinucleotide repeat and the through an earlier, clinically inapparent borderline or adjacent HpaII sites in the ®rst exon of the AR . The radiolabeled PCR products were loaded onto a 6% polyacryla- lower grade counterpart. This hypothesis is also mide gel and electrophoresed at 1800 V for 2 h supported by the identi®cation of a high percentage of K-ras in both BOT and IEOC, particularly in the mucinous subtype (Mok et al., the tumor tissue of cases 336, 384, 386, 490, 528 and 1993). 545 was two times greater than that of the By using HpaII digestion, we attempted to determine corresponding allele in the normal tissue. whether the remaining allele is active or not. The relatively CpG-dense promoter of the AR gene subject to X-chromosome inactivation is hypermethylated and, Discussion therefore, resistant to digestion with methylation sensitive restriction enzymes such as HpaII (Vogel- We have identi®ed a one centimorgan smallest stein et al., 1987). Using PCR analysis combined with common region of allelic loss on Xq11.2-q12 in HpaII digestion, the active and inactive AR alleles can borderline tumors and invasive epithelial ovarian be distinguished (Allen et al., 1992). These results are carcinomas. This common region of loss contains the important in the interpretation of the LOH data as AR locus and is ¯anked by the primers DXS1161 and well as in the construction of a model. If the lost allele PGKP1. We also observed a higher percentage of LOH is always the active allele, then this is equivalent to at the AR locus for high grade carcinomas when inactivation of both alleles, suggesting that the compared to borderline tumors and low grade candidate gene in the deleted region is a tumor carcinomas. suppressor gene. On the other hand, if the lost allele We have found a molecular genetic abnormality is always the inactive allele, the LOH data could ®t which is common in borderline tumors, low grade either a tumor suppressor gene or proto-oncogene Deletion mapping of chromosome Xq in ovarian cancer MI Edelson et al 201 model, depending on whether there is reduplication summarized in Figure 1. The forward primer of each set and of the remaining allele. If there is was end labeled with gamma 32P-ATP (ICN, Irvine, CA) reduplication of the remaining active allele without and polynucleotide kinase (Boehringer Mannheim, India- an inactivating mutation, the net e€ect of this genetic napolis, IN). The reaction mix was then diluted into a ®nal alteration would be to double the gene dosage. Thus, volume of 320.5 ml of primer-PCR mixture containing 40 ml 106PCR Bu€er (0.1 M Tris-HCl, 0.5 M KCl, pH 8.3), 20 ± the candidate gene could be an oncogene with 50 ml4mMMgCl2,20ml1.25mMdNTP, 2.0 ml (10 units) expression of two copies of the gene instead of only of Taq Polymerase (Perkins-Elmer Cetus, Norwalk, CT) one. However, if there is inactivating mutation in the and 1 ml of each primer. Four mlofthisPCRmixturewas remaining active allele, the candidate gene could still be mixed with each DNA sample. Ampli®cation was carried a tumor suppressor gene. Six out of our nine cases with out using 50 ng of genomic DNA using 35 cycles of PCR LOH showed loss of the inactive allele. Of those six with denaturation at 948Cfor30s,annealingat45±668C cases, four showed duplication of the remaining allele. for 30 s and extension at 728C for 30 s. PCR products were Thus, these data are inconclusive in determining if the diluted in 45 ml of loading bu€er containing 95% candidate gene is a tumor suppressor gene or an formamide, 20 mM EDTA, 0.05% bromophenol blue and oncogene. It will eventually require the identi®cation of 0.05% xylene cyanol FF (Sigma, St Louis, MO). Three microliters of this mixture was then loaded onto a 6% the candidate gene and its mutational analysis before acrylamide denaturing gel and electrophoresed at a any ®nal conclusion can be drawn. constant 1700 volts. The gel was transferred onto a As far as the possibility of the candidate gene(s), 3 MM paper, dried and autoradiographed with Kodak X- there are relatively few known genes in the minimally OMAT AR ®lms for 3 ± 4 h at room temperature. The deleted region. The single highest incidence of LOH is autoradiographs were then developed and examined for found at the AR locus for both borderline and evidence of LOH. invasive ovarian tumors, thus making AR a likely Cases were considered to be informative when hetero- candidate gene. The only reported role of the zygosity was detected in normal tissue samples. LOH was androgen receptor in cancer is in the hormonal de®ned as a visible reduction of 50% or more in the band response of prostate cancer and male breast cancer intensity of one of the tumors sample allele when compared to the normal tissue control. Homozygous alleles in the (Schoenberg et al., 1994; Newmark et al., 1992; normal tissue samples were labeled as uninformative. Lobaccaro et al., 1993; Tilley et al., 1996; Taplin et al., 1995). Ampli®cation of the androgen receptor gene has been found in recurrent hormone-refractory X chromosome inactivation studies prostate tumors, but not in primary prostate tumors Two micrograms of tumor and normal DNA samples from (Visakorpi et al., 1995). It is not clear whether the cases with known LOH at the AR locus were digested with androgen receptor could contribute to the oncogenesis 20 units of HpaII restriction endonuclease (Boehringer of tumor in the genital tract. Work is now Mannheim, Indianapolis, IN) in a total volume of 20 ml. ongoing in our laboratory to search for mutations in Control reactions consisting of normal and tumor DNA the AR gene in ovarian cancer as well as the role of samples incubated in the digestion bu€er without androgens in the growth regulation of both normal endonuclease were also performed. After digestion over- ovarian epithelial cells and ovarian cancer cells in nightat378C, 1 ml of DNA from each reaction was used as vitro. templates for PCR ampli®cation using the AR primers as described above.

Materials and methods Multiplex PCR analysis

Tumor collection and DNA extraction Multiplex PCR analysis of microsatellite polymorphism was conducted to determine whether allelic imbalances Surgical specimens of human ovarian tissue were obtained observed at the AR locus were due to total deletion or from 73 patients under a protocol approved by the Human deletion with duplication of the homozygous allele. Besides Subjects Committee of the Brigham and Women's the AR primer set, another primer pair (D7S523 on Hospital. Archival material consisted of 33 paran chromosome 7q or D11S1334 on chromosome 11p) was blocks. The corresponding normal tissues consisted of included in the PCR reaction as an internal control. The segments of normal fallopian tube, uninvolved round control primers were chosen based on their retention of ligament, or peripheral blood. All histopathologic diag- heterozygosity and the size of the microsatellite markers noses of ovarian epithelial tumors were con®rmed by a used. The number of PCR cycles were varied between 25 gynecologic pathologist. Grade 1 and grade 2 invasive and 35 before an optimal protocol allowed for consistent tumors were categorized as low-grade tumors, while high- ampli®cation in repeated assays. Three ml of the PCR grade tumors included grade 3 and grade 4 invasive mixture was then loaded onto a 6% acrylamide denaturing tumors. All tumors were surgically staged according to gel and electrophoresed as described above. A second gel FIGO criteria (International Federation of Gynecology was rerun on diluted samples which showed identical allelic and Obstetrics, 1987). DNA was extracted using previously intensities on both normal and tumor samples in the published methods (Tsao et al., 1993). control markers. The allelic intensities of both AR alleles in tumor DNA and normal DNA were then compared and PCR and LOH studies quanti®ed by a densitometer. LOH was performed by PCR ampli®cation of tandem repeats located at eight loci on chromosome Xq11.2-q12. Statistical analysis The map position and genetic distance between primers are Comparison of allelic loss frequencies and clinicopatholo- based on the Genethon chromosome X map and a report gic parameters was assessed by Chi-Square and Fisher's on the sixth international workshop on X chromosome exact test when an expected cell value was ®ve or less. mapping (Gyapay et al., 1994; Nelson et al., 1995; Weeks Statistical signi®cance was considered when P values were et al., 1995). The di€erent loci and their map positions are less than 0.05. Deletion mapping of chromosome Xq in ovarian cancer MI Edelson et al 202 Acknowledgements R01CA63381 from the National Institute of Health, This work was supported by Public Health Service Grant Department of Health and Human Services.

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