Genomic imbalances in ovarian borderline serous and mucinous tumors

Cancer Genetics and Cytogenetics 139 (2002) 18–23

Genomic imbalances in ovarian borderline serous and mucinous tumors Jie Hua,b,*, Vinish Khannab, Mirka M.W. Jonesc, Urvashi Surtib,c,d,e a

Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA b Department of Genetics, UPMC Magee-Womens Hospital, Pittsburgh, PA, USA c Department of Pathology, UPMC Magee-Womens Hospital, Pittsburgh, PA, USA d Magee-Womens Research Institute, Pittsburgh, PA, USA e Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA Received 24 January 2002; received in revised form 29 April 2002; accepted 29 April 2002

Abstract

We analyzed 25 ovarian borderline tumors (13 serous and 12 mucinous tumors) by comparative genomic hybridization (CGH). Genomic imbalance was detected in 85% of serous tumors and 75% of mucinous tumors. Different patterns of genomic alterations were identified in serous and mucinous tumors. Gain of the X chromosome was common in both serous (30%) and mucinous (42%) tumors. However, gain of chromosome 8 was detected exclusively in 38% of serous and mixed sero-mucinous tumors, but not in any pure mucinous tumors. According to the present and previous studies, gain of chromosome 8 is the most common abnormality in borderline serous tumors. Gain of the same chromosome is also common in high grade and advanced stage serous carcinomas, but uncommon in early stage serous carcinomas. In addition gain of chromosome X is common in borderline serous and mucinous tumors, while loss of chromosome X is predominant in invasive carcinomas. These findings do not support the multi-step progression theory from borderline tumor to high-grade, advanced stage carcinoma, but indicate that the borderline ovarian tumor is a distinct entity. Genes in chromosome 8 may be critical for the development and the differentiation of borderline serous tumors. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction Epithelial tumors are the most common ovarian tumors. These tumors are classified according to the cell type, serous, mucinous, or endometrioid and according to the biological behavior, for example, benign, borderline (low malignant potential), and invasive (carcinomas). The borderline tumors are placed between clearly benign and obviously malignant tumors, because they exhibit some, but not all of the morphologic features of malignancy. All borderline tumors are characterized by the presence of malignant cytologic features and by the absence of stromal invasion. Borderline tumors are more often found in the young population with a mean age of 40 for both serous and mucinous types. In general patients with borderline tumors have a favorable prognosis. The 10-year survival rate for patients with serous and mucinous borderline tumors is estimated to be 70–90%, respectively [1]. However, the mortality for patients with advance-stage borderline tumors is 30–40% regardless of the histological type [2]. It is not clear, whether ovarian carcinomas develop from preexisting borderline tumors.

* Corresponding author. Magee-Womens Hospital, Department of Genetics, 300 Halket Street, Pittsburgh, PA 15213. Tel: (412) 641-1736; fax: (412) 641-2255.

A study reviewing 953 borderline tumors disclosed malignant transformation in only 0.7% [1]. However, recent molecular studies of invasive carcinomas with contiguous benign, borderline, and invasive areas have suggested that the tumor progresses from benign through borderline to invasive carcinoma [3]. Therefore, it is still questionable whether the borderline tumor is a distinct clinical and histological entity or a precursor of invasive carcinoma. Little is known about the molecular features and the biology of borderline tumors. There is only a handful of genetic data available. The lack of the genetic data in borderline tumors is mainly due to their histologic nature. Specimens of ovarian borderline tumors often contain a mixture of both normal and tumor cell populations, which makes it difficult to obtain pure tumor cells for molecular and cytogenetic analysis. To our knowledge, cytogenetic studies have been reported in less than 40 ovarian borderline tumors. Fifty percent of these tumors showed normal karyotype [4–10]. Only 32 ovarian borderline tumors have been studied by comparative genomic hybridization (CGH), and genomic imbalances have been detected in only 16 of the 32 tumors [3,11–13]. To gain an insight into the molecular mechanism of the development and progression of the ovarian epithelial tumors, we studied 25 ovarian borderline tumors by CGH. To our knowledge this is the first report studying a relatively

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large number (25 cases) of ovarian borderline tumors by CGH and it is the first report of comparing genomic alterations between borderline serous and mucinous tumors by CGH analysis. This study provides valuable additional information to the tumor database of genomic alterations in ovarian borderline tumors. 2. Materials and methods 2.1. Tumor specimens All 25 ovarian borderline tumors were examined histologically to confirm the diagnosis. Tumors containing more than 70% tumor-cellularity were included in the study. A total of 13 serous borderline tumors (10 pure serous and three seromucinous tumors) and 12 mucinous tumors were studied. Table 1 summarizes the clinical information. Figures 1a and 1b show the examples of hematoxylin-eosin stained sections of borderline serous and borderline mucinous tumors. 2.2. CGH High-molecular-weight–DNA samples were extracted from 19 frozen and 6 paraffin-fixed archival tumor tissues by routine methods with salt precipitate after proteinase K digestion. Tumor DNA was labeled by nick translation with SpectrumGreen dUTP, and normal male reference DNA was labeled with SpectrumRed dUTP. Approximately 300 ng of each labeled tumor and reference DNA were coprecipitated with 20 g of human Cot 1 Table 1 Clinical information of 25 ovarian borderline tumors No. of tumors 2 3 5 6 7 12 14 15 16 19 24 18 25 9 10 13 1 21 22 4 8 11 20 23 17

Stage

Type of tumor

Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ia Ib Ib Ib Ib II II III III III III III III

BMT BMT BMT BMT BMT BMT BMT BMT BMT BMT BMT BST BST BST BST BST BSMT BSMT BST BST BST BST BST BST BMT

Laterality

Size (mm)

Age of patients (y)

L L R R L R R L L L R L R B B B B B L B L B R B B

140 215 170 120 210 215 195 95 220 27 90 220 140 80 70 150 180 90 100 145 75 85 100 180 130

56 26 69 41 37 41 17 68 39 35 72 79 58 72 46 78 32 60 72 42 58 55 34 38 34

Abbreviations: B, bilateral; BMT, borderline mucinous tumor; BSMT, borderline serous-mucinous tumor; BST, borderline serous tumors; L, left ovary; R, right ovary; y, years.

Fig. 1. Representative photographs of hematoxylin-eosin–stained tissue sections showing (a) borderline serous tumor and (b) borderline mucinous tumor.

DNA, and the resulting pellet was dissolved in 13 L of hybridization buffer (50% formamide, 2 SSC, 20% dextrin sulfate, pH 7). The probe and target slides were denatured. The hybridization was carried out at 37C for 3 days. 2.3. Digital analysis Grey-level images were acquired from at least 15 metaphase cells with an Olympus microscope and a CCD camera. The hybridization was analyzed with the Applied Imaging Cytovision System. Chromosomes were identified by DAPI banding. Background fluorescence was subtracted. A ratio of 1 represents the balanced state of the chromosomal copy number. We used upper and lower thresholds more than 1.2 and less than 0.8 to interpret gain or loss of chromosomal materials. To evaluate gain of the X chromosome in tumors we used a normal female as a control and used upper and lower thresholds of more than 1.7 and less than 1.3 to interpret gain or loss of X chromosomal materials. 3. Results Genomic alterations were detected in 85% (11/13) of the serous tumors and 75% (9/12) of the mucinous tumors (Tables 2

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Table 2 Genomic alterations in 13 borderline serous tumors

4. Discussion

Case no.

4.1. Serous versus mucinous borderline tumor

1

4 8 9 10

11 13 18 20 21 22 23 25

Results rev ish enh(Xq21→q28,7,8q,12,14q),dim(1p33→p36,8p,9q34, 11p11.1q12.12,16p,17,19,21,22) rev ish enh(X) rev ish enh(X,16,22p13→q12) rev ish enh(8,15q11.1q15,18q) rev ish enh(1q22→q44,2,7,8p22→p23,8q21→q24,13q14→q34), dim(X,1p33→p36,6,15,17,20p11.2→q11.2,22) rev ish enh(1q22→q44,8p12→q24,12p13→q14) rev ish enh(5) rev ish enh(12q11→q15) rev ish enh(8) rev ish enh(X),dim(1p36) rev ish dim(22) Normal Normal

and 3). The abnormalities involved 19 chromosomes. Gain of chromosome X was found in 30% of borderline serous tumors and in 42% of borderline mucinous tumors. Gain of chromosome 8 was detected in 38% of borderline serous tumors, including pure serous and sero-mucinous tumors, but not in any of the pure borderline mucinous tumors. Gain of chromosome 12 was identified in 23% of borderline serous tumors and in 8% of borderline mucinous tumors. Loss of 1p was found in 23% of serous tumors and in 17% of mucinous tumors. A summary of chromosomal regions with significant gains and losses of DNA copy number in serous and mucinous tumors is illustrated in Fig. 2. A representative partial CGH profile with gains of chromosome 7, 8, and 12 (case 1) is shown in Fig. 3. The CGH profile of X chromosomes from a normal female control and representative profiles from tumors with gain of X chromosome is shown in Fig. 4.

Table 3 Genomic alterations in 12 borderline mucinous tumors Case no.

Results

2 3 5 6 7 12 14 15 16

rev ish enh(4p15.1q13),dim(1p33→p36,19,22p13) rev ish enh(X,9,12,19,22q) rev ish enh(X) rev ish enh(X) Normal rev ish enh(9p13→q13),dim(Xp11.2→q13) rev ish enh(X) Normal rev ish enh(Xp21→p22,Xq22q28,6q11→q22,13q21→q22), dim(1p32→p36,7p11.2q11.2,16p,17,19,22) rev ish enh(1p33→p36,16p,16q23→q24,17p12p13,17q24→q25), dim(Xp11.2q13,2p22→q34,3p14→q13.3,4p15.3q32,5→ p14q23,6p21.1q23,13q14→q31,18q11→q12) Normal rev ish enh(1q21→q44)

17

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Serous and mucinous tumors are the two most common histologic types of borderline tumors of the ovary. In addition to the histological differences, serous tumors are more often bilateral and mucinous tumors are predominantly unilateral. Patients with borderline mucinous tumors have better survival rate than patients with borderline serous tumors. The 10-year survival rate for the patients with borderline mucinous tumor is 90% whereas for the patients with borderline serous tumors is 70% [1]. Divergence of DNA copy number changes in serous, mucinous, and endometrioid ovarian carcinomas has been reported in literature, and various pathways of tumorigenesis for these subtypes of tumors have been suggested [14]. The cytogenetic and molecular cytogenetic data in ovarian borderline tumors are extremely limited. To our knowledge less than 40 tumors have been reported with karyotypes [4–10, 15]. A literature review of cytogenetic findings in borderline serous and mucinous tumors is presented in Tables 4 and 5, respectively. Based on the previous studies, trisomy 12 was the common abnormality in both serous and mucinous tumors; trisomy 8 was detected only in serous tumors but not in any mucinous tumors; and trisomy 7 was predominantly identified in mucinous tumors. Of the 10 borderline serous tumors reported in literature with CGH analysis [3] seven tumors did not show any abnormality, while two of the three abnormal cases had gain of chromosome 8. One of two mucinous tumors had gain of 5p and 9p and the remaining one had balanced CGH profile in another CGH study [12]. Additional 18 borderline tumors (14 serous and 4 mucinous) were reported with CGH results by Blegen et al. [13], 11 of which showed genomic imbalances, and the most common chromosome alterations were gains of 8q (52%), 3q (44%), 5p (32%), Xq (32%), and 12p (28%), and losses of 22q (28%) and 17p (32%). However, the histology of each tumor was not specified. We studied 25 ovarian borderline tumors by CGH. Genomic alterations were detected in 20 of the 25 tumors. The number of genomic alterations ranged from one to 13, and the majority of the tumors (16/20) had less than five chromosomal alterations. The most common regions of gain involved chromosomes X, 8, and 12, or chromosome regions 12q and 8q. The most common region of loss was 1p33→ pter or 1p36→pter. These alterations were also identified by previous CGH studies [3,13]. CGH profiles for borderline serous and mucinous tumors were found to be different in the present study. Gain of chromosome 8 (or 8q) was identified in 38% of borderline serous tumors. Gain of the same chromosome was detected in 29% of borderline serous tumors in the previous cytogenetic studies. Interestingly, it was not detected in any pure mucinous tumors either in the present study or in previous studies (Table 3.2). The present study detected increase in DNA copy number of chromosome 12 in 23% (3/13) of the borderline serous tumors and in 8% (1/12) of the borderline mucinous

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Fig. 2. Schematic diagram of gains and losses in 13 borderline serous tumors (wide bars) and 12 borderline mucinous tumors (narrow bars).

tumor. Review of the literature revealed gain of chromosome 12 in 31% (8/29) of borderline serous tumors and in 29% (2/7) of borderline mucinous tumors (Table 3.2). Our results of CGH study in borderline serous tumors are consistent with that of previous classical cytogenetic studies. Although 16/32 borderline tumors reported earlier showed genomic alterations, the histologic types of the tumor were not correlated with the results in any of these previous studies. Therefore, the different patterns of genomic alterations be-

Fig. 3. The partial CGH profile from tumor 1 showing gains of chromosomes 7 and 12, 8q and the 1:1 ratio is represented by the vertical black line. The lines to the right (green) and to the left (red) of the 1:1 ratio line represent 0.25 increments of gains and losses, respectively. The pink tracings are generated by image software analysis and present the mean green:red ratio along the chromosome. The tan tracing reflects the standard error of the mean; while n  number of chromosomes counted to provide the mean.

tween serous and mucinous tumors in previous CGH studies are unknown. Our findings and analysis of previous cytogenetic findings indicate that the increased DNA copy number of chromosome 8 may play an important role in the development and differentiation of serous borderline tumors; such alterations are not part of borderline mucinous tumors. The critical genes located on chromosome 8 cannot be predicted at this time, because the overlapping region of gain (q13→qter) covers almost the entire q arm of the chromosome 8. Gain of chromosomes X and 12 may be involved in the pathogenesis of both border-

Fig. 4. X chromosome CGH profiles from normal female control and tumors with gain of the X chromosome.

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Table 4 Literature review of cytogenetic findings in 29 serous borderline tumors Case no. 1 2 3a 4

5 6 7 8 9 10 11 12 13 14 15 16a 17a 18 15 19a 20 21–29

Cytogenetic findings

References

4749,XX,7,8,mar[c]19]/46,XX[1] 47,XX,7[3]/46,XX[16] 4546,XX,6[7]/46,XX[12] Right ovary: 4748,XX,7,7[cp20] Left ovary: 4749,XX,7,7,7[cp20] Ommentum: 4649,XX,t(1;6)(p10;p10),7,7,7,22[cp20] Left ovary: 47,XX,10 Right ovary: 47,XX,10 49,XX,2,7,12 Left ovary: 47,XX,12 Right ovary: 47,XX,12 47,XX,12 47,XX,r(1)(p36q21),der(11)t(1;11)(q21;q24) 46,XX 46,XX 47,XX,12 47,XX,12 46,XX 47,XX,7[6]/48,XX,7,8[5]/46,XX,7,8[cp11] 5758[2n],XX,3,4,5,7,8,11,12,14,16,20[cp18] 46,XX,r(1)(p23p36),8,12,t(14;16)(q31;q13)[7] 46,XX 47,XX,12 47,XX,5,8,12 46,XX,r(1)(p36q42) 46,XX

Deger et al., 1997 [10]

Knoerr-Gaerytner et al., 1977 [4] Crickard et al., 1986 [5] Yang-Feng et al., 1991 [7]

Jenkins et al., 1993 [15]

Thompson et al., 1994 [8]

Izutsu et al., 1996 [16] Pejovic et al., 1996 [9]

a

Karyotypes copied from previous original reports in the literature.

line serous and mucinous tumors. The smallest overlapping region of gain of chromosome 12 was q11→q14, which harbors the oncogene GLI (glioma-associated oncogene homolog), ERBB3 (V-ERB B2 avain erythroblastic leukemia viral oncogene homolog 3), SAS (sarcoma amplified sequence), MDM2 (mouse double minutes 2 homolog), and CDK4 (cyclin-dependent kinase 4). The common region of gain of X was q22→qter. Increase in DNA copy number of chromosome 7 was detected in borderline serous tumors (serous or sero-mucinous in the present and previous studies [5,8,10]. Although the present study did not find gain of chromosome 7 in any pure mucinous tumor, previous cytogenetic studies identified trisomy or tetrasomy in three of seven borderline mucinous tumors [6,9,10,16]. Therefore, gain of chromosome 7 is common in both serous and mucinous tumors. If the tumor has less than 50% of cells with trisomy 7, such as the tumor with trisomy 7 in three of 20 cells in the case reported by Tharapel

et al. [6] (Table 3), this abnormality may not be detected by CGH. Loss of 1p was found in 23% of serous tumors and in 17% of mucinous tumors. Deletion of 1p36 resulting from ring chromosome 1 was reported in three serous borderline tumors by previous studies [7–9]. Chromosome arm 1p contains a few tumor suppressor genes (TSG) [17,18] including NOEY2, of which one of the alleles is imprinted and loss of the functional allele was found in ovarian and breast carcinomas, and neuroblastomas. 4.2. Genomic alterations and tumor stages

Case no. Cytogenetic findings

References

1 2 3 4 5 6

Tharapel et al., 1996 [6] Pejovic et al., 1996 [9]

A total of six advance-stage borderline tumors and 19 early-stage borderline tumors were analyzed in the present study (Table 1). All of 12 mucinous tumors, but one, were stage 1a, whereas five of 13 serous tumors were stage III. Due to the small number of tumors in each histologic type, a correlation between particular genomic alterations and clinical stages could not be established at the present time. Although almost all of stage III tumors (5/6) were serous and gain of chromosome 8 was found only in serous tumors, gain of chromosome 8 is not correlated with this stage, because it has been found in both early stage and advanced stage borderline serous tumors.

Izutsu et al., 1996 [16]

4.3. Borderline tumors and invasive carcinomas

Deger et al., 1997 [10]

Whether borderline tumor is the precursor of ovarian invasive carcinoma is in question. The histologic finding of invasive carcinoma arising in the background of borderline

Table 5 Literature review of cytogenetic findings in mucinous borderline tumors

7

47,XX,7[3]/46,XX[27] 47,XX,12 47,XX,7 47,XX,7 46,XX,del(14)(q24q32) 4748,XX,del(1)(q25),12[cp3]/ 46,XX[4] 46,XX

Yang-Feng et al., 1991 [7]

J. Hu et al. / Cancer Genetics and Cytogenetics 139 (2002) 18–23

tumor supports the precursor theory. Some chromosomal aberrations, such as gain of chromosome 8 commonly found in ovarian borderline tumors and carcinomas were also used as an evidence to support this theory [3]. The present study found that gain of chromosome 8 was the most common abnormality in borderline serous tumors regardless of the clinical stage. This abnormality was also detected in invasive carcinomas, but predominantly in high-grade and advancestage serous carcinomas and rarely in early stage carcinomas [19,20]. Therefore, it cannot be used as a strong evidence to support the progression theory. Gain of the X chromosome was a common abnormality in borderline serous and mucinous tumors detected by the present study and the previous reports [13] whereas loss of X chromosome has been detected in 50% of carcinomas in our more recent study (submitted for publication). In addition, loss of X chromosome has been also frequently reported in ovarian carcinomas by other investigators [11,13]. Furthermore, a previous study found that non-random germ-line X chromosome inactivation is prevalent among individuals who develop invasive ovarian carcinomas, but not borderline tumors [21,22]. Taken together, these findings do not support a multistep progression theory from borderline tumor through lowgrade to high-grade carcinoma, but support the theory that borderline tumor is a distinct entity. However, it cannot be ruled out that a small percent of borderline tumors may progress to invasive carcinoma. 5. Conclusion Ovarian borderline tumor appears to be a genetically distinct entity from invasive carcinoma. Gain of chromosome 8 is the most common genomic imbalance in serous borderline tumors, but not observed in borderline mucinous tumors. Gains of chromosomes X and 12 and loss of 1p are common findings in both serous and mucinous borderline tumors. Genes located in chromosome 8 may play an important role in the development and differentiation of serous borderline tumors. Appendix 1 This project was funded by the Magee-Womens Health Foundation Research Fund. References [1] Stevens A, Lowe J. Gynaecological and obstetric Pathology. In: Stevens A, Lowe J, editors. Pathology. London: Mosby, 1995 p. 361–86. [2] Kurman RJ, Trimble CL. The behavior of serous tumors of low malignant potential: are they ever malignant? Int J Gynecol Pathol 1993; 12:120–7. [3] Wolf NG, Abdul-Karim FW, Farver C, Schrock E, du Manoir S, Schwartz S. Analysis of ovarian borderline tumors using comparative genomic hybridization and fluorescence in situ hybridization. Genes Chromosomes Cancer 1999;25:307–15.

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