REPORT

Molecular Evidence for the Independent Origin of Extra-ovarian Papillary Serous Tumors of Low Malignant Potential

Jian Gu, Lawrence M. Roth, Cheryl Younger, Helen Michael, Fadi W. Abdul-Karim, Shaobo Zhang, Thomas M. Ulbright, John N. Eble, Liang Cheng

Affiliations of authors: J. Gu, L. M. Roth, C. Younger, H. Michael, S. Zhang, T. M. Ulbright, J. N. Eble, L. Cheng, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis; F. W. Abdul-Karim, Case Western Reserve University School of Medicine, Cleveland, OH.

Correspondence to: Liang Cheng, M.D., Department of Pathology and Laboratory Medicine, Indiana University Medical Center, University Hospital 3465, 550 N. University Blvd., Indianapolis, IN 46202 (e-mail: lcheng{at}iupui.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Molecular data suggest that peritoneal tumors in women with advanced-stage ovarian papillary serous adenocarcinoma are monoclonal in origin. Whether the same is true for ovarian tumors of low malignant potential is not known. We compared peritoneal and ovarian tumors from women with advanced-stage ovarian papillary serous tumors of low malignant potential to determine whether the peritoneal tumors arose from the same clone as the ovarian tumors. Methods: We studied the clonality of 73 peritoneal and ovarian tumors from 18 women with advanced-stage ovarian papillary serous tumors of low malignant potential. Formalin-fixed, paraffin-embedded tumors and representative normal tissues were sectioned and stained with hematoxylin–eosin, representative sections from separate tumors were manually microdissected, genomic DNA was extracted from the microdissected tumors, and the polymerase chain reaction was used to amplify a CAG polymorphic site in the human androgen receptor locus on the X chromosome to determine the inactivation pattern of the X chromosome and the clonality of the tumors. Results: The pattern of X-chromosome inactivation could be determined from the tumors of 13 of 18 patients. Of the 13 patients, seven (54%) had nonrandom inactivation of the X chromosome, and six of the seven had different inactivation patterns in the peritoneal and ovarian tumors. Three of these patients also had different patterns of nonrandom X-chromosome inactivation in tumors from each ovary. The remaining six patients had random patterns of X-chromosome inactivation in the peritoneal and ovarian tumors. Conclusions: Our data suggest that peritoneal and ovarian tumors of low malignant potential arise independently.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Epithelial ovarian tumors of low malignant potential, also known as borderline tumors, account for approximately 15% of all epithelial ovarian cancers. These tumors, characterized by a proliferation of epithelial cells forming papillary projections or tufts, lack stromal invasion. Patients with ovarian tumors of low malignant potential may have peritoneal tumors that are morphologically identical to the primary ovarian tumors. However, studies (14) have suggested that the presence of these peritoneal tumors does not imply a poor patient prognosis unless the tumors have an invasive growth pattern. Indeed, the 5-year survival rate for patients with ovarian tumors of low malignant potential is greater than 90%, compared with approximately 30% for patients with invasive ovarian cancers.

Although ovarian tumors of low malignant potential and the associated peritoneal tumors are often similar histologically, it is unknown whether the ovarian and peritoneal tumors are of common origin (i.e., arise from the same neoplastic clone). Several studies (57) have addressed the clonality of advanced-stage ovarian carcinomas and found these tumors to be monoclonal at ovarian and extra-ovarian sites. By contrast, papillary serous carcinomas of the peritoneum have been shown to be multifocal in origin (8). Understanding the clonal origin of peritoneal tumors in patients with ovarian tumors of low malignant potential may have important biologic and clinical implications for tumor prevention, tumor classification, and treatment.

The clonality of a tumor can be determined by X-chromosome-linked and non-X-chromosome-linked analysis, such as loss of heterozygosity (LOH), gene rearrangements, and point mutations. The most consistent informative marker of the clonal composition of neoplastic and preneoplastic disorders in women is the cellular pattern of X-chromosome inactivation. In women, normal somatic cells contain two X chromosomes, one of which is inactivated during early embryogenesis. X-chromosome inactivation occurs randomly and results in somatic mosaicism in normal tissues, with an equal mix of cells inactivating the X chromosome of either maternal or paternal origin. Throughout the life of the cell, the same maternal or paternal X chromosome will be inactivated in any subsequent cell division. Because the fidelity of the X-chromosome inactivation is retained, if the ovarian and peritoneal tumors arise from the same neoplastic clone, they should have identical inactive X chromosomes. Identical patterns of nonrandom X-chromosome inactivation would, therefore, suggest that the ovarian and peritoneal tumors are monoclonal in origin, implying that the peritoneal tumors metastasized from the primary ovarian tumor. Different patterns of nonrandom X-chromosome inactivation would suggest that the tumors are independent in origin.

Although there are several methods for assessing X-chromosome inactivation, such as X-linked DNA polymorphism of hypoxanthine phosphoribosyl transferase (HPRT) or phosphoglycerate kinase, etc., we took advantage of the polymorphic CAG repeats near the methylation-sensitive sites of the HhaI restriction endonuclease in exon 1 of the androgen receptor (AR) gene, located in chromosome Xq11–12 (9,10). The frequency of genetic polymorphism for the human AR gene is more than 90%, compared with 29% for that of the HPRT gene. The methylation status of the HhaI restriction endonuclease site corresponds with the inactivation status of the X chromosome and thus allows for the distinction between the active and inactive X chromosomes. In this report, we assessed whether ovarian and peritoneal tumors of low malignant potential were of common origin.


    PATIENTS AND METHODS
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Patients

Eighteen women, ranging in age from 18 to 86 years, were diagnosed with advanced-stage ovarian papillary serous tumors of low malignant potential and underwent staging laparotomies and resection of the ovaries and extra-ovarian tumor sites at Indiana University Hospitals and University Hospitals of Cleveland from 1991 to 1999. All samples were procured after obtaining a signed informed consent form in accordance with the Institutional Committee for the Protection of Human Subjects. All patients had both ovarian and peritoneal papillary serous tumors of low malignant potential. All tumors were staged according to the criteria of the International Federation of Gynecology and Obstetrics for ovarian carcinoma (11). None of the tumors in our series had a micropapillary pattern (12). Such tumors have also been referred to as micropapillary serous carcinoma by some investigators (13).

Tumor Samples and Microdissection

Histologic sections were prepared from formalin-fixed, paraffin-embedded blocks and stained with hematoxylin–eosin for histopathologic review and the X-chromosome inactivation analysis. In total, 73 separate tumors were obtained from the 18 patients. Tumors were obtained from both ovarian and peritoneal sites for each patient. One control sample (i.e., not involved with the tumor) was obtained from the normal stromal tissue for each patient.

Tumors were microdissected from serial sections as described previously (14,15). Briefly, cells of interest were selected under direct light microscopic visualization (Olympus, Tokyo, Japan) and gently scraped with the use of a sterile 28-gauge needle until the selected cells were detached from the deparaffinized slides. The cells were then picked up by the needle and transferred into a single-step extraction buffer (see below). Approximately 400–600 cells were microdissected per sample. For examples of tumor sections before and after microdissection, see Fig. 1Go.



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Fig. 1. Sequential microdissection of ovarian serous borderline tumors (case 8). Panel A: tumor from right ovary before microdissection. Panel B: tumor from right ovary after microdissection. Panel C: tumor from the left ovary before microdissection. Panel D: tumor from the left ovary after microdissection. Original magnification x400.

 
Detection of X-Chromosome Inactivation

DNA samples were prepared from distinctly separate tumors from the same patient. The dissected cells were placed in 15 µL of buffer (i.e., 10 mM Tris–HCl, 1 mM EDTA, 1% Tween 20, and 0.2 mg/mL of proteinase K [pH 8.3]) and incubated overnight at 37 °C (14,15). The solution was boiled for 10 minutes to inactivate the proteinase K and used directly for subsequent clonal analysis without further purification. Aliquots (8 µL) of the DNA extract were digested overnight at 37 °C with 1 U of HhaI restriction endonuclease (New England Biolabs Inc., Beverly, MA) in a total volume of 10 µL. Equivalent aliquots of the DNA extracts were also incubated in the digestion buffer without HhaI endonuclease as control reactions for each sample. After the incubation, 3 µL of digested or nondigested DNA was amplified in a 25-µL polymerase chain reaction (PCR) volume containing 0.1 µL 32[P]{alpha}-labeled deoxyadenosine triphosphate (dATP) (3000 Ci/mmol), 4 µM AR-sense primer (5' TCCAGAATCTGTTCCAGAGCGTGC3'), 4 µM AR-antisense primer (5'GCTGTGAAGGTTGCTGTTCCTCAT3') (9), 4% dimethyl sulfoxide, 2.5 mM MgCl2, 300 µM deoxycytidine triphosphate, 300 µM deoxythymidine triphosphate, 300 µM deoxyguanosine triphosphate, 300 µM dATP, and 0.13 U Taq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT). Each PCR amplification had an initial denaturation step of 95 °C for 8 minutes, followed by 32 cycles at 95 °C for 40 seconds, at 63 °C for 40 seconds, and at 72 °C for 60 seconds and then followed by a single final extension step at 72 °C for 10 minutes. The PCR products were then diluted with 4 µL of loading buffer containing 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanole FF (Sigma Chemical Co., St. Louis, MO). The samples were heated to 95 °C for 5 minutes and then placed on ice. Three microliters of the reaction mixture was loaded onto 6.5% polyacrylamide-denaturing gels without formamide, and the PCR products were separated by electrophoresis at 1600 V for 4–7 hours. The bands were visualized after autoradiography with Kodak X-OMAT AR film (Eastman Kodak Company, Rochester, NY) for 8–16 hours.

Analysis of X-Chromosome Inactivation

The cases were considered to be informative if two AR allelic bands were detected after PCR amplification in normal control samples that had not been treated with HhaI. Only informative cases (i.e., those without a skewed pattern of X-chromosome inactivation after being treated with HhaI in normal control samples) were included in the analysis. In tumor samples, nonrandom X-chromosome inactivation was defined as a complete or a nearly complete absence of an AR allele after HhaI digestion, which indicated a predominance of one allele (1618).

Tumors were considered to be monoclonal if the same AR allelic inactivation pattern was detected in different tumors from the same patient (see Fig. 2Go, Pattern X). Tumors were considered to be multiclonal if alternate predominance of AR alleles after HhaI digestion (different allelic inactivation patterns) was detected in different tumors from the same patient. Tumors with different allelic inactivation patterns were considered to be of independent origin (see Fig. 2Go, Pattern Y).



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Fig. 2. Schematic of determining X-chromosome-inactivation patterns. Nonrandom methylation of X chromosomes may occur in women by the hypermethylation of exon 1 of the androgen receptor (AR) gene on the X chromosome. These genetic alterations (both the hypermethylation and the X-chromosome inactivation) will be maintained in subsequent cell doublings. Clonal analysis was performed by a polymerase chain reaction-based endonuclease restriction enzyme-dependent method (9,16), which took advantage of an X-chromosome-linked polymorphic marker, a CAG–nucleotide repeat of the AR gene (HUMARA), and several methylation-sensitive HhaI (GCGC) endonuclease sites, to detect the nonrandom inactivation of X chromosomes. Tumors with identical allelic loss patterns (Pattern X) are of monoclonal origin. Tumors with different allelic inactivation patterns (Pattern Y) are of independent origin.

 

    RESULTS
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
To determine the origin of peritoneal and ovarian tumors of low malignant potential, we assessed X-chromosome inactivation in 73 separate tumor samples from 18 patients. All patients had papillary serous ovarian tumors of low malignant potential with extra-ovarian site involvement (Table 1Go). Twelve patients had bilateral ovarian tumors of low malignant potential; these tumors were morphologically similar. Tumor samples were obtained from the ovaries and one or more peritoneal sites from each patient. Six patients (cases 3, 10, 11, 12, 13, and 17) had invasive implants, and two (cases 5 and 6) had noninvasive desmoplastic implants.


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Table 1. Summary of patient data and X-chromosome inactivation pattern*

 
In the tumors from five of 18 patients (cases 6, 7, 11, 14, and 16), the patterns of X-chromosome inactivation for all sites were noninformative. Of the remaining 13 informative cases, six (cases 2, 3, 5, 10, 13, and 18) showed random inactivation of the X chromosome, with two allelic bands present after digestion with HhaI; six (cases 1, 8, 9, 12, 15, and 17) had different nonrandom X-chromosome-inactivation patterns in the ovarian and peritoneal tumors (Fig. 3Go), and one (case 4) had the same pattern of nonrandom X-chromosome inactivation in both the ovarian and peritoneal tumors (Table 1Go). In cases 8 (Fig. 3Go, A), 15, and 17, the tumor samples from the left and right ovary had different patterns of nonrandom X-chromosome inactivation. In case 9, each tumor from the left and right ovary and the right fallopian tube had a different pattern of X-chromosome inactivation. In case 12, the tumor sampled from the peritoneal nodule had a different pattern of nonrandom X-chromosome inactivation than the tumors sampled from the omentum and small bowel mucosa. Of the six cases (cases 3, 10, 11, 12, 13, and 17) with invasive implants, two (cases 12 and 17) had nonrandom X-chromosome inactivation. The tumors that were monoclonal were histologically indistinguishable from those that had a random X-chromosome-inactivation pattern.



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Fig. 3. Representative results of inactivation of X-chromosome analysis. Panel A: Tumors (case 8) from the left and right ovary showed different patterns of X-chromosome inactivation (Pattern Y), consistent with independent origin. Panel B: Case 1 shows that the peritoneal tumor is of independent origin and that the ovarian tumors are of monoclonal origin. Panel C: Case 12 shows that the left ovarian tumor and two of the peritoneal implants (OM and SBS) are of monoclonal origin, and one peritoneal nodule (PN) is of independent origin. DNA was prepared from tumor and normal tissues, digested with (+) or without (-) the HhaI methylation-specific restriction enzyme, and amplified by polymerase chain reaction. Arrows point to the allelic bands. N = normal tissue (control); LO = left ovary; LO1 = left ovary, sample 1; LO2 = left ovary, sample 2; RO = right ovary; P = peritoneum (case 1); OM = omentum; SBS = small bowel serosa; and PN = peritoneal nodule (case 12).

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
The monoclonal origin of advanced epithelial ovarian carcinomas and their concurrent metastatic tumors is well documented (57,19). Jacobs et al. (20) analyzed LOH, p53 mutations, and X-chromosome inactivation status and found that tumors from multiple sites in patients with advanced serous ovarian carcinoma were monoclonal in origin. However, whether the peritoneal tumors in cases of ovarian papillary serous tumor of low malignant potential arise as multiple primary tumors or through the metastatic spread from primary ovarian tumors is unknown. We used exon 1 of the AR gene in chromosome Xq11–12 for the detection of nonrandom X-chromosome inactivation. This particular exon was chosen because of a highly polymorphic marker, the CAG–nucleotide repeat, and the methylation-sensitive restriction endonuclease HhaI sites close to the CAG repeat (9). We detected nonrandom X-chromosome inactivation in seven (54%) of 13 informative cases, indicating that these tumors were derived clonally. We detected different patterns of nonrandom X-chromosome inactivation in the ovarian and peritoneal tumors in six cases, suggesting that these tumors were derived independently. Furthermore, three cases had different patterns of nonrandom X-chromosome inactivation in tumors derived from contralateral ovaries. These data indicate that the multiple ovarian tumors of low malignant potential from different sites arise independently in some patients, supporting the "field effect" hypothesis of ovarian tumorigenesis (21).

There are several striking differences between ovarian tumors of low malignant potential and ovarian carcinomas. For example, in contrast to ovarian carcinomas, ovarian tumors of low malignant potential lack destructive stromal invasion (7,22, 23). LOH or microsatellite instability (24,25) and p53 mutations are uncommon in ovarian tumors of low malignant potential (26). In addition, K-ras mutations are more common in ovarian tumors of low malignant potential than in ovarian carcinomas (26,27). Although these characteristics may, in part, be responsible for the better prognosis and 5-year survival rate for women diagnosed with ovarian tumors of low malignant potential (1,2833), they suggest that ovarian carcinomas and ovarian tumors of low malignant potential might arise through different mechanisms of carcinogenesis (24,25).

Several theories have been put forth to explain the clonality of ovarian cancers. In the monoclonal theory of carcinogenesis, a single malignant cell expands clonally to form a primary malignancy and its metastases. However, the monoclonal model of carcinogenesis cannot easily explain the clinical observations of multifocal, synchronous, or metachronous tumors in the ovaries. Therefore, other models that consider field effect have evolved. During ovarian carcinogenesis, a field effect may promote the independent transformation of epithelial cells at different locations. Indeed, Buller et al. (21) proposed that multiple recurrent tumors arose de novo and were different clonally from primary ovarian tumors. An alternative theory, suggested by Segal and Hart (34), was based on the observation that most patients with advanced serous borderline tumors had ovarian cortical surface involvement. Segal and Hart (34) proposed that neoplastic epithelial cells become detached from the papillary excrescences growing on the external surfaces of the ovary and subsequently implant on the peritoneum, omentum, and serosal surfaces of other visceral organs, resulting in multifocal disease (34). However, earlier, Russell (23) argued that peritoneal tumors are not truly metastatic in nature but arise independently (in situ) in response to the same tumorigenic agents that are responsible for the ovarian tumors.

Approximately 30%–40% of patients with serous ovarian tumors of low malignant potential have bilateral or multifocal lesions at the time of diagnosis (28,29, 31,32,35). Determining the clonal origin of peritoneal and bilateral ovarian tumors of low malignant potential may have important biologic and clinical implications. Multiple tumors that originate from a single tumor clone through metastasis may indicate an aggressive clinical course and thus warrant the designation of a higher tumor stage. On the other hand, tumors arising independently in the peritoneum would not necessarily have the same clinical relevance. In this study, we found that tumor samples from the left and right ovaries showed different patterns of nonrandom X-chromosome inactivation, suggesting that bilateral ovarian tumors of low malignant potential arise independently from different clones. In some cases, tumor samples from the ovaries and peritoneum showed different patterns of nonrandom X-chromosome inactivation. These results suggest that the peritoneal tumors in patients with multifocal ovarian tumors of low malignant potential are of independent origin. Lu et al. (16) hypothesized that peritoneal tumors in patients with ovarian tumors of low malignant potential may be independent early papillary serous tumors of the peritoneum, which have been shown to have a multiclonal origin (8). Because data suggest that some peritoneal tumors in advanced-stage ovarian serous tumors of low malignant potential are derived from different clones than those that give rise to the ovarian tumors, the designation of peritoneal tumors as "implants" may not be appropriate. So-called invasive implants may actually represent primary peritoneal carcinomas. It is of interest to note that, in case 17, the invasive implants in the peritoneal tumors had a different nonrandom pattern of X-chromosome inactivation than that of the ovarian tumors, indicating the polyclonal origin of these tumors. Furthermore, the different pattern of nonrandom X-chromosome inactivation observed in different peritoneal tumors, as in case 12, strongly suggests that these peritoneal tumors arise independently as a result of a field effect.

The incidence of nonrandom X-chromosome inactivation (54%) in the current study is higher than reported previously (36). This difference may be explained by the methods of sample collection and the different types of malignancy studied. For example, we used tissue microdissection for the procurement of the DNA samples, and none of our patients had invasive ovarian cancer.

In summary, we used X-chromosome inactivation to determine the clonality of advanced-stage papillary serous ovarian tumors of low malignant potential. Some patients with bilateral ovarian tumors of low malignant potential had two primary tumors instead of one ovarian tumor with metastases to the other ovary. Our results indicate that the peritoneal tumors associated with ovarian tumors of low malignant potential may arise independently from their own primary tumor clones.


    REFERENCES
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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Manuscript received February 9, 2001; revised May 28, 2001; accepted June 4, 2001.


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