Molecular genetic changes that are associated with the initiating stage of tumor development are important in tumorigenesis. Ovarian serous borderline tumors (SBTs), putative precursors of low-grade serous carcinomas, are among the few human neoplasms with a high frequency of activating mutations in BRAF and KRAS genes. However, it remains unclear as to how these mutations contribute to tumor progression. To address this issue, we compared the mutational status of BRAF and KRAS in both SBTs and the adjacent epithelium from cystadenomas, the presumed precursor of SBTs. We found that three of eight SBTs contained mutant BRAF, and four SBTs contained mutant KRAS. All specimens with mutant BRAF harbored wild-type KRAS and vice versa. Thus, seven (88%) of eight SBTs contained either BRAF or KRAS mutations. The same mutations detected in SBTs were also identified in the cystadenoma epithelium adjacent to the SBTs in six (86%) of seven informative cases. As compared to SBTs, the cystadenoma epithelium, like ovarian surface epithelium, lacks cytological atypia. Our findings provide cogent evidence that mutations of BRAF and KRAS occur in the epithelium of cystadenomas adjacent to SBTs and strongly suggest that they are very early events in tumorigenesis, preceding the development of SBT.

It has been shown that tumors result from an accumulation of genetic alterations that result in uncontrolled cellular proliferation. Identification of the alterations that occur early in tumor development is critical to understanding carcinogenesis and can provide insight into potential markers for early detection (1, 2, 3). Ovarian cancer is one of the most lethal neoplasms in women, and serous carcinoma is the most common type (4), but the molecular events that underlie the development of ovarian serous carcinoma are largely unknown. Recent studies have shown that ovarian serous carcinoma develops along two distinct pathways, and we have proposed a model of ovarian carcinogenesis that reflects this concept (5, 6, 7). In one pathway, invasive low-grade serous carcinoma develops from a noninvasive (or in situ) tumor that has traditionally been termed “serous borderline tumor” (SBT; ref. 8). The progression of SBT to invasive low-grade carcinoma mimics the adenomacarcinoma sequence in colorectal carcinoma (1). Detailed analysis of SBTs shows that SBTs consist of two tumors at different stages of tumor progression, a benign tumor termed “atypical proliferative serous tumor,” and an intraepithelial low-grade (noninvasive micropapillary serous) carcinoma, the immediate precursor of invasive low-grade serous carcinoma (5, 7). SBTs are frequently associated with serous cystadenomas that develop from ovarian surface epithelium through a hyperplastic process (9). Like ovarian surface epithelium, the epithelial cells of a cystadenoma do not show cytological atypia, and their proliferation index is extremely low (9). In the second pathway, high-grade serous carcinoma develops from ovarian surface epithelium or from surface inclusion cysts (10), but precursor lesions have not been well characterized. Accordingly, this process has been described as “de novo(5).

Molecular genetic analysis has shown that SBTs and invasive low-grade serous carcinomas are characterized by mutations of BRAF and KRAS in 61 to 68% of cases (6, 7, 11, 12), but p53 mutations are rare (12, 13, 14). In contrast, high-grade serous carcinomas frequently contain p53 mutations (>50%) but rarely BRAF and KRAS mutations (6, 7, 13, 14, 15, 16, 17). These studies analyzed advanced stage tumors in which putative precursor lesions may have been obliterated by the tumor. In this study, we confined our analysis to small SBTs and associated cystadenomas to delineate the early molecular genetic events in their pathogenesis. Specifically, we compared the mutational status of BRAF and KRAS in SBTs and the adjacent nontransformed epithelium of serous cystadenomas.

A total of eight small SBTs (corresponding to what has been classified as atypical proliferative tumor) and the associated cystadenomas were collected. The acquisition of tumor samples was approved by the Johns Hopkins institutional review board. The SBTs ranged from 8 to 20 mm (average 16 mm) in greatest dimension and associated cystadenomas ranged from 5 to 8 cm (average 6.8 cm). The SBTs occupied 5 to 15% of the total surface area of the cystadenomas. Only a small number of cases were studied because although cystadenomas and SBTs are not uncommon, cystadenomas containing synchronous small SBTs are relatively rare. Microscopically, the SBTs contained a hierarchical branching papillae lined by epithelial cells with mild to moderate cellular atypia (Fig. 1). The epithelium of the SBTs merged abruptly with the cystadenoma epithelium that was composed of a single layer of flat to columnar cells without atypia (Fig. 1). The Palm laser capture microdissection microscope (Zeiss) was used to separately collect the epithelium from the SBTs and adjacent cystadenoma. The PicoPure DNA extraction kit (Arcturus, Mountain View, CA) was used to prepare genomic DNA. PCR was then done, and an ABI 3100 sequencer (ABI, Foster City, CA) was used to do nucleotide sequencing. Exon 1 of KRAS and exon 15 of BRAF were both sequenced as each exon harbors almost all of the mutations in both genes (6, 7, 11, 18). The primers for PCR and sequencing were as follows: for BRAF, 5′-tgcttgctctgataggaaaatga-3′ (forward); 5′-ccacaaaatggatccagacaac-3′ (reverse); and 5′-gaaaatgagatctactgttttccttta-3′ (sequencing); for KRAS, 5′-taaggcctgctgaaaatgactg-3′ (forward); 5′-tggtcctgcaccagtaatatgc-3′ (reverse); and 5′-ctgcaccagtaatatgcatattaaaac-3′ (sequencing). The Lasergene program (DNASTAR, Madison, WI) was used to analyze the sequences.

The results of the mutational status correlated with the SBT or cystadenoma component of the tumors are shown in Table 1. We found that four SBTs (cases 1, 4, 5, and 8) contained activating KRAS mutations at codon 12 (three mutations of GGT to GAT and one mutation of GGT to GTT) and three SBTs (cases 3, 5, and 6) had BRAF mutations at codon 599 (all of T1796A mutation). As in our previous report (6), the presence of KRAS and BRAF mutations was mutually exclusive. Thus, seven (88%) of eight SBTs had either a BRAF or a KRAS mutation. Case 2 contained wild-type KRAS and BRAF. Analysis of the mutational status in the epithelium from the cystadenomas adjacent to the SBTs revealed that both the cystadenoma and SBT components contained identical mutations in six of seven informative cases. Representative sequence analyses are shown in Fig. 2. The frequent mutations of KRAS and BRAF in small SBTs are consistent with previous reports showing mutations in either BRAF or KRAS in 66 to 68% of large SBTs (6, 11). The higher frequency of mutations (88%) in the current report is probably because of the use of purer tumor cell samples obtained by laser capture microdissection or may have resulted from the small sample size in the present analysis.

The findings in this study provide important insights into the molecular pathogenesis of low-grade ovarian serous tumors (Fig. 3). Because we only analyzed a single time point in the sequence of cystadenoma to SBTs, we can only infer that the findings truly describe the events in early tumor progression. However, the coexistence of a cystadenoma with a SBT strongly suggests that the latter arises from the former (Fig. 3). Accordingly, the presence of identical mutations in the cystadenoma epithelium that displayed no evidence of cytological atypia strongly suggests that mutations of BRAF and KRAS occur before the development of a SBT and indicates that cystadenomas are the precursors of SBTs. Our results support the view that mutations of BRAF and KRAS (or NRAS) are early events associated with tumor initiation as occurs in melanoma (19) and colorectal carcinoma (20).

We have recently studied 30 consecutive pure cystadenomas without SBTs and have shown an absence of BRAF and KRAS mutations in all of them (9). The frequency of mutations in BRAF and KRAS in cystadenomas associated with SBTs was significantly higher than those without SBTs (P < 0.001, Fisher’s exact test; Table 2). This finding together with the fact that SBTs are relatively uncommon as compared to cystadenomas (21, 22, 23, 24, 25) suggests that only a small proportion of serous cystadenomas are neoplastic with the potential to progress to SBTs. Finally, our findings suggest a “gatekeeper” role of BRAF and KRAS genes in the development of low-grade serous carcinomas (26). This is supported by the observation that activating mutations in these genes are oncogenic in experimental cell culture systems (19, 27, 28) probably through a constitutive activation of mitogen-activated protein kinase (29, 30). Future experiments will determine whether mutations of BRAF and KRAS are sufficient to initiate the development of SBTs or additional genetic “hits” are required in tumorigenesis. Because mutations of BRAF and KRAS in serous cystadenomas are associated with the development of SBTs, detection of BRAF and KRAS mutations could facilitate the differentiation of cystadenomas with a high risk of progression from the vast majority of cystadenomas that lack BRAF or KRAS mutations and have a very low risk of progression. Development of molecular assays (31, 32) that can detect such mutations (in cyst fluid, for example) could play an important role in the management of patients with ovarian cystadenomas, particularly young women who would prefer fertility-sparing treatment.

Fig. 1.

A, small serous borderline tumor and associated serous cystadenoma (case E). The tumor measures 0.8 cm in greatest dimension and is composed of hierarchical branching papillae lined by cells with mild to moderate atypia (left inset). The adjacent cystadenoma epithelium is composed of a single layer of epithelium without cytological atypia (right inset). B, laser capture microdissection with minimal contamination from the underlying stromal cells were used to isolate epithelial cells lining the cystadenoma (between arrows).

Fig. 1.

A, small serous borderline tumor and associated serous cystadenoma (case E). The tumor measures 0.8 cm in greatest dimension and is composed of hierarchical branching papillae lined by cells with mild to moderate atypia (left inset). The adjacent cystadenoma epithelium is composed of a single layer of epithelium without cytological atypia (right inset). B, laser capture microdissection with minimal contamination from the underlying stromal cells were used to isolate epithelial cells lining the cystadenoma (between arrows).

Close modal
Fig. 2.

Chromatograms of nucleotide sequences of BRAF and KRAS in two representative cases. Left panel (case 1) shows a point mutation in the KRAS gene in both SBT and the adjacent cystadenoma (Cyst) of the same specimen. Right panel (case 6) shows a point mutation in the BRAF gene in both the serous borderline tumor and the corresponding cystadenoma.

Fig. 2.

Chromatograms of nucleotide sequences of BRAF and KRAS in two representative cases. Left panel (case 1) shows a point mutation in the KRAS gene in both SBT and the adjacent cystadenoma (Cyst) of the same specimen. Right panel (case 6) shows a point mutation in the BRAF gene in both the serous borderline tumor and the corresponding cystadenoma.

Close modal
Fig. 3.

Schematic representation of tumor progression in low-grade serous carcinoma. Mutations of BRAF and KRAS occur in a small proportion of cystadenomas that may contribute to the development of a SBT. Some serous borderline tumors progress further to intraepithelial and then to invasive low-grade serous carcinoma. APST, atypical proliferative serous tumor.

Fig. 3.

Schematic representation of tumor progression in low-grade serous carcinoma. Mutations of BRAF and KRAS occur in a small proportion of cystadenomas that may contribute to the development of a SBT. Some serous borderline tumors progress further to intraepithelial and then to invasive low-grade serous carcinoma. APST, atypical proliferative serous tumor.

Close modal

Grant support: Supported by a research grant OC010017 from the United States Department of Defense.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Ie-Ming Shih, The Johns Hopkins Medical Institutions, 1503 E. Jefferson Street, Room B-315, Baltimore, MD 21231. Phone: 410-502-7774; Fax: 410-502-7943; E-mail: [email protected]

Table 1

Mutational status of KRAS and BRAF genes in eight small serous borderline tumors and associated cystadenomas

GeneCase 1Case 2Case 3Case 4Case 5Case 6Case 7Case 8
KRAS         
 SBT G35A* WT WT G35T G35A WT WT G35A 
 G12D   G12V G12D   G12D 
 Cyst G35A WT WT WT G35A WT WT G35A 
 G12D    G12D   G12D 
BRAF         
 SBT WT WT T1796A WT WT T1796A T1796A WT 
   V599E   V599E V599E  
 Cyst WT WT T1796A WT WT T1796A T1796A WT 
   V599E   V599E V599E  
GeneCase 1Case 2Case 3Case 4Case 5Case 6Case 7Case 8
KRAS         
 SBT G35A* WT WT G35T G35A WT WT G35A 
 G12D   G12V G12D   G12D 
 Cyst G35A WT WT WT G35A WT WT G35A 
 G12D    G12D   G12D 
BRAF         
 SBT WT WT T1796A WT WT T1796A T1796A WT 
   V599E   V599E V599E  
 Cyst WT WT T1796A WT WT T1796A T1796A WT 
   V599E   V599E V599E  

Abbreviation: WT, wild-type.

*

Alteration in nucleotide sequence.

Alteration in amino acid sequence.

Table 2

Frequency of mutations in BRAF and KRAS in cystadenomas with associated serous borderline tumor and pure cystadenomas

Cystadenoma associated with SBTPure cystadenoma
Mutations in BRAF or KRAS 
Wild-type in BRAF and KRAS 30 
Total cases 30 
Cystadenoma associated with SBTPure cystadenoma
Mutations in BRAF or KRAS 
Wild-type in BRAF and KRAS 30 
Total cases 30 

We gratefully acknowledge the technical support of the Oncology Imaging Core at the Johns Hopkins Medical Institutions for the photomicrographs and the laser capture microdissection.

1
Kinzler KW, Vogelstein B .
The genetic basis of human cancer
1998
McGraw-Hill Toronto
2
Kinzler KW, Vogelstein B Landscaping the cancer terrain.
Science (Wash D C)
1998
;
280
:
1036
-7.
3
Kern SE Advances from genetic clues in pancreatic cancer.
Curr Opin Oncol
1998
;
10
:
74
-80.
4
Seidman JD, Horkayne-Szakaly I, Haiba M, et al The histologic type and stage distribution of ovarian carcinomas of surface epithelial origin.
Int J Gynecol Pathol
2004
;
23
:
41
-4.
5
Shih I-M, Kurman RJ Ovarian tumorigenesis- a proposed model based on morphological and molecular genetic analysis.
Am J Pathol
2004
;
164
:
1511
-8.
6
Singer G, Oldt R, 3rd, Cohen Y, et al Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma.
J Natl Cancer Inst (Bethesda)
2003
;
95
:
484
-6.
7
Singer G, Kurman RJ, Chang H-W, et al Diverse tumorigenic pathways in ovarian serous carcinoma.
Am J Pathol
2002
;
160
:
1223
-8.
8
Burks RT, Sherman ME, Kurman RJ Micropapillary serous carcinoma of the ovary. A distinctive low-grade carcinoma related to serous borderline tumors.
Am J Surg Pathol
1996
;
20
:
1319
-30.
9
Cheng EJ, Kurman RJ, Wang M, et al Molecular genetic analysis of ovarian serous cystadenomas.
Lab Investig
2004
;
84
:
778
-84.
10
Yang DH, Smith ER, Cohen C, et al Molecular events associated with dysplastic morphologic transformation and initiation of ovarian tumorigenicity.
Cancer (Phila)
2002
;
94
:
2380
-92.
11
Sieben NLG, Macropoulos P, Roemen G, et al In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumors.
J Pathol
2004
;
202
:
336
-40.
12
Cuatrecasas M, Erill N, Musulen E, et al K-ras mutations in nonmucinous ovarian epithelial tumors: a molecular analysis and clinicopathologic study of 144 patients.
Cancer (Phila)
1998
;
82
:
1088
-95.
13
Zheng J, Benedict WF, Xu HJ, et al Genetic disparity between morphologically benign cysts contiguous to ovarian carcinomas and solitary cystadenomas [see comments].
J Natl Cancer Inst (Bethesda)
1995
;
87
:
1146
-53.
14
Teneriello MG, Ebina M, Linnoila RI, et al p53 and Ki-ras gene mutations in epithelial ovarian neoplasms.
Cancer Res
1993
;
53
:
3103
-8.
15
Leitao MM, Soslow RA, Baergen RN, et al Mutation and expression of the TP53 gene in early stage epithelial ovarian carcinoma.
Gynecol Oncol
2004
;
93
:
301
-6.
16
Kappes S, Milde-Langosch K, Kressin P, et al p53 mutations in ovarian tumors, detected by temperature-gradient gel electrophoresis, direct sequencing and immunohistochemistry.
Int J Cancer
1995
;
64
:
52
-9.
17
Singer G, Stohr R, Dehari R, et al. Patterns of p53 mutations separate ovarian serous borderline tumors, low- and high-grade carcinomas and provide support for a new model of ovarian carcinogenesis. Am J Surg Pathol. In press 2004.
18
Davies H, Bignell GR, Cox C, et al Mutations of the BRAF gene in human cancer.
Nature (Lond)
2002
;
417
:
949
-54.
19
Pollock PM, Harper UL, Hansen KS, et al High frequency of BRAF mutations in nevi.
Nat Genet
2003
;
33
:
19
-20.
20
Chan TL, Zhao W, Leung SY, et al BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
Cancer Res
2003
;
63
:
4878
-81.
21
Mink PJ, Sherman ME, Devesa SS Incidence patterns of invasive and borderline ovarian tumors among white women and black women in the United States. Results from the SEER Program, 1978–1998.
Cancer (Phila)
2002
;
95
:
2380
-9.
22
Conway C, Zalud I, Dilena M, et al Simple cyst in the postmenopausal patient: detection and management.
J Ultrasound Med
1998
;
17
:
369
-72; quiz 3734.
23
Oyelese Y, Kueck AS, Barter JF, et al Asymptomatic postmenopausal simple ovarian cyst.
Obstet Gynecol Surv
2002
;
57
:
803
-9.
24
Christensen JT, Boldsen JL, Westergaard JG Functional ovarian cysts in premenopausal and gynecologically healthy women.
Contraception
2002
;
66
:
153
-7.
25
Seidman JD, Russell P, Kurman RJ Surface epithelial tumors of the ovary Kurman RJ eds. .
Blaustein’s pathology of the female genital tract
edition 5. 
2002
791
-904. Springer Verlag New York
26
Kinzler KW, Vogelstein B Cancer-susceptibility genes. Gatekeepers and caretakers [news; comment].
Nature (Lond)
1997
;
386
:
761, 763
27
Peyssonnaux C, Eychene A The Raf/MEK/ERK pathway: new concepts of activation.
Biol Cell
2001
;
93
:
53
-62.
28
Malumbres M, Barbacid M RAS oncogenes: the first 30 years.
Nat Rev Cancer
2003
;
3
:
459
-65.
29
Allen LF, Sebolt-Leopold J, Meyer MB CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK).
Semin Oncol
2003
;
30(5 Suppl 16)
:
105
-16.
30
Satyamoorthy K, Li G, Gerrero MR, et al Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation.
Cancer Res
2003
;
63
:
756
-9.
31
Vogelstein B, Kinzler KW Digital PCR.
Proc Natl Acad Sci USA
1999
;
96
:
9236
-41.
32
Dressman D, Yan H, Traverso G, et al Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations.
Proc Natl Acad Sci USA
2003
;
100
:
8817
-22.