[CANCER RESEARCH 54, 33-35, January 1,1994]

Advances in Brief

Mutation of K-ras Protooncogene Is Associated with Histological Subtypes in

Human Mucinous Ovarian Tumors 1

Y o s h i h i r o I c h i k a w a , M a s a t o N i s h i d a , H i r o m i c h i S u z u k i , S a d a o Y o s h i d a , H a j i m e T s u n o d a , T a k e s h i K u b o ,

K a z u h i k o U c h i d a , 2 a n d M a s a n a o M i w a

Department of Biochemistry, Institute of Basic Medical Sciences [Y L, H. S., S. Y, K. U., M. M.]; Department of Obstetrics and Gynecology, Institute of Clinical Medicine [M. N., H. T, Z K.], University of Tsukuba, Tsukuba 305, Japan

Abstract Materials and Methods

A series of 57 mucinous and 47 serous ovarian tumors (adenomas, tumors of borderline malignancy, and carcinomas) were examined by polymerase chain reaction-single strand conformation polymorphism analysis and direct sequencing for mutations in codons 12, 13, and 61 of K-ras gene. Higher incidence of K-ras mutations was observed in muci- nous tumors compared to serous ones. Mutations were detected in 4 of 30 mucinous adenomas (13%), in 4 of 12 mucinous tumors of borderline malignancy (33%), and in 7 of 15 mucinous carcinomas (46%). Only 1 of 17 serous carcinomas (6%) had a mutation of K-ras in serous ovarian tumors. All mutations identified were in codon 12. Detailed analysis re- vealed that more K-ras mutations in mucinous adenomas were observed in intestinal type (identified in 4 of 13) than in endocervical type (identified in 0 of 17). Thus, K.ras gene codon 12 mutations in mucinous ovarian adenomas appear to be associated with the occurrence of intestinal type adenomas.

Introduct ion

A series of genetic alterations detected in premalignant states of

carcinomas and in malignant tumors provide important information

about the mechanism of carcinogenesis (1). In colorectal neoplasia,

alterations in the cellular protooncogenes leading to disordered cell

growth and differentiation have been identified (1, 2). The common

epithelial ovarian neoplasms are classified on histogenetic principles

mainly in serous, mucinous, endometrioid, and clear cell tumors. The

origin of serous ovarian tumors is the surface epithelium (3). Muci-

nous ovarian tumors are considered to originate from the ovarian

surface epithelium (a miillerian origin) or from a teratoma, but this

histogenesis of the mucinous ovarian tumors has not been clearly

elucidated (3). Recently, mutations of K-ras gene have been detected

more frequently in mucinous ovarian carcinomas and tumors of bor-

derline malignancy than in serous ones (4). But the association of

K-ras mutations with histological subtypes of the ovarian mucinous

tumors has not been determined. Additionally, the genetic alterations

involved in ovarian adenomas are still poorly understood. In the

present study, we investigated the possible involvement of K-ras

mutations in the development of mucinous ovarian adenomas, tumors

of borderline malignancy and carcinomas, and serous tumors, and

discuss the association of K-ras mutations with histological subtypes

in mucinous ovarian tumors.

Specimens. A total of 52 cases of ovarian adenomas (30 mucinous and 22 serous tumors), 20 ovarian tumors of borderline malignancy (12 mucinous and 8 serous tumors), and 32 ovarian adenocarcinomas (15 mucinous and 17 serous tumors) were analyzed in this study. All the specimens were obtained from the University of Tsukuba Hospital (Ibaraki, Japan). Before DNA extraction from these specimens, histological diagnosis was confirmed by microscopic exami- nation of the hematoxylin-eosin-stained sections according to the WHO criteria (5). All cases of mucinous adenomas and tumors of borderline malignancy were classified into two types according to the lining of the epithelium. The intestinal-type epithelium contains goblet cells and resembles the epithelium of the intestine or stomach. The endocervical-type epithelium resembles the epi- thelium of the endocervix of the uterus. The samples containing both types of epithelium in the same section were excluded.

DNA Extraction. The 10-/xm thick sections from formalin-fixed paraffin- embedded tissue blocks were deparaffinized by washing with xylene followed by absolute ethanol. The deparaffinized tissues were incubated overnight at 55~ in 30 ~I of digestion buffer (1 mg/ml proteinase K, 0.1 M Tris-HC1, pH 8.8). Then DNA was extracted by twice adding an equal volume of phenol/ chloroform/isoamylalcohol mixture and was recovered by ethanol precipita- tion.

PCR-SSCP 3 Analysis. Mutation of K-ras gene was examined by using PCR-SSCP analysis (6) with slight modifications. Two sequences containing codons 12 and 13, and codon 61 of K-ras gene were amplified by using oligonucleotide primers as follows: 5'-GGCCTGCTGAAAATGACTGA-3' (forword) and 5'-ATrCGTCCACAAAATGATI'C-3' (reverse) for codons 12 and 13, 5'-TTCCTACAGGAAGCAAGTAG-3' (forword) and 5'-CA- CAAAGAAAGCCCTCCCCA-3' (reverse) for codon 61. After 40 cycles of PCR (93 ~ 57 ~ and 72~ for 1, 1, and 1.5 min, respectively) from 1 /xg of sample DNA in a 5-/xl volume containing 10 mM Tris-HC1 (pH 8.3), 50 mM KCI, 1.75 mM MgC12, 0.02% gelatin, 0.05% Tween 20, 25 p,M concentrations of each deoxyribonucleotide triphosphate, 0.125 unit of Taq DNA polymerase (Wako, Osaka, Japan), 0.02 pmol of each primer, and 2 ~Ci of [a-32p]dCFP (3000 Ci/mmol, Amersham, United Kingdom); the PCR products were ana- lyzed by a 6% polyacrylamide gel with or without 5% glycerol at 30 W at 25~ Autoradiography of the gel was analyzed with a Fujix BAS2000 Imaging Analyzer (Fuji Photo film, Tokyo, Japan).

Sequencing of PCR Products. The direct sequencing of PCR products was performed by using a double-stranded DNA Cycle Sequencing System (GIBCO BRL, Gaithersburg, MD). The excess primers were removed by centrifugation, using Suprec-02 (Takara Biomedicals, Kyoto, Japan). The primers used for PCR-SSCP analysis were end-labeled with [T-32p]ATP (5000 Ci/mmol, Amersham) and used for sequencing. The K-ras mutations were confirmed by sequencing both strands in an 8% polyacrylamide gel containing 7 M urea.

Results

Received 10/12/93; accepted 11/12/93. 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.

1 This study was supported in part by Grants-in-Aid for Scientific Research, Cancer Research, and Tsukuba Project Research from the Ministry of Education, Science and Culture, Japan.

2 To whom requests for reprints should be addressed, at Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305, Japan.

33

Ovarian mucinous and serous adenomas, tumors of borderline ma- lignancy, and carcinomas were examined for point mutations in codon 12, 13, or 61 of K-ras gene by PCR-SSCP analysis. The mobility patterns in the SSCP gel are shown in Fig. 1. Mobility shifts indicating

3 The abbreviations used are: PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism.

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K-RAS MUTATION AND HISTOLOGICAL TYPES OF OVARIAN TUMORS

1 N 2 N31 N 47 45 N 88

Fig. 1. PCR-SSCP analysis of K-ras gene codon 12 and 13 mutations in human ovarian tumors. Top ordinate, case numbers, N, normal human placenta. Arrowheads, bands showing mobility shifts.

the presence of point mutations in codon 12 or 13 were found in 4 of 30 mucinous adenomas (13%), in 4 of 12 mucinous tumors of bor- derline malignancy (33%), and in 7 of 15 mucinous carcinomas (46%). In serous tumors, however, only 1 of 17 carcinomas (6%) showed an altered migration. All other serous adenomas, tumors of borderline malignancy, and carcinomas represented the wild-type pat- tern. In all cases examined, no altered migration indicating the pres- ence of a point mutation in codon 61 was detected.

Every case showing a mobility shift in SSCP was examined by direct sequencing to confirm a mutation. There was a complete con- cordance between the mobility patterns in SSCP and the results of direct sequencing. Typical DNA sequencing autoradiographs are shown in Fig. 2. The results are summarized in Table 1. All 16 cases confirmed by sequencing involved a second base change of codon 12. Eight cases involved a base change from GGT to GTT (Gly to Val), 7 from GGT to GAT (Gly to Asp), and 1 from GGT to GCT (Gly to Ala). A high frequency of K-ras mutation in mucinous tumors includ- ing adenomas compared to serous tumors was observed. K-ras mu- tations were detected in stages Ia and Ib of mucinous tumors of borderline malignancy and carcinomas (Table 1). Two cases, cases 43 and 44, which showed stage IIIc and IIa, respectively, involved a base change from GGT to GRIT. Only one case of serous carcinoma with a K-ras mutation was in stage Ia (case 88).

Analysis of correlation between histological subtypes of ovarian tumors and K-ras mutations is summarized in Table 2. Mutation in codon 12 of K-ras gene was observed in 15 of 57 (26%) mucinous tumors, but in 1 of 47 (2%) serous ones. The higher frequency of K-ras mutation in mucinous tumors than in serous ones is statistically significant (P < 0.0005; Fisher exact test). In mucinous tumors, the statistical difference of the incidence of K-ras mutation was found between adenomas (4 of 30) and carcinomas (7 of 15) (P < 0.03).

In mucinous adenomas, mutation in codon 12 of K-ras gene was associated with the lining of the epithelium. The incidence of K-ras mutation in mucinous adenoma was significantly higher in intestinal type (4 of 13) than in endocervical type (0 of 17) (P < 0.03). However, in mucinous tumors of borderline malignancy, a higher mutation fre- quency was observed in endocervical type (2 of 5) compared to intestinal ones (2 of 7), but this was not statistically significant (P = 0.15) (Table 2).

Discussion

The present study showed a higher incidence of mutations in codon 12 of K-ras gene was associated with intestinal type-mucinous ad- enoma. This correlation was not observed in mucinous tumors of borderline malignancy. The origin of intestinal-type mucinous tumors is considered as a teratoma. The finding of goblet cells, argentaffin cells, Paneth cells, and cystic teratomas, which are present in about 5% of mucinous tumors, suggests that some mucinous neoplasms may represent monomorphic endodermal differentiation of a teratoma (7). In this study, all the mucinous adenomas with a mutated K-ras gene

were classified as intestinal type. Therefore, mutation of K-ras gene may be involved in tumorigenesis of intestinal-type mucinous adeno- mas originating from teratomas. K-ras mutation was not found in endocervical type of adenomas, but was found in both intestinal type and endocervical type of tumors of borderline malignancy. Mutations of K-ras might be involved in the development of endocervical-type mucinous tumors of borderline malignancy. The genetic events in- cluding K-ras mutation are possibly associated with the determination of histological subtypes during ovarian tumor development.

Our study is the first comprehensive analysis of K-ras mutation in ovarian mucinous adenomas. The higher incidence of K-ras mutations in ovarian mucinous tumors of borderline malignancy and carcinomas than in serous ones is consistent with previous reports (4, 8). The rate of positive cases (2%) in serous carcinomas was lower than that reported previously (20-30%) in serous carcinomas (4, 8). Serous carcinomas with mutated K-ras gene reported previously have all been in stage III or IV (4, 8), but in our study, the mutation-positive case was in stage Ia, and no K-ras gene mutations were found in 10 cases of stage III and in 2 cases of stage IV serous carcinomas. The reason for this discrepancy remains unknown, but racial differences might be one possibility.

Recent studies revealed that the ras family of protooncogenes has been involved not only in cellular carcinogenesis but also in control of cellular growth and differentiation (9). In certain human neoplasms, K-ras gene mutations have been suggested to play a role in the cellular pathway of mucinous differentiation (10, 11). In our study, K-ras mutations were preferentially identified in mucinous carcinomas, tu- mors of borderline malignancy, and adenomas compared to serous ones. These results suggest that the K-ras mutations are an early genetic event in a subset of ovarian mucinous tumors.

In colorectal tumors, K-ras mutations are detected at a similar incidence in both adenomas and carcinomas (2, 12). In lung adeno- carcinomas, alterations in codon 12 of K-ras gene occur early and irreversibly during their development (13). In gynecological tumors, the incidence of K-ras mutations in endometrial hyperplasias is simi- lar to the incidence in carcinomas (14). These findings suggest that

T G C A T G C A

J G G

O

N 1

T G C A

/ G G -->" T

~ T

T G C A

G G - ~ A T

45 88 Fig. 2. Mutations in K-ras gene codon 12 in human ovarian tumors. N, normal human

placenta. Numbers correspond to the case numbers in Table 1. From top to bottom of the gel, the sequences correspond to the 5' to 3' direction in the transcribed strand. Mutations were confirmed by sequencing of both strands.

34

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K-RAS MUTATION AND HISTOLOGICAL TYPES OF OVARIAN TUMORS

Table 1 K-ras gene codon 12 mutations in mucinous and serous ovarian tumors

Case no. Age Histology (epithelial lining) Stage a

K-ras gene codon 12 mutation

Base change Amino acid change

1 69 2 31 3 52 4 33

5 - 1 3 27 c 14- 30 41 c

31 59 32 34 33 26 34 32

3 5 - 3 9 47 c 4 0 - 4 2 40 c

43 22 44 66 45 53 46 32 47 36 48 30 49 49

5 0 - 5 7 57 c

5 8 - 7 9 50 c 8 0 - 8 7 39

88 56 89-104 47 c

Mucinous adenoma (intestinal) Mucinous adenoma (intestinal) Mucinous adenoma (intestinal) Mucinous adenoma (intestinal) Mucinous adenoma (intestinal) Mucinous adenoma (endocervical)

Mucinous borderline tumor (intestinal) Mucinous borderline tumor (intestinal) Mucinous borderline tumor (endocervical) Mucinous borderline tumor (endocervical) Mucinous borderline tumor (intestinal) Mucinous borderline tumor (endocervical)

Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma

Serous adenoma Serous borderline tumor Serous carcinoma Serous carcinoma

NA b NA NA NA NA NA

Ib Ia Ia Ia Ia-Ic Ia-Ic

IIIc IIa Ia Ia Ia Ia Ia Ia-IIIc

GGT to GCI" Gly to Ala GGT to GrIT Gly to Val GGT to GAT Gly to Asp GGT to GAT Gly to Asp WT

GGT to GTT Gly to Val GGT to GTI" Gly to Val GGT to GTF Gly to Val GGT to GTF Gly to Val WT WT

GGT to GrIT Gly to Val GGT to GTT Gly to Val GGT to GTF Gly to Val GGT to GAT Gly to Asp GGT to GAT Gly to Asp GGT to GAT Gly to Asp GGT to GAT Gly to Asp WT

NA WT Ia-Illb WT Ia GGT Ia-IV WT

to GAT Gly to Asp

a International Federation of Gynecology and Obstetrics (FIGO) stage. b NA, not applicable; WT, wild type. c Median age.

Table 2 Correlation between histological parameters and K-ras gene codon 12 mutations in mucinous and serous ovarian tumors

Histological parameters K-ras gene codon 12 mutation

Tumor histology Mucinous tumor 15/57 (26) ' ' b

Adenoma 4/30 (13) Borderline tumor 4/12 (33) Carcinoma 7/15 (46) c

Serous tumor 1/47 (2) Adenoma 0/22 (0) Borderline tumor 0/8 (0) Carcinoma 1/17 (6)

Epithelial lining of mucinous tumor Mucinous adenoma

Intestinal type 4/13 (31) a Endocervical type 0/17 (0)

Mucinous borderline tumor Intestinal type 2/7 (29) e Endocervical type 2/5 (40)

a Numbers in parentheses, percentage. b Significantly higher than in serous tumor (P < 0.0005). c Significantly higher than in mucinous adenoma (P < 0.03). d Significantly higher than in endocervieal type of mucinous adenoma (P < 0.03). e Not significantly lower than in endocervical type of mucinous borderline tumor

(P = 0.15).

the K-ras gene may play an important role in an early step of carcinogenesis. On the other hand, in ovarian tumors, a lower inci- dence of K-ras mutation in serous adenomas compared to mucinous ones was observed in our study, and also in a previous report (15). These results indicate possible pathways, other than K-ras gene acti- vation, in the development of ovarian neoplasms. More detailed studies are required to define the ovarian adenoma-carcinoma se- quence, and to clarify other genetic alterations in mucinous ovarian carcinogenesis.

Acknowledgments

We thank Dr. M. H o d for preparat ion o f Lu65 cells for SSCP analyses. We

also thank S. Hanai for advice in sequencing and T. Mogi for preparat ion o f

sections f rom tissue blocks.

35

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1994;54:33-35. Cancer Res Yoshihiro Ichikawa, Masato Nishida, Hiromichi Suzuki, et al. Histological Subtypes in Human Mucinous Ovarian Tumors

Protooncogene Is Associated withrasMutation of K-

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