Are DNA mismatch repair deficiencies responsible for accumulation of genetic alterations in epithelial ovarian cancers?

Cancer Genetics and Cytogenetics 124 (2001) 152–158

Are DNA mismatch repair deficiencies responsible for accumulation of genetic alterations in epithelial ovarian cancers? Mitsuaki Suzukia,*, Michitaka Ohwadaa, Yasushi Sagaa, Susumu Saitob, Ikuo Satoa a

Department of Obstetrics and Gynecology, Jichi Medical School, 3311 Yakushiji, Minamikawachi, Kawachi, Tochigi, Japan b SRL, Inc., 5-6-50 Shinmachi, Hino, Tokyo, Japan Received 21 April 2000; received in revised form 25 July 2000; accepted 1 August 2000

Abstract

To investigate the association of DNA mismatch repair deficiencies in the development and/or progression of epithelial ovarian cancers, the relationship between replication errors (RERs) and genetic alterations in three genes (p53, c-erbB2, K-ras) and loss of heterozygosity (LOH) on 6q27 was investigated in 70 patients with epithelial ovarian cancers. The presence of RERs was examined by PCR using five microsatellite markers. Mutations of p53 were analyzed by PCR-SSCP and sequencing. Amplification of c-erbB2 was analyzed by Southern blot hybridization. Point mutations of K-ras codon 12 were identified by PCR-PHFA, while 6q27LOH was examined by Southern blot hybridization. As a result, 18 of 70 patients with epithelial ovarian cancers (26%) were RER-positive and 52 patients (74%) were RER-negative. Tumors with two or three genetic alterations accounted for 28% and 33% of RER-positive tumors, respectively, and these were significantly more frequent than in the RER-negative tumors (17% and 6%, respectively)(P ⫽ .002). These results are consistent with mismatch repair deficiencies being involved in the development and/or progression of a proportion of epithelial ovarian cancers through accumulation of genetic alterations. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction Recent studies suggest that DNA mismatch repair deficiencies are partially responsible for the development and/ or progression of epithelial ovarian cancers. It has recently been reported that replication errors (RERs) as a phenotype of mismatch repair deficiencies are observed in 12–37% of epithelial ovarian cancers [1–5]. However, it is almost unknown how mismatch repair deficiencies are involved in the development and/or progression of epithelial ovarian cancers. The most frequent genetic alteration in epithelial ovarian cancers is seen in the p53 gene at present [4,6,7]. There is no other genetic alteration occurring frequently, but alterations in c-erbB2 [8,9] and K-ras [10–13] were occasionally observed. In epithelial ovarian cancer, a relatively high incidence of loss of heterozygosity (LOH) is observed on chromosomes 3p, 6p, 6q, 11p, 17p and 17q, and the presence of putative tumor suppressor genes for ovarian cancers at these

* Corresponding author. Tel.: ⫹81-285-58-7376; fax: ⫹81-285-448505.

positions was suggested [14–17]. We and some other researchers have a particular interest in LOH on chromosome 6q27 [18–22]. In this study, we investigated the relationship between mismatch repair deficiencies and genetic alterations including 6q27LOH. In addition, we investigated whether mismatch repair deficiencies are involved in accumulation of these genetic alterations. 2. Patients and methods 2.1. Patients The study population included 70 patients with epithelial ovarian cancers who underwent surgery at the Department of Obstetrics and Gynecology, Jichi Medical School between 1988 and 1997. The histologic type of ovarian cancer was classified as serous adenocarcinoma (SAC) in 37 cases, mucinous adenocarcinoma (MAC) in 18 cases, clear cell adenocarcinoma (CCC) in 9 cases and endometrioid adenocarcinoma (EMC) in 6 cases. Tumors were staged using the International Federation of Gynecology and Obstetrics (FIGO) classification system, producing the following results: stage I disease in 17 patients; stage II in 8; stage III in 28; and stage IV in 17.

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2.2. Tissue preparation and DNA extraction DNA from tumors and corresponding blood samples was prepared as described previously [20]. Formalin-fixed and paraffin-embedded tissues from the ovarian tumors were prepared for RER investigation. Sections were mounted on glass slides and stained with hematoxylin. Three or more samples from both carcinoma and normal control tissues were collected in pairs under a stereoscope from each patient. DNA was extracted according to the method described by Wright et al. [23]. 2.3. DNA · RERs Five sets of microsatellite primers (TP53 [24], D2S123 [25], D3S1029 [26], D3S1611 [25] and D18S34 [27]) were utilized for PCR amplification, using the method of Senba et al. [28]. Amplification was for 30 cycles of denaturation (1 min at 94⬚C), annealing (1 min at 55⬚C), and extension (2 min at 72⬚C). PCR products were diluted with formamide dye solution (Amersham Life Science, Inc., Cleveland, OH, USA), heated for 3 min at 90⬚C, electrophoresed on 8% polyacrylamide gels containing 7M urea, and transferred to nylon membranes. The membranes were incubated with Fab fragments of antidigoxigenin antibody (Boehringer Mannheim Biochemica, Mannheim, Germany) conjugated to alkaline phosphatase. Following the addition of CSPD solution (Boehringer Mannheim Biochemica), the membranes were exposed to X-ray film for about 2 h. Samples in which PCR products were detected at four or more of the five microsatellite loci were considered informative. An RER was denoted by the occurrence of band shifts or extra bands in tumor samples compared to matched normal tissue; any tumor containing a band shift or extra band at one or more microsatellite loci was considered positive for RER. In tumors considered RER-positive, PCR was repeated and the reproducibility of band shifts or extra bands was re-analyzed. When two or more of the five microsatellite primers showed instability, the tumor was judged high-frequency RER, and when only one of the five primers showed instability, the tumor was judged low-frequency RER. 2.4. p53 gene analysis DNA samples were screened for the presence of p53 gene mutations in the most frequently affected exons (5 to 8) of the gene using single-strand conformation polymorphism (SSCP) analysis. Primers were used for PCR amplification of DNA essentially as described by Hsu et al. [29]. SSCP analysis for the detection of p53 gene mutations has been described previously [30]. Samples that showed mobility differing from that of healthy controls on PCR-SSCP gels were additionally analyzed by sequencing. To sequence the p53 gene, a DNA fragment containing a single exon was obtained by PCR amplification using the SSCP primers. The fragment was then cloned using the TA Cloning System, Version C (Invitrogen Corp., San Diego,

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CA, USA). Plasmid isolation and purification were done using pLASmix (TALENT, Trieste, Italy). We used the following-sequence primers described elsewhere [29]. The sequence of both DNA strands from mixed recombinant colonies was determined by the dideoxy ribonucleotide chain termination method using a T7-Sequencing kit (Pharmacia Biotech, Uppsala, Sweden). 2.5. Detection of c-erbB2 amplification DNAs were digested with the HindIII restriction endonuclease and analyzed by Southern blotting according to the method described previously [31]. cDNA probe for the c-erbB2 was used for hybridization. Signal intensities on autoradiographs were measured using an Imaging Densitometer Model GS-670 (Bio-Rad, Hercules, CA, USA). The relative c-erbB2 copy numbers were obtained by dividing c-erbB2 signal intensities in tumor DNAs by the intensity of the signal in human placenta. Samples showing more than two-fold increase in copy numbers were considered positive. 2.6. Detection of K-ras codon 12 mutations Activated K-ras genes were identified by polymerase chain reaction dependent preferential homoduplex formation assay (PCR-PHFA) [32]. The first exon of the K-ras gene was amplified. Double labeled standard DNAs (wild type and six kinds of mutant probes) and unlabeled PCR products were hybridized respectively at 89⬚C, then cooled slowly to 80⬚C. Hybridization mixture, 25␮l, was transferred to a streptavidincoated microtiter well with 100␮l of anti-DNP antibody conjugated alkaline phosphatase. After incubation for 30 min at 25⬚C, the mixture was aspirated and the well was washed three times with 350␮l of washing solution. The coloring reaction proceeded at 25⬚C for 30 min and the absorbance at 405 nm was read using a microtiter plate reader. The index value was calculated using the following formula: Index (%) ⫽ 100⫻ (absorbance of sample/absorbance of wild type) Samples correspond to the index less than 66.7% were considered positive. 2.7. Allelic loss analysis Southern blot hybridization was carried out according to the method described previously [33]. DNA (5␮g) from blood-tumor pairs was digested overnight at 37⬚C with PstI or MspI, electrophoresed in 0.8% agarose gels, transferred to nylon membranes, and then fixed using ultraviolet crosslinking. Subsequently, the membranes were prehybridized overnight at 65⬚C in a hybridization solution containing 7% polyethylene glycol 8000 and 10% sodium dodecyl sulfate (SDS) with 200 mg/ml human placental DNA. The membranes were hybridized in the same solution containing

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P-labeled probes at 65⬚C overnight, washed twice at 65⬚C with 0.1 ⫻ standard saline citrate (0.15M NaCl, 0.015M sodium citrate) containing 0.1% SDS, and exposed to X-ray film at ⫺70⬚C for 2 days. The following probes and restriction enzymes were used: CI6-111 (D6S193), MspI or PstI; CI6-24 (D6S149), PstI; A12, PstI; and A2, PstI. The source of these probes has been described previously [22]. 32

2.8. Statistical methods The significance of differences between groups was analyzed using ␹2 test or U test. A P value of ⬍.05 was considered significant. 3. Results Of the 70 patients with epithelial ovarian cancers, 18 patients (26%) were RER-positive (Fig. 1), and the remaining 52 patients (74%) were RER-negative. Of 18 patients with RER-positive, the tumor was high-frequency RER in 6 and low-frequency RER in 12. There were no significant differences in histologic type, disease stage, or patient age between patients with RER-positive tumors and those with RER-negative tumors. On analysis of the p53 sequence, mutations were detected in 29 patients (41%). Four patients had frame shift mutations: 1-base pair deletion in two (exon 6, codon 211,212; exon 8, codon 276), 3-base pair deletion in one (exon 7, codon 255-256), and 17-base pair deletion in one (exon 5, codon 141-147). Twenty-five patients had

Fig. 2. Results of Southern blot analysis of samples from patients with epithelial ovarian cancers with partial deletions of chromosome 6q27. T and N indicate matched DNA samples isolated from tumor tissue and peripheral lymphocytes, respectively. Cases 7 and 12 had lost one allele at the S149 and A12 loci, respectively.

point mutations: non-sense mutations in 5 and mis-sense mutations in 20. There was no difference in types of p53 mutations between RER-positive and RER-negative tumors. Amplification of c-erbB2 was detected in 15 patients (21%). The point mutation in K-ras codon 12 was detected in 14 patients (20%). Transversions from GGT to GAT and GGT to GTT were detected in eight and six, respectively. 6q27LOH was found in 25 of 59 informative cases (42%)(Fig. 2). Mutations of p53, c-erbB2 amplification, point mutations of K-ras codon 12, and 6q27LOH found in 70 patients with epithelial ovarian cancers were divided into RER-positive tumors and RER-negative tumors, and are shown in Fig. 3. These findings are also summarized in Table 1. In RER-positive tumors, the incidence of genetic alterations was generally higher than that in RER-negative tumors in all of these genes, but significance was reached only for the K-ras mutations. Table 2 shows the relationship between the RER status and the number of genetic alterations demonstrated in epithelial ovarian cancers. In RER-positive tumors, many cases showed multiple genetic alterations. Cases having two and three genetic alterations accounted for 28% and 33%, respectively, in RER-positive tumors, which was significantly higher than those in RER-negative tumors (17% and 6%, respectively)(P ⫽ .002). 4. Discussion

Fig. 1. RER in tumor samples from patients with epithelial ovarian cancers. The presence of extra bands in the tumor sample (T), absent from matched normal tissue from the same patient (N), indicates the presence of an RER.

Association of mismatch repair deficiencies with genetic alterations has been investigated in colorectal cancers and endometrial cancers. In colorectal cancers, association of

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Fig. 3. Status of p53 mutations, c-erbB2 amplification, point mutations at codon 12 of K-ras, and 6q27LOH in 70 patients with epithelial ovarian cancers. Many RER-positive tumors possessed genetic alterations in multiple genes.

mutations or overexpression of the p53 gene and K-ras gene with RERs has mainly been investigated, concluding that there is almost no association of these genetic alterations with the RER status [34–37]. Similarly, no association of RERs with p53 gene and K-ras gene was observed for endometrial cancers, in several investigations reported [38–41]. However, in investigations of the PTEN gene, it was reported that the frequency of PTEN gene mutations is significantly higher in RER-positive tumors than in RERnegative tumors in endometrial cancers [42–44], and the PTEN gene has been attracting attention as a target of mismatch repair deficiencies. Regarding ovarian cancers, Sood et al. [45] reported a high frequency of p53 insertion/deletion mutations in RERpositive tumors, but there are almost no other reports re-

garding the relationship between RER status and genetic alterations. In this study, although the frequencies of alterations in RER-positive ovarian tumors were higher in all of the genes examined than those in RER-negative tumors, the difference was significant only in K-ras mutations. However, interestingly, it was revealed that many RER-positive tumors possessed alterations in multiple genes. The incidence of tumors with two or three genetic alterations was significantly higher in RER-positive tumors than in RERnegative tumors. This finding suggests a mechanism involving the accumulation of genetic alterations in various genes as one way of involving mismatch repair deficiencies in the development and/or progression of epithelial ovarian cancers. Although basic research studies in vitro and in vivo have shown that mismatch repair deficiencies trigger vari-

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Table 1 Association between RER status and genetic alterations demonstrated in epithelial ovarian cancers RER status

A. p53 mutation Present Absent B. c-erbB2 amplification Present Absent C. K-ras point mutation Present Absent D. 6q27LOH Present Absent

Positive (%)

Negative (%)

P value

11 (61) 7 (39)

18 (35) 34 (65)

.091

7 (39) 11 (61)

8 (15) 44 (85)

.078

7 (39) 11 (61)

7 (13) 45 (87)

.047

8 (53) 7 (47)

17 (39) 27 (61)

.489

ous genetic alterations [46,47], there have been no previous reports of this phenomenon in clinical cases. In colorectal cancers and endometrial cancers, it is reported that mutations in the genes of transforming growth factor ␤ receptor type II (TGF-␤, RII) [48–51], insulin-like growth factor II receptor (IGF-IIR) [49–51] and Bcl2-associated X protein (BAX)[50,51] are often observed among RER-positive tumors, suggesting that mismatch repair deficiencies are involved in cancer development and/or progression through mutations in these growth-related factors. In ovarian cancers, there has been almost no investigation of these growth-related factors. Colella et al. [52] reported investigations of BAX gene mutations in 20 ovarian cancer patients in whom mismatch repair proteins of hMLH1 and hMSH2 were expressed, and heterozygous frameshift mutation was found in only one patient. At present, there is almost no association between mismatch repair deficiencies and mutations of growth-related factor in the development and/or progression of ovarian cancers. In “The International Workshop on Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition” held in December 1997, definitions of RER and guidelines on methodology were presented [53]. Among microsatellite primers recommended for RER detection, only D2S123 was used in this study. However, the primers recommended in the workshop are appropriate for colorectal cancers and it is not clear if these primers are also appropriate for gynecological cancers. In our previous studTable 2 Association between RER status and number of genetic alterations demonstrated in epithelial ovarian cancers RER status No. of genetic alterations

Positive (%)

Negative (%)

0 1 2 3 Total

2 (11) 5 (28) 5 (28) 6 (33) 18 (100)

17 (33) 23 (44) 9 (17) 3 (6) 52 (100)

P value

.002

ies on endometrial and ovarian cancers, primers TP53 and D3S1611 showed good sensitivity [5,54]. Selection of appropriate primers for RER detection in gynecological cancers is an important issue for the future. For RER detection in formalin-fixed, paraffin-embedded materials, PCR failure is one potential problem. We repeated PCR and confirmed the reproducibility of RER-positivity. However, as recommended in the International Workshop, it may be better to strictly define tumors confirmed to be instable by two or more of the five primers as RER-positive tumors. In conclusion, this study of RER status and alterations in the four species of genes showed that RER-positivity, that is, mismatch repair deficiencies, is involved in the development and/or progression of epithelial ovarian cancers through the accumulation of genetic alterations. However, involvement of genetic alteration in development and/or progression of sporadic ovarian cancers other than p53 gene alteration is not known at present. It cannot be simply concluded from the results regarding the four types of gene alteration in this study that mismatch repair deficiency is involved in the development and/or progression mechanism of this disease. Although in colorectal cancers in which the relationship between genetic alteration and carcinogenesis has been elucidated fairly well, according to the Vogelstein group, involvement of mismatch repair deficiencies in the development and/or progression mechanism of sporadic colorectal cancers has almost not been elucidated, nor were there findings showing that mismatch repair deficiencies facilitate accumulation of genetic alterations [34,37]. Therefore, development and/or progression mechanism of various cancers including ovarian cancers with mismatch repair deficiencies may be very complex. In ovarian cancers, mismatch repair deficiencies and its involvement in genetic alteration will not be clarified until novel protooncogenes and tumor suppressor genes closely associated with cancer development and/or progression are discovered and identified in the future. References [1] Fujita M, Enomoto T, Yoshino K, Nomura T, Buzard GS, Inoue M, Okudaira Y. Microsatellite instability and alterations in the hMSH2 gene in human ovarian cancer. Int J Cancer 1995;64:361–6. [2] King BL, Carcangiu M-L, Carter D, Kiechle M, Pfisterer J, Pfleiderer A, Kacinski BM. Microsatellite instability in ovarian neoplasms. Br J Cancer 1995;72:376–82. [3] Arzimanoglou II, Lallas T, Osborne M, Barber H, Gilbert F. Microsatellite instability differences between familial and sporadic ovarian cancers. Carcinogenesis 1996;17:1799–804. [4] Sood AK, Skilling JS, Buller RE. Ovarian cancer genomic instability correlates with p53 frameshift mutations. Cancer Res 1997;57: 1047–9. [5] Ohwada M, Suzuki M, Saga Y, Sato I. DNA replication errors are frequent in mucinous cystadenocarcinoma of the ovary. Cancer Genet Cytogenet 2000;117:61–5. [6] Milner BJ, Allan LA, Eccles DM, Kitchener HC, Leonard RCF, Kelly KF, Parkin DE, Haites NE. p53 mutation is a common genetic event in ovarian carcinoma. Cancer Res 1993;53:2128–32.

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