YGENO-08651; No. of pages: 7; 4C: Genomics xxx (2014) xxx–xxx

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Microsatellite instability, promoter methylation and protein expression of the DNA mismatch repair genes in epithelial ovarian cancer Shilpa V. a,1, Rahul Bhagat a,1, Premalata C.S. b,1, Pallavi V.R. c,1, Lakshmi Krishnamoorthy a,⁎ a b c

Department of Biochemistry, Kidwai Memorial Institute of Oncology, Dr. M.H. Marigowda Road, Bangalore 560029 Karnataka, India Department of Pathology, Kidwai Memorial Institute of Oncology, Dr. M.H. Marigowda Road, Bangalore 560029 Karnataka, India Department of Gynaec Oncology, Kidwai Memorial Institute of Oncology, Dr. M.H. Marigowda Road, Bangalore 560029 Karnataka, India

a r t i c l e

i n f o

Article history: Received 17 April 2014 Accepted 26 August 2014 Available online xxxx Keywords: LOH MMR MSI Ovarian cancer Promoter methylation

a b s t r a c t The role of defective mismatch repair (MMR) system in ovarian carcinoma is not well defined. The purpose of the study was to determine the relationship between microsatellite instability (MSI), promoter methylation and protein expression of MMR genes in epithelial ovarian carcinoma (EOC). MSI and promoter methylation of MLH1, MSH2 and PMS2 genes were studied using PCR methods in the study cohort. A small subset of samples was used to analyze the protein expression by immunohistochemistry (IHC). MSI was observed in N60% of tumor samples and 47% of normal ovaries. MLH1 was methylated in 37.5% and 64.3% EOCs and LMP tumors. The loss of immunoexpression of MMR genes was not seen in ovarian tumors. There was no correlation between MSI, promoter methylation and protein expression of the MMR genes suggesting that each may function independently. MSI is a common event in ovarian carcinoma and may increase the clinical awareness of the subset of tumors. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Ovarian cancer takes first place as a cause of death from gynecological malignancies and it is the fifth leading cause of cancer deaths among women [1]. Both environmental and genetic factors play an important role in the etiology but the causal relationship between these and tumor development is not clear [2]. Alternative modes of inactivation of genes during cancer development include an epigenetic process marked by promoter region hypermethylation associated with transcriptional loss leading to gene silencing [3]. Epigenetic changes occurring in the DNA repair genes impair the stability of DNA making it more vulnerable to damage and increasing the mutation rates. It has been suggested that the impairment of the MMR activity is an important step in carcinogenesis common to several types of cancer [4]. Significant proportions of carcinomas develop through DNA MMR deficiency and exhibit frequent MSI [5].

Abbreviations: EOC, epithelial ovarian cancer; IHC, immunohistochemistry; LOH, loss of heterozygosity; LMP, low malignant potential; MSP, methylation specific PCR; MMR, mismatch repair; MSI, microsatellite instability; MSI-H, microsatellite instability — high; MSIL, microsatellite instability — low; MSS, microsatellite stable; NCI, National Cancer Institute; TMA, tissue microarray; WHO, World Health Organization. ⁎ Corresponding author at: Consultant Biochemist, Sri Shankara Cancer Hospital & Research Centre, 1st cross, Shankarpuram, Basavanagudi, Bangalore 560004, Karnataka India. E-mail addresses: [email protected] (S. V.), [email protected] (R. Bhagat), [email protected] (P. C.S.), [email protected] (P. V.R.), [email protected] (L. Krishnamoorthy). 1 Fax: +91 80 26560723.

The human DNA MMR family is a highly conserved group of proteins that function in genome stabilization and mutation avoidance. The DNA MMR system consists of many genes including: MSH2, MLH1, MSH3, PMS1, PMS2 and MSH6 [6]. These genes are very important in distinguishing and repairing mispairing and slippage errors in DNA synthesis. MMR inactivation leads to the occurrence of unrepaired deletions in mono- and dinucleotide repeats resulting in variable lengths of these repeats. This is called MSI and is used as a marker for MMR deficiency [7]. Following its discovery in the hereditary non-polyposis colon cancer (HNPCC) syndrome, MMR deficiency was also identified in several other cancer sites, including the uterus and the ovaries [8]. Microsatellite instability has been reported in many studies although the reported frequency of MSI has shown great variability [9–11]. Data on the mismatch repair gene alterations in ovarian cancer among the Indian population is not available. This study uses the panel of NCI markers with the aim of evaluating the role of MSI and MMR expression and promoter methylation of MLH1, MSH2 and PMS2 genes in patients with epithelial ovarian carcinoma, LMP tumors and benign cystadenomas compared with normal ovaries and the correlation of these with the clinicopathological parameters. 2. Results 2.1. Analysis of MSI and LOH The results for MSI phenotype obtained using the panel of NCI markers are presented in Fig. 1. The frequency of instability at each individual microsatellite locus for the five markers studied is shown in

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Please cite this article as: S. V., et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.08.016

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Fig. 1. Microsatellite analysis using NCI markers. For each marker, the first graph represents normal DNA and second graph represents tumor DNA.

Table 1. A comparison of the frequency of instability seen in carcinomas, LMP tumors and benign ovarian tumors with normal ovaries showed that the dinucleotide markers D2S123, D5S346 and D17S250 and the

Table 1 Analysis of MSI in the study group. Microsatellite markers

Tumor type Carcinomas (88)

LMP (14)

Benign (20)

Normal (15)

BAT25 (S) BAT25 (I) BAT25 (L) p-value BAT26 (S) BAT26 (I) BAT26 (L) p-value D2S123 (S) D2S123 (I) D2S123 (L) p-value D5S346 (S) D5S346 (I) D5S346 (L) p-value D17S250 (S) D17S250 (I) D17S250 (L) p-value

34 (38.6%) 26 (29.8%) 28 (31.8%) 0.383 14 (15.9%) 20 (22.7%) 54 (61.4%) 0.000⁎

8 (57.2%) 3 (21.4%) 3 (21.4%) 0.781 2 (14.3%) 4 (28.6%) 8 (57.1%) 0.020⁎

10 (50%) 1 (5%) 9 (45%) 0.032⁎ 2 (10%) 8 (40%) 10 (50%) 0.012⁎

14 (15.9%) 35 (39.8%) 39 (44.4%) 0.005⁎ 14 (15.9%) 31 (35.2%) 43 (48.9%) 0.000⁎

2 (14.3%) 11 (78.5%) 1 (7.1%) 0.015⁎ 2 (14.4%) 6 (42.8%) 6 (42.8%) 0.017⁎

7 (35%) 8 (40%) 5 (25%) 0.619 1 (5%) 15 (75%) 4 (20%) 0.000⁎

34 (38.6%) 12 (13.6%) 42 (47.8%) 0.000⁎

8 (57.2%) 0 (0%) 6 (42.8%) 0.006⁎

15 (75%) 0 (0%) 5 (25%) 0.05⁎

8 (53.3%) 5 (33.4%) 2 (13.3%) – 8 (53.3%) 5 (33.4%) 2 (13.3%) – 8 (53.3%) 4 (26.7%) 3 (20%) – 10 (66.7%) 3 (20%) 2 (13.3%) – 15 (100%) 0 (0%) 0 (0%) –

S — Stable, I — Instable and L — LOH. ⁎ Significant — p-value N0.05 (p-values are for combination of S, I and L).

mononucleotide marker BAT 26 were the most significantly altered markers with p b 0.01 for the four markers. LOH was analyzed using the same panel of NCI markers (Table 1). The LOH values for the markers BAT 25, BAT 26, D2S123, D5S346 and D17S250 were 31.8%, 61.4%, 44.4%, 48.9% and 47.8% in EOC; 21.4%, 57.1%, 7.1%, 42.8% and 42.8% in LMP tumors and 45%, 50%, 25%, 20% and 25% respectively in benign tumors. Using the panel of NCI markers, 60/88 (68.2%) MSI-H and 16/88 (18.2%) MSI-L were observed in EOCs. 9/14 (64.3%) MSI-H and 2/14 (14.3%) MSI-L were seen in LMP tumors and 10/20 (50%) MSI-H and 5/20 (25%) MSI-L were observed in benign tumors. MSS was identified in 13.6% EOCs, 21.4% LMP tumors and 25% benign tumors respectively. A statistical significance among the three study groups was seen when compared to normal ovarian tissue samples (p b 0.01 for EOCs, p = 0.020 for LMP tumors and p b 0.01 for benign tumors respectively). A correlation of the MSI frequency and phenotype with the clinicopathological variables showed that D2S123 and D17S250 were significantly associated with histological subtype of the tumor (p b 0.01 and p = 0.029) and the BAT 26 marker was significantly associated with the presence of ascites in carcinomas (p b 0.01). The MSI-H phenotype showed significant association with the presence of ascites in carcinomas (p b 0.01) [data not shown]. 2.2. Promoter hypermethylation of MMR genes Representative gel pictures are shown in Fig. 2. Table 2 gives the methylation frequencies of the DNA repair genes studied. Promoter

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Fig. 2. Methylation analysis of MMR genes. Agarose gel showing representative MSP products of MMR genes in epithelial ovarian tumors. UM — unmethylated allele and M — methylated allele. +UM — positive control for unmethylated allele. +M — positive control for methylated allele. −ve UM — negative control for unmethylated allele. −ve M — negative control for methylated allele. C011 and C087 — carcinomas, B010 — benign tumor, and N003 — normal ovarian tissue. Note: Results of only 3 cases [one carcinoma (C011), one benign (B010) and one normal (N003) ovaries] are represented for MSH2 gene.

Table 2 Methylation status in the subject studied. Genes

MLH1 (U) MLH1 (M) p-value MSH2 (U) MSH2 (M) p-value PMS2 (U) PMS2 (M) p-value

Table 3 Correlation of promoter methylation of MMR genes with microsatellite phenotype in the study group.

Tumor type Carcinomas (88)

LMP (14)

Benign (20)

Normal (15)

55 (62.5%) 33 (37.5%) 0.027⁎

5 (35.7%) 9 (64.3%) 0.000⁎

14 (70%) 6 (30%) 0.005⁎

81 (92%) 7 (8%) 1.000 87 (99%) 1 (1%) 1.000

14 (100%) 0 (0%) – 14 (100%) 0 (0%) –

19 (95%) 1 (5%) 0.382 20 (100%) 0 (0%) –

15 (100%) 0 (0%) – 15 (100%) 0 (0%) – 15 (100%) 0 (0%) –

U — unmethylated and M — methylated. ⁎ Significant — p-value N 0.05.

methylation was absent in all the three genes for normal ovarian tissues. Promoter methylation of MLH1 gene was observed in 33 (37.5%) EOC's, 9 (64.3%) LMP tumors and 6 (30%) benign tumors which were statistically significant (p = 0.027, p b 0.01 and p b 0.01 respectively) when compared to normal ovarian tissues individually. Methylation in MSH2 promoter was present in 7 (8%) patients with EOC, while for PMS2 gene only one case showed methylation. None of the cases showed methylation in all three genes studied, while 4 (4.5%) cases showed methylation in two of the genes. 36 (40.9%) cases had promoter methylation in at least one of the three genes studied. Promoter methylation by MSP revealed no statistically significant association with the MSI markers studied and clinicopathological parameters. A significant association between MLH1 promoter hypermethylation and MSI-H phenotype was observed (p = 0.027) [Table 3]. 2.3. MMR protein expression MMR protein expression was exclusively nuclear. A complete loss of MLH1 expression was detected in 4.7% cases of EOC and none in LMP and benign tumors. MSH2 imunoreactivity was observed in all

Gene

Tumor type

Methylated cases

MSI-H

MSI-L

MSS

p-value

MLH1

Carcinomas LMP Benign Carcinomas Benign Carcinomas

33 9 6 7 1 1

17 (51.5%) 5 (55.6%) 3 (50%) 4 (57.1%) 1 (100%) 1 (100%)

10 (30.3%) 2 (22.2%) 2 (33.3%) 2 (28.6%) 0 (0%) 0 (0%)

6 (18.2%) 2 (22.2%) 1 (16.7%) 1 (14.3%) 0 (0%) 0 (0%)

0.027⁎ 0.492 0.621 0.744 0.703 0.789

MSH2 PMS2

MSI-H — microsatellite instability — high, MSI-L — microsatellite instability — low, and MSS — microsatellite stable. ⁎ Significant — p-value b 0.005 (p-values are for combination of MSI-H, MSI-L and MSS).

examined cases of ovarian cancer. A complete loss of PMS2 expression was detected in 23.4% cases of EOC and 10% of LMP tumor and none in benign tumors (Table 4). The immunoexpression of MMR proteins did not correlate either with the MSI markers or with MSI phenotype. Representative photomicrographs are shown in Fig. 3. In the current study, we found that loss of MMR protein expression was not significantly correlated with either promoter methylation or the microsatellite instability phenotype. 2.4. Follow-up of patients A follow-up analysis was performed in 102 patients (88 EOC's and 14 LMP tumors). Median progression-free survival (PFS) was 17 months. From the follow-up information available for the 102 ovarian carcinoma patients, 40.9% (n = 36) were alive without disease progression, 23.8% (n = 21) remained alive with disease progression, 9.1% (n = 8) were lost to follow-up and 26.1% (n = 23) patients died of recurrence or of the disease without remission. MLH1 promoter hypermethylation showed a significant correlation with the survival of the patients (p = 0.014). The median survival time for unmethylated group was

Table 4 Immunoexpresssion of the MMR genes. MLH1 expression

MSH2 expression

PMS2 expression

No of cases

Positive

Intermediate

Negative

Positive

Intermediate

Negative

Positive

Intermediate

Negative

Carcinomas (64) LMP (10) Benign (10) Normal (5) p-value

44 (69%) 10 (100%) 10 (100%) 5 (100%) 0.130

17 (26.6%) 0 (0%) 0 (0%) 0 (0%)

3 (4.7%) 0 (0%) 0 (0%) 0 (0%)

60 (93.8%) 10 (100%) 10 (100%) 5 (100%) 0.659

4 (6.2%) 0 (0%) 0 (0%) 0 (0%)

0 (0%) 0 (0%) 0 (0%) 0 (0%)

34 (53.2%) 4 (40%) 6 (60%) 5 (100%) 0.092

15 (23.4%) 5 (50%) 4 (40%) 0 (0%)

15 (23.4%) 1 (10%) 0 (0%) 0 (0%)

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26 months (95% CI 24.2 — 27.7) and for methylated group was 21 months (95% CI 15.7 — 26.2). No significance was observed between the clinical response of the patients and methylation of MSH2 and PMS2 genes or with MSI-H phenotype. 3. Discussion Despite an increasing knowledge of the genetics and biochemistry of MMR, little is known about the factors that contribute to the etiology and tissue specificity of MSI-associated carcinogenesis. MSI has been implicated in the pathogenesis of sporadic colon, endometrial, ovarian and gastric carcinomas which are associated with defects in the MMR pathway [12,13]. Genetic as well as epigenetic changes in the MMR genes are likely to diminish mismatch repair, leading to a higher mutation rate facilitating carcinogenesis. In contrast to several reported studies, we found a relatively high percentage (N60%) of MSI in our study. Ethnically diverse population and racial background of the patients enrolled in the study may be one of the factors responsible for high MSI frequency observed. MSI

was found to be more frequent in women between the ages of 45– 50 years. Women who have undergone repeated ovulatory cycles are more prone to have MSI phenotype owing to repeated injuries to the ovarian epithelium. The continuous ovulatory process may result in genetic alterations that compromise the MMR system [14]. An interesting observation from our study was the high percentage (47%) of MSI seen in normal ovaries (as compared to normal peripheral lymphocytes from the same subject). The normal healthy controls also belonged to the same age group as our cases and thus the high MSI frequency observed in normal controls could be explained. Our results suggest that MSI may not solely be a feature of malignant ovarian cancer [15]. In our study, we also observed that MSI was more frequent in the dinucleotide markers and the mononucleotide marker BAT 26. The highest frequency of MSI was found in D2S123 (39.8%) followed by D5S346 (35.2%). A systematic review done by Megan et al. on the observed frequency of MSI in ovarian cancer among different studies done using the NCI markers also showed a higher MSI frequency in the dinucleotide markers as compared to mononucleotide markers [16]. MSI frequency in BAT 26 marker in our study was 22.7%, a finding

Fig. 3. IHC staining for MMR genes (MLH1, MSH2 and PMS2) in epithelial ovarian carcinomas. A. Positive. B. Negative.

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similar to the results of Gras et al. who has shown that the reliability of the mononucleotide markers BAT 25 and BAT 26 is so high that most MSI could even be predicted by evaluating the two markers exclusively [17]. An association between MSI status and instability of the BAT 26 locus was in concordance with the results observed for colon cancer [18]. Several studies have been done using the panel of markers recommended by NCI to study MSI in ovarian cancers and results have been highly variable. Sood et al. were the first to study MSI in ovarian carcinoma using the NCI markers and have reported a frequency of 19% in EOC patients [19]. Lu et al. have reported a MSI frequency of 53% whereas Yoon et al. reported a frequency of only 8% [20,21]. The MSI rate increased from 19% to 36% when additional MSI markers were added in a study carried out by Sood et al. [19]. This probably suggests that the marker chosen for MSI study could possibly be tissue specific or unique for a certain type of cancer tissue. Hence each type of cancer could demand testing of a unique set of markers. MSI has been reported in 0–77% ovarian cancers (most commonly 10–20%) and is regarded as a component of the molecular pathways underlying the development of borderline tumor and mainly the better differentiated tumors [11,22–24]. The wide discrepancy in the reported frequency of MSI reflects differences between studies in age of patients, sample size, tumor histotype, choice of MSI markers, criteria for defining MSI positivity and ethnically diverse populations and racial background of the patients. Further studies on MSI markers are needed that can lead to an understanding of the choice or combination of MSI markers that can serve as a definitive diagnostic, predictive or even a prognostic tool. Allen et al. have reported that LOH is found frequently in ovarian cancer and may play an important role in ovarian carcinogenesis [25]. Pyat et al. have reported allelic variation in BAT 26 locus in 13% of African-American individuals [26]. Interestingly, results from this study showed the complete loss of one allele in BAT 26 in 61.4% of cases, 44.4% in D2S123, 48.9% in D5S346 and 47.8% in D17S250. The results from the present study are novel as other studies have reported a high LOH only in the dinucleotide marker D17S250 [27]. The experimental method used in our study was similar to the method adopted by Demokan et al. [28]. Since the results were different from the reported data, the results obtained were rechecked by randomly repeating 20 samples for each marker and concordant results were obtained for all markers. The NCI markers are highly polymorphic in nature and mutations in these markers may also contribute to the high MSI frequency observed and further studies are clearly warranted. Several studies have shown that the DNA MMR pathway is particularly affected by abnormal function of MLH1 and MSH2 genes [29]. A high frequency of promoter hypermethylation of MLH1 gene of 37.5% in malignant cases and 64.3% in the borderline cases was observed in our study. The finding of higher promoter methylation of MLH1 gene in borderline tumors than the malignant tumors suggests that promoter methylation may be involved in the development of early stages of ovarian carcinogenesis and could be a frequent mechanism by which key genes are inactivated. Aberrant methylation of the MLH1 gene is potentially a very important mechanism leading to the MSI phenotype in colon tumors [30]. Consistent with these observations, the MLH1 gene was preferentially methylated in our study and significantly correlated with the MSI-H phenotype. Geisler et al. have reported 47.6% of MSI-H phenotype in ovarian carcinomas along with reduced or absent MLH1 mRNA [31]. Promoter hypermethylation of MSH2 was 8% while the PMS2 gene promoter showed only 1% (negligible) hypermethylation. Studies on endometrioid and colorectal cancers have shown that hypermethylation of the MSH2 gene promoter seems to occur in familial but not in sporadic cases and our results are consistent with these reports [32,33]. A variety of techniques have been used for the identification of tumors with MMR deficits ranging from PCR-based to immunohistochemistry. Several reports have demonstrated that IHC of MMR genes may be used as an alternative method for the detection of MSI status in tumor samples

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[34]. In our study, promoter hypermethylation, MSI and protein expression were not correlated, a finding similar to the results reported by Diniz et al. in Wilms' tumor who found that there was no concordance with the frequency of tissue expression of MMR proteins and MSI [35]. Our results are in contrast to the results obtained for colorectal cancer where a positive correlation was seen between loss of MMR protein expression and MSI status [36]. The results from our study indicate that these pathways may function independently and are not mutually exclusive in promoting tumorigenesis. Greater than half of MSI-H tumors were not explained by MLH1 and MSH2 CpG island hypermethylation, perhaps the CpG island chosen for the study may not be associated with MSI-H. A gene mutation in MLH1 and MSH2 genes may explain the high frequency of MSI in these tumors which may be supported by the current understanding of MSI and MMR defects in other tumor types [37]. The phenomenon of MSI in ovarian cancer may be different from MSI in colorectal cancers. Sporadic colorectal carcinomas with MSI are characterized by a specific phenotype and better prognosis. The data from our study is suggestive that ovarian carcinomas with MSI are characterized by advanced stage, poor differentiation, grade and poor prognosis. Endometrial carcinomas and cancers with endometrioid histology with MSI have been shown to be associated with a shorter disease free interval [38]. Promoter methylation of MLH1 gene was more frequent in non-serous tumors. Assessing the role of hypermethylation of DNA repair genes in different histological subtypes of ovarian tumor will be interesting as it may pave the way to understand the underlying mechanisms of ovarian carcinogenesis and could find use as early detection markers. 4. Conclusions Microsatellites in cancer cells undergo expansion or contraction at high frequencies within genes and provide a mechanism for inactivation of tumor suppressor genes during tumor progression [39]. These results suggest that microsatellite instability plays an important role in the pathogenesis of sporadic ovarian carcinoma. To the best of our knowledge, we are the first to report the results of a defective MMR system in ovarian cancer for the Indian population. These findings are clinically relevant as they may increase the clinical awareness of the subset of tumors, having potential implications for medical management. This evidence highlights the importance of further studies of this understudied group of cancers. 5. Materials and methods 5.1. Patients Fresh ovarian tumor samples of 122 consecutive patients admitted to the Department of Gynec Oncology, Kidwai Memorial Institute of Oncology, Bangalore, India were collected. The patients had no prior treatment. The samples included 88 primary EOC samples, 14 LMP tumor samples and 20 benign ovarian tumors samples. Metastatic carcinoma to the ovary and tumors of germ cell and stromal cell were excluded from the study. 15 normal ovarian tissues were obtained from women without a family history of ovarian and breast cancer undergoing oophorectomy, during total abdominal hysterectomy. Tissue specimens were snap frozen at −80 °C immediately after surgery and stored. The tumors were classified histologically according to the criteria of the World Health Organization (WHO) and staged according to the Federation of International Gynecological Oncologists (FIGO) system. The mean age of the patients was 48.8 ± 9.81 years (range 22–72) for carcinomas, 40 ± 9.13 years (range 26–72) for LMP tumors, 51.4 ± 10.67 years (range 51–65) for benign tumors and 52.3 ± 7.33 years (range 41–58) for non-cancer patients. Most of the tumors were grade III [57 (65%)] and advanced stage [stage III, {64 (73%)}]. Serous tumors were the most common histological type [52 (59%)]. Tissue immunoexpression of MLH1, MSH2 and PMS2

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genes was studied in a subset of 64 malignant cases, 10 LMP tumors, 10 benign cystadenomas and 5 normal ovaries. The study was approved by the Institutional Review Board and Medical Ethics Committee and a written informed consent was taken from the patients enrolled into the study. For patients with EOC, information regarding clinical response was retrieved from the medical records. 5.2. DNA isolation: MSI analysis and methylation-specific PCR Genomic DNA was extracted from 25 mg of tissue using QIAamp DNA Mini Kit (Qiagen, CA, USA). The extracted DNA was quantified and stored at −20 °C until processed. 5.3. Microsatellite analysis MSI and LOH were concomitantly detected by PCR amplification of the microsatellites. MSI was evaluated in the tumor tissue and normal ovarian tissue and lymphocyte DNA using a panel of five markers: BAT 25, BAT 26, D2S123, D5S346 and D17S250. These specific microsatellite loci were derived from the NCI reference and alternative loci panel in order to ensure standardized findings [40]. Primer sequences of the PCR assay chosen for the study have been published previously [41]. The forward primer of each pair was labeled with one of the three dyes (NED, HEX, 6-FAM) and the fourth dye (TAMRA) was reserved for the size standard. The PCR was performed in a total volume of 7.5 μL, containing 0.6 μL (50 ng/μL) DNA, 4.5 μL of true allele PCR premix, 0.5 μL of primer mix and 1.9 μL of sterile distilled water. PCR was carried out in a thermal cycler, beginning with a 12 min denaturation at 95 °C, followed by 10 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 15 s and extension at 72 °C for 30 s and 20 cycles of denaturation at 89 °C for 15 s, annealing at 55 °C for 15 s and extension at 72 °C for 30 s with a final 10 min extension at 72 °C. The fluorescent labeled products were analyzed by capillary electrophoresis on an Applied Biosystems 3700 DNA analyzer. 1 μL of pooled PCR product was mixed with 9 μL of deionized formamide and 0.5 μL of Gene Scan TAMRA 500 Size Standard (Applied Biosystems). The mixture was denatured at 94 °C for 5 min, cooled on ice and then loaded on the DNA analyzer. The data on the size of PCR products and the amount of fluorescent signal collected were analyzed with the GeneScan 3.1 and Genotyper analysis programs (Applied Biosystems). The presence of MSI was confirmed when monomorphic or polymorphic variants identified in microsatellite DNA in ovarian cancer samples were not present in normal ovarian tissue samples. The level of MSI was classified as high (MSI-H) when two or more markers tested demonstrated instability, low (MSI-L) when one of the markers tested demonstrated instability and stable (MSS) when no instability was detected. LOH was defined as an identical pattern in one of the two alleles, but complete loss in the second allele and was scored for all the markers [42]. Repeatability was confirmed by randomly rerunning 20 samples and verifying the results obtained. 5.4. Methylation specific polymerase chain reaction (MSP) MSP was used to determine the promoter methylation after the DNA (~600 ng) was modified with sodium bisulfite using the EZ DNA Methylation KitTM (Zymo Research Corp, California, USA). Nested MSP was used to determine the promoter methylation of MLH1 and MSH2 genes. PMS2 gene promoter methylation was determined by single step conventional MSP. The first step primers in the nested MSP, recognize the bisulfite modified template and the resulting PCR products were diluted ten-fold and subjected to second step PCR with primers specific for methylated and unmethylated promoters. Amplification was performed in a VeritiTM Thermal Cycler (Applied Biosystems) by an initial denaturation at 95 °C for 5 min, followed by

35 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s and a final extension at 72 °C for 5 min. The primer sequences of MMR genes used for nested MSP, methylated and unmethylated PCR assay and PCR conditions have been published previously [43]. The reactions were performed in a 50 μL volume with template DNA, 1× PCR buffer containing 1.5 mM MgCl2, 10 pM/L primer mix, 200 μM of each dNTP and 1 U Taq polymerase (New England Biolabs Inc, England) and the amplification was carried out for 35 cycles. Controls without DNA were included. CpGenome Universal Methylated DNA (Zymo Research Corp, California, USA) and peripheral blood lymphocyte DNA were used as positive and negative control for methylated and unmethylated promoters respectively. 10 μL of PCR product was separated electrophoretically on 2% agarose gel containing 0.1% ethidium bromide and analyzed using a gel documentation system (Gbox F3, Syngene) with a 100 bp DNA ladder (Fermentas, Germany) as a molecular weight standard. 5.5. Immunohistochemical staining To analyze the expression of the repair proteins MLH1, MSH2 and PMS2 an immunohistochemical analysis was performed on a subset of 64 EOCs, 10 LMP tumors, 10 benign tumors and 5 normal ovarian tissues using tissue microarray (TMA). 5.5.1. Construction of tissue microarray (TMA) The TMAs were constructed from formalin-fixed paraffin embedded tissue blocks. Hematoxylin-and-eosin-stained sections were reviewed by a Pathologist to select representative areas of tumor with adequate adjacent normal cells to acquire cores for the TMA. A tissue-arraying instrument (Beecher Instruments, Sun Prairie, WI) was used to acquire cylindrical 2 mm tissue core areas from donor blocks. Two identical core areas were selected for each tumor. 5.5.2. Immunohistochemical study Immunohistochemical staining was carried out on 5 μm sections from the TMA slides. The primary antibodies used for immunohistochemical analyses were mouse monoclonal anti-human MLH1 (pre-diluted; M 3640, clone ES05, Dako), mouse monoclonal anti-MSH2 (1:50 dilution, CM 219Aa, clone FE11, Biocare Medical) and mouse monoclonal anti-PMS2 (1:50 dilution, CM 344AK, clone A 16-4, Biocare medical). Immunostaining was done using a peroxidase kit (Biogenex Laboratories Inc, CA) with 3,39 diaminobenzidine as chromogen. The sections were counterstained with hematoxylin, dehydrated and mounted with Dpx. Colorectal carcinoma tissue section stained with primary antibody was used as a positive control. Nuclear staining was considered as a positive expression. Internal positive control was established by analyzing nuclear staining in the normal cells adjacent to the tumor. IHC results were scored by a pathologist without the knowledge of methylation status or the MSI status of the tumors. The stain intensity was graded as no staining, weakly positive and strongly positive and the percentages of cancer cells that stained positively were also graded as 0–20%, 20–80% and 80–100% [44]. 5.6. Statistical analysis Chi-square test or Fischer's exact probability test was used to analyze the association between MSI status, LOH, methylation frequencies and IHC. Contingency table analysis was used to analyze the correlations among ovarian carcinomas with and without microsatellite instability, tumors with and without hypermethylation, tumors with and without MMR protein expression and clinicopathological parameters. All statistical analysis was performed using a software package SPSS version 21.0 (Chicago, IL, USA). P b 0.05 was considered statistically significant.

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Please cite this article as: S. V., et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.08.016