Get 20M+ Full-Text Papers For Less Than $1.50/day. Subscribe now for You or Your Team.

Learn More →

Methylation Status and Immunohistochemistry of BRCA1 in Epithelial Ovarian Cancer

Methylation Status and Immunohistochemistry of BRCA1 in Epithelial Ovarian Cancer Background: Cancer initiation and progression are controlled by genetic and epigenetic events. One epigenetic process which is widely known is DNA methylation, a cause of gene silencing. If a gene is silenced the protein which it encodes will not expressed. Objectives: 1. Identify the methylation status of BRCA1 in patients with epithelial ovarian cancer (EOC)and assess BRCA1 protein expression in tumor tissue. 2. Examine whether BRCA1 gene methylation and BRCA1 protein are associated with survival of epithelial ovarian cancer patients. Methods: The study design was a prospective-cohort study, conducted at Sardjito hospital, Yogyakarta, Indonesia. Results: A total of 69 cases were analyzed in this study. The data showed that the methylation status of BRCA1 in EOC was positive in 89.9%, with clear protein expression of BRCA1 in 31.9%. Methylation status and expression of BRCA1 were not prognosticators of EOC patients. Menarche, CA125 level, clinical stage and residual tumor were independent factors for prognosis. Keywords: EOC - methylation - BRCA1 - prognosis factors - survival Asian Pac J Cancer Prev, 15 (21), 9479-9485 of expression is said to be regulated by the transcriptional Introduction implications of methylation of CpG nucleotide promoter. Cancer develops through a multistep process in which Esteller et al. research in the methylation of some the genomes of the new cancer cells undergo mutations in suppressor genes in various types of cancer showed that some groups of the specific genes such as protooncogen, the methylation of BRCA1 in sporadic ovarian cancer tumor suppressor genes and other genes that directly was 19% (11/58) (Esteller et al., 2001), in Vietnam found or indirectly control a cell proliferation. Cancer cells the methylation status of BRCA1 in women with ovarian also have a genetic instability that allows cells to get cancer patients was 11/59 (18.6%) (Lan et al., 2013). other changes all the time. One of the characteristics of The examination of the profile of BRCA1 methylation in malignant tumors is its heterogeneity. Cancers are caused ovarian cancer is very crucial to understand the molecular by the accumulation of mutations of several categories pathology of ovarian cancer, which in the end it will be of genes, the initiation and the progression of cancer are very useful in the clinical management, due to methylation controlled by the genetic and epigenetic events. is a reversible procces. The most known epigenetic process is a DNA As it was known that the use of azacitidine (AZA), methylation. Methylation is adding four atoms on cytosine, an inhibitor of DNA methylation, has been approved by one of four DNA nucleotides. This additional atom blocks the FDA for the treatment of myelodysplastic syndrome the protein that transcribes genes. DNA methylation is (MDS), a precancerous condition of acute myeloid an epigenetic mechanism that becomes very clear in the leukemia (Issa et al., 2005). Different combinations of recent years that there is a synergy between the genetic genetic changes are found in genomes of more than 100 and epigenetic changes. different types of human cancers. Thus, each cancer The expression of BRCA1 protein is ubiquitous in may be unique and the spectrum of genetic changes that humans, located in the nucleus, whereas the highest levels initiates the incidence of cancer may have many variations. are obtained in the ovarian, testis and thymus. It is a There is no single rule that underlies the occurrence of tumor suppressor and the reduced expression is associated all cancer and cancer is a phenomenology of infinite with the transformation procedures and the etiology of complexity. Hahn and Weinberg made an alternative view sporadic breast cancer and ovarian cancer. The reduction that the pathogenesis of cancer in humans was governed 1 2 3 Department of Obstetric and Gynecology, Department of Histology and Biomolecular, Department of Pathology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia *For correspondence: pradjatmo@yahoo.com; herupradjatmo@ gmail.com Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9479 Heru Pradjatmo et al by a number of genetic and biochemical rules applied in (Figure 2). Tumor were catagorised as having aberrant most types of human cancers (Hahn and Weinberg, 2002). BRCA1 expression for very low to no staining (<10%); This study was conducted to determine the methylation 0 or 1 score) and normal BRCA1 expression for >10% status of BRCA1 gene in epithelial ovarian cancer and the BRCA1 staining (2-4 score). (Thrall et al., 2006; Lesnock expression of BRCA1 protein in the tumor whether the et al., 2013). proportion of expression appropriate with the proportion of methylation. Does the proportion of methylation Results status of BRCA1 genes and the proportion of expression of BRCA1 protein influence survival of patients with The cases of ovarian tumors had been collected epithelial ovarian cancer? found 69 cases were malignant epithelial ovarian cancer, which were then used for the study of this manuscript. The patient’s survival was followed up and the longest Materials and Methods follow-up was 54 months and the shortest was 12 months. The study design was a prospective cohort, it was Several clinicopathological characteristic which were conducted at Sardjito Hospital, Yogyakarta, Indonesia in associated with risk factors of ovarian tumors such as which patients with ovarian tumors when the results of patients age, menarche, parity, menopausal age, nutritional frozen section showed malignant ovarian neoplasm, then status (BMI), the CA125 level before operation, residual some of the tumors were stored at -70 C, then the tissues tumor during operation, histopathological type and grade were used as the sample of the study to examine the of differentiation were recorded and analyzed. Those methylation status by methylation specific PCR (MSP). clinicopathological characteristic of the patients were While the other tumor tissues were examined at pathology clasified in two or three group. Table 1 showed the results anatomy to determine the type of histopathology, the of the methylation of BRCA1 genes in the tissue of EOC degree of differentiation and examined the expression of was 62/69 cases (89.9%) without methylation 7/69 cases BRCA1 in the epithelial ovarian cancer. (10.1%). IHC staining of BRCA1 normal (positive) was 22/69 cases (31.9%) and aberrant (negative) was 47/69 BRCA1 methylation status examination (68.1%). The agreement between methylation status of DNA tissue extraction was performed with QIAamp the BRCA1 gene and the expression of BRCA1 protein in DNA Mini Kit (Qiagen, Germany) as appropriate with the the tumor was -0.019 its mean that there is no agreement manual kit. The conversian procedure of DNA bisulphite between methylation and expression of BRCA1. This used protocol of MethylEasy Xceed Rapid DNA Bisulphite result can be proved either in the electrophoresis seen modification Kit. For PCR reaction, 1μl DNA which was that although some cancer cells the BRCA1 gene was converted with sodium bisulphide add 18μl PCR mix found methylated some cells found unmethylated either which contain 15PCR buffer, optimum concentration and these cells still express BRCA1 protein (Figure. Mg2+ for each primer, 0.4μl primer and 0.1μl Taq 1). Distribution of methylation status and IHC BRCA1 polymerase. The primer length which were used for staining correlated with clinicopathologial factors were BRCA1_M75 bp, BRCA1_U76 bp, with a nukleotid base not statistically significant different with p>0.05 (Table sequence as follows: BRCA1_MF: 5’-TCG TGG TAA CGG AAA AGC GC-3’, BRCA1_MR: 5’-AAA TCT CAA CGA ACT CAC GCC G-3’, BRCA1_UF: 5’-TTG GTT TTT GTG GTA ATG GAA AAG TGT-3’, BRCA1_UR : 5’-AAA AAA TCT CAA CAA ACT CAC ACC A-3’, Immunohistochemistry (IHC) for BRCA1 expression and scoring. Paraffin block section 3-5µm thick of ovarian tumor tissue on glass slide was examined for IHC. The IHC assay for BRCA1 expression used the MS110 clone monoclonal antibody (Biocare Medical, LLC, 4040, Pike Lane Concord, CA 94520, LA, USA) that reacts with N-terminal portion of the BRCA1 protein. The possitive BRCA1 expression of breast cancer tissue were used for Figure 2. IHC Examination of BRCA1: (B1-P2) Show positive control, and negative control used the same tissue Positive 2 Staining, (B1-P3) Show Positive 3, (B1-P4) and the staining without BRCA1 antibody. The percentage Show Positive 4 of staining was determined by an independent pathologist Table 1. Kappa Statistic of the Methylation Status of he was blinded to the identity and clinical outcome of the BRCA1 Gene and Expression of BRCA1 Protein samples. The ascribes score was based on the number of cells with nuclear staining. The score was classified Methylation Status I H C p value Kappa as Thrall et al. used: slides was score as 0 if there was Aberrant<10%(-) Normal≥10%(+) no staining, 1 if there was scattered staining (<10%). 2 Methylated (+) 42 (60.9%) 20 (29.0%) 0.84 -0.019 if 10-50% of the cells were stained, 3 if 50-90% of cells Unmethylated (-) 5 (7.2%) 2 (2.9%) Total 47 (68.1%) 22 (31.9%) were stained and 4 if nearly all cells (>90%) were stained 9480 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 100.0 6.3 12.8 10.1 20.3 10.3 25.0 30.0 75.0 75.0 46.8 56.3 51.1 51.7 54.2 50.0 31.3 30.0 25.0 38.0 33.1 31.3 31.3 30.0 27.6 25.0 23.7 Newly diagnosed without treatment Newly diagnosed with treatment Persistence or recurrence Remission None Chemotherapy Radiotherapy Concurrent chemoradiation DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9479 2). In the survival analysis by bivariable analysis found was 19% (11/58) (Esteller et al., 2001), 10% of 49 patients that menarche, CA125 level, stage of disease and residual ovarian cancer (Rathi et al., 2001), 31% (Wang et al., tumor during operation were significantly influence the 2004), Lan et al (2013) in Vietnamese women suffer from survival of the EOC patients with HR 2.55 (p=0.03), 4.42 ovarian cancer was 18,6%. It was considered whether (p=0.01), 3.77 (p=0.01), 2,4 (p=0.04) respectively, but in there was a possibility of technical error, for checking multivariable analysis there were no factors influence the and proving the absence of errors in the examination of survival of the patients, even methylation status of BRCA1 this research, it had carried out an internal validation with gene and expression of BRCA1 protein as well (Table 3). positive and negative controls using positive and negative From the results of the present research, its were controls on each of the electrophoresis examination found that the methylation levels of BRCA1 was 89.9% (Figure 1). We also conducted an external validation in the compared with the results of other studies elsewhere was way of re-examining randomly samples of the study. We high. Methylation of BRCA1 in sporadic ovarian cancer also did the examination to some samples of other normal Table 2. Characteristic of Epithelial Ovarian Cancer and Comparibility of the Methylation Status of BRCA1 Gene and IHC Result of BRCA1 Protein Variable Methylation Status BRCA1 IHC BRCA1 protein Methylated (%) Unmethylated (%) p value Aberrant/- (%) Normal/+ (%) p value Age of patients: < 40 years 17 (24.6) 1 (1.4) 0.45 12 (17.4) 6 (8.7) 0.87 ≥ 40 years 45 (65.2) 6 (8.7) 35 (50.7) 16 (23.2) Age of menarche < 15 years 42 (60.9) 5 (7.2) 0.84 32 (46.4) 15 (21.7) 0.99 ≥ 15 years 20 (29.0) 2 (2.9) 15 (21.7) 7 (10.1) Parity: < 2 30 (43.5) 3 (4.3) 0.78 23 (33.3) 10 (14.5) 0.78 ≥ 2 32 (46.4) 4 (5.8) 24 (34.8) 12 (17.4) Menopauze: Not menopauze 33 (47.8) 2 (2.9) 0.21 22 (31.9) 13 (18.8) 0.34 Menopauze 29 (42.0) 5 (7.2) 25 (36.2) 9 (13.0) B M I: < 25 kg/m 47 (68.1) 5 (7.2) 0.79 35 (50.7) 17 (24.6) 0.8 ≥ 25 kg/m 15 (21.7) 2 (2.9) 12 (17.4) 5 (7.2) CA 125 level ≤ 70 IU/ml 21 (30.4) 3 (4.3) 0.63 17 (24.6) 7 (10.1) 0.72 > 70 IU/ml 41 (59.4) 4 (5.8) 30 (43.5) 15 (21.7) Clinical Stage: Early Stage 20 (29.0) 3 (4.3) 0.57 18 (26.1) 5 (7.2) 0.2 Late Stage 42 (60.9) 4 (5.8) 29 (42.0) 17 (24.6) Histophatological type: Serous 18 (26.1) 1 (1.4) 0.5 10 (14.5) 9 (13.0) 0.23 Musinous Ca. 30 (43.5) 5 (7.2) 26 (37.7) 9 (13.0) Others 14 (20.3) 1 (1.4) 11 (15.8) 4 (5.8) Grade of Differentiation Well differentiated 30 (43.5) 5 (7.2) 0.3 24 (34.8) 11 (15.9) 0.69 Moderate 15 (21.7) 0 (0.0) 9 (13.0) 6 (8.7) Poor differentiated 17 (24.6) 2 (2.9) 14 (20.3) 5 (7.2) Residual tumor: Optimal operation 36 (52.2) 4 (5.8) 0.96 29 (42.0) 11 (15.9) 0.35 Not optimal 26 (37.7) 3 (4.3) 18 (26.1) 11 (15.9) Table 3. Survival Analysis for Bivariable and Multivariable Analysis with Cox’s Regression Variable Bivariable analysis Multivariable analysis HR (95% CI) p-value HR (95% CI) p-value Age of patients: < 40 years Ref Ref. ≥ 40 years 1.54 (0.52-4.59) 0.43 1.38 (0.35-5.33) 0.63 Menarche < 15 years Ref. Ref. ≥ 15 years 2.55 (1.07-6.03) 0.03 2.64 (0.93-7.48) 0.06 Parity: < 2 Ref. Ref. ≥ 2 2.00 (0.80-4.96) 0.13 2.28 (0.91-7.79) 0.22 Menopauze: Not menopauze Ref. Ref. Menopauze 1.07 (0.45-2.53) 0.86 0.74 (0.26-2.15) 0.59 B M I: < 25 kg/m Ref. Ref. ≥ 25 kg/m 0.67 (0.22-2.01) 0.48 0.41 (0.11-1.49) 0.17 CA 125 ≤ 70 IU/ml Ref. Ref. >70 IU/ml 4.42 (1.29-15.10) 0.01 3.01 (0.74-12.19) 0.12 Clinical Stage: Early Stage Ref. Ref. Late Stage 3.77 (1.11-128.6) 0.01 3.15 (0.58-17.15) 0.18 Histophatological type: Serous 0.84 (0.25-2.78) 0.77 0.78 (0.20-2.98) 0.71 Musinous Ca. 0.82 (0.30-2.27) 0.71 1.75 (0.35-7.99) 0.46 Others (Clear cell+ Ref. Ref. Endometrioid) Grade of Differentiation Well differentiated Ref. Ref. Moderate 1.98 (0.66-5.92) 0.22 2.25 (0.52-9.63) 0.27 Poor differentiated 2.29 (0.82-6.36) 0.11 3.83 (0.71-20.69) 0.11 Residual tumor: Optimal operation Ref. Ref. Not optimal 2.45 (1.02-5.89) 0.04 0.81 (0.24-2.73) 0.74 Methylation status BRCA1 Methylated Ref. Ref. Unmethylated 1.02 (0.23-4.41) 0.97 2.11 (0.38-11.71) 0.39 IHC BRCA1 Protein Aberrant Ref. Ref. Normal 0.83 (0.32-2.15) 0.7 0.39 (0.11-1.36) 0.14 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9481 Heru Pradjatmo et al and would pause the mRNA translation process if the protein that was resulted from the translation of gene was no longer needed by binding to the mRNA so mRNA would be silent or undergo degradation. The protein expression of BRCA1 and others were regulated by various molecular devices within the cell. The setting was at the level of Figure 1. The Result Electrophoresis of BRCA1 transcription, translation and post translation. In the result Methylation of Some Sample of the Epithelial Ovarian of the latest research, it was reported that the causes and Cancer the early incidence of EOC were governed by a group of genes including microRNA genes, which could be cells, namely leukocytes cells of the normal people and modulated at four levels, namely the level of genomic, the result were same. The above validation checks proved transcriptional regulation, post-translation modification that the possibility of technical errors in the examination and regulatory processes of miRNA (Li et al., 2011). The could be ruled out or the results of the examination in the position of miRNA genes had been mapped to be scattered study were reliable. The results of this study might show in chromosome 1 to 22, and the X and Y sex chromosomes. characteristics of ovarian cancer patients in Indonesia, Apparently, around 16% miRNA was regulated at the particularly in areas of southern Central Java. level of post-translational. Changes in the pair of A-T base As it was known that the process of carcinogenesis became the pair of A-I base (inosine that acts on RNA was caused by various factors as described in the editing) caused a conversion of miRNA biogenesis, the literature. Likewise, promoter methylations of suppressor changes of miRNA product and the targeting changes of genes could be caused by various factors, namely, specific miRNA (Kawahara et al., 2009). environment, nutrition, chemicals, infections, and so on. It had also been reported that lin28, a repressor let7 For Indonesians, these factors were different to those in miRNA when a change of the base pairs occurred, caused Western countries or the other more developed countries. a Dicer enzyme that did not function and the degradation So, it would greatly affect the methylation status of the of pre-miRNA; consequently, the target protein of mRNA genes in these individuals in our population. had over-expression (Heo et al., 2008). This might explain why there was a difference of expression profile of BRCA1 and they had methylations, but the expression of the Discussion proteins in the tissue was high. As mentioned by Hilton et The data showed that frequency methylation status al. that the universal inactivation of BRCA1 had multiple of the patients were 89.9% and the expression of mechanisms that were not only because of mutation and BRCA1 were 31.9%. If all of the tumor cells underwent hypermethylation but there were still other factors or methylations in the BRCA1 genes, certainly, the tumor mechanisms (Hilton et al., 2002). These mechanisms cells would not express the BRCA1 protein but on the could include: first, the mutations of BRCA1 promoter, examination it obtained 22/69 cases (31.9%) that still so failing to identify the correct CpG island of BRCA1 showed the expression of BRCA1 protein and 47/69 promoter; second, the loss of the function of gene products case (68.1%) showed no expression of BRCA1. It meant required for the transcription of BRCA1, as well as no loss that not all ovarian tumor cells in a patient who on the of heterozygosity (LOH) as the third cause (Hilton et al., examination had positive methylation status had cells 2002). Similarly, not all cancer cells on these examination without methylation either. This was confirmed on the showed the negative expression of BRCA1 although the electrophoresis examination of any sample even it gained results of the examination of the methylation of BRCA1 a band that showed the positive methylation genes but on gene were positive. the other hand there was also a band which showed the There were no correlation among BRCA1 methylation presence of the genes not methylated (Figure 1). there and BRCA1 expression with several clinocopathological was almost no agreement between methylation status of variables such as; age, menarche, parity, menopauze BRCA1 genes and expression of BRCA1 protein in EOC status, BMI, CA125 level, clinical stage of disease, of this study as the result of Kappa statistic=0.01 (Table histopathological type, grade and residual tumor during 1). The methylation of BRCA1 promoter is important in surgery as seen in the distribution of methylation status of silencing BRCA1 in sporadic EOC. The loss of expression BRCA1 gene and expression of BRCA1 protein in EOC of tumor suppressor genes such as BRCA1 is known tissue statistically were not different with Chi square test occur through biallelic inactivation. The sugestion is that p>0.05 (Table 2). Lan et al (2013) found that association somatic mutation of BRCA1 is rare in sporadic EOC, thus, between methylation status of BRCA1, RASSF1A and the loss of BRCA1 expression is considered to be due to ER genes with the clinical and pathological parameters of a combination of allelic loss and methylation (Esteller women suffering from ovarian cancer in Vietnam. Other et al., 2001). Other authors suggested that the silent or studied find expression of BRCA1 protein in EOC was inactive BRCA1 gene on the tumor development were 40% and no association between BRCA1 expression with not only because of the epigenetic or genetic processes tumor grade, stage and overall survival of EOC patients (gene mutation), but also because of a variety of other (Shawky et al., 2014). As already were mentioned that mechanisms that might occur together, as the role of micro methylation of promoter gene or aberrant methylation RNA (miRNA) where miRNA was an RNA with a short was influenced by several environmental agents. Aberrant nucleotide chain length less than 22 bases of nucleotide DNA methylation currently is recognized as a common 9482 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9479 molecular abnormality in cancer and its become potential cancer. BRCA1 was a tumor suppressor that reduced the molecular marker for diagnosis, prognosis and treatment expression associated with the transformation process and (Laird, 2003). Previous studies regarding the association the etiology of breast cancer and sporadic ovarian cancers. between BRCA1 gene methylation and patient survival Reduced expression of BRCA1 was quite possible related remain controversial. Yang et al. and Montovan et al. to the presence of gene methylation. The present study of indicated that BRCA1 methylation was not assosiated with the examination of BRCA1 gene methylation obtained patient survival (Yang et al., 2011; Montovan et al., 2012). as much as 89.9%. Thus, 68.1% of epithelial ovarian Chiang et al. demonstrated survival disavantage in patients tumors without the BRCA1 protein expressed was very whose neoplasm were methylated at BRCA1 (Chiang likely because of methylation while the remaining 31.9% et al., 2006). However, reported significant association expressed with the positive levels (≥10%) or normal. between BRCA1 gene methylation and improved survival Therefore, it was very likely that the lack of BRCA1 rate in patients with advanced stage (stage III-IV) (Bai protein expression was because of the presence of down et al., 2014). The inconsistency of these results may be regulation due to the methylation process. due to the varying population and different environtment It was said that ovarian cancers caused by the germline condition that were involved in the studies. mutation had frequency of 10-15% while the rest were the In the present study demonstrated that there was sporadic very likely due to the role of lost BRCA1 gene no significant association between methylation status because of the presence of methylation on the promoter of BRCA1 with survival of the patients. Even though, to be inactive. Thrall et al (2006) found 84% of cases of the data from this studied show methylation status of EOC were still expressed positives, statistically there BRCA1 at EOC was high (89.1%) in this population. By was a significant correlation (p <0.001) between the acquiring a fact that the proportion of methylation BRCA1 expression of BRCA1 in the tumor tissue and the tumor gene was high, the population of patients with epithelial stage, where the expression seemed in all stage I and ovarian cancer in Indonesia especially in Central Java stage II, completely negative in 16% of tumor in stage would be used as the basic rationale for the presence of a III, or 65% of tumors in stage III had minimal expression strong indication of the use of gene targeted therapy with until completely negative (0-<10%) compared with 22% inhibitors of DNA methylation such as DMT inhibitors in stage I and 14% in stage II. So, overall there was a (azacitidine) and HDAC inhibitors in the therapy of significant decrease in BRCA1 protein expression with ovarian cancer in the future in Indonesia when the use of increasing stages of epithelial ovarian cancer (Bast et this therapy is already approved by authorized institutions al., 2000). The data studied found that early stage (stage for being used in ovarian cancer. As it was known that I) compare to late stage (stage II - IV) no significance the use of azacitidine (AZA), an inhibitor of DNA different between the two group p=0.20. Expression of methylation, has been approved by FDA since May 2004 BRCA1 protein in tumor cells seem to have advatages to for the therapy of myelodysplastic syndrome (MDS), a the survival of EOC patients independently or adjusted precancerous condition of acute myeloid leukemia. This even though it was not statistically significance, where was an example of drug pioneer in which the target was with expression were positive HR=0.83 (p=0.70) and HR “epigenetic gene silencing”, a mechanism that occurred 0.39 (p=0.14) respectively. in cancer cells to inhibit the expression of genes where The data of this studied also found that menarche, the effect inhibited a malignancy phenotype (Issa et al., CA125 level, clinical stage, residual tumor during surgery 2005). Because ovarian cancer was a highly heterogeneous were independently as prognostic factors for survival of cancer in which it has many types of the histopathology EOC with p=0.03, p=0.01, p=0.01, p=0.04 respectively. and malignancy with a progression ranging from slow to However, after adjusted for other variables they were not very fast and the patient could die quickly. Thus, using significant as a prognostic factors (p>0.05). As far we the molecular profiles of ovarian cancer to reduce the researched no study mentioned menarche corellated with heterogeneity will be important for the patient selection prognosis of EOC. Age of menarche usually corellated in determining the therapy. It also happened in the MDS with risk of ovarian cancer pathogenesis. However, in this (Myelo Dysplastic Syndrome), a heterogeneous disease study menarche was independently as prognostic factor group in which the outcomes differed greatly depending on for survival. The levels of CA125 in this studied was the profile of clinical pathology from chronic and slow to classified into two groups with the cut off point of 70 U/ aggressive with a short survival. The results of this study ml due to the level of CA125 was still normal is <35U/ml. got that the high frequency of BRCA1 methylation was a There are several conditions of non neoplastic conditions new fact for us in Indonesia. Hypermethylation in BRCA1 CA125 level are increase, nearly 6% of women without gene in the epithelial ovarian cancer had largely various ovarian cancer had CA125 levels more than 35mlU/ml levels of variation and it might likely be very different (Bast et al., 2000; Urban, 2003), then in this study of in each country because of differences in environmental malignancy condition espescially EOC the level of CA125 factors, nutrition, chemical exposures and polution factors, twice of normal limit used as the cut of high level. The as these greatly affected the occurrence of promoter data showed that CA125 was as independent prognostic methylation of suppressor genes and other genes. factor of the survival of EOC of all stage with HR 4.42 Without the BRCA1 protein expressed in ovarian and p=0.01, after adjusted to other clinicopathological tumors it showed the presence of dysfunction of BRCA1 factors HR 3.01 (95% CI 0.74-12.18) and p=0.12. Other gene due to either genetic or epigenetic changes that could studied found that CA125 were as prognostic factor for lead to the occurrence of the transformation of cells into EOC stage I (Nagele et al., 1995; Paramasivam et al., Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9483 Heru Pradjatmo et al 5-years survival estimates at 78.8% vs 58.8% and 35.3% 2005; Petri et al., 2006). A review of 15 studies showed respectively (p<0.001), even after adjusted for race, stage, that CA125 levels increased in 50% of patients with stage grade and surgical treatment the difference between the I of the disease, 90% in stage II, 92% in stage III, 94% age group persist (Chan et al,. 2006). In this studied in the stage IV of disease (Jacob and Bast, 1989). It was show that older women ≥ 40 years had lower survival also reported a positive correlation between the increased than younger women <40 years, but statistically not serum levels of CA125 and the expression levels of CA125 significant with HR 1.54 (p=0.43) and after adjusted for in the epithelial ovarian cancer tissues. However, in the other variables HR 0.93 (p=0.83). The present study show epithelial ovarian cancer of serous type the expression that parity and more than one child had less chances of was significantly more positive than the other types of survival compared to patients with no or one child as seen epithelial ovarian cancer. It was also found significantly independently as well as adjusted analysis HR more than shorter survival in the patients with ovarian cancer of stage 100.0 100.0 twice though statistically not significant, Other factor in III and IV without the expression of CA125 compared with 6.3 6.3 12.8 12.8 10.1 10.1 20.3 20.3 this study were histopathological type, menopauze status the patient of ovarian cancer in stage III and IV with the 10.3 10.3 apparently were not influential to the survival of EOC expression of CA125 in the tumor (Hogdall et al., 2007). 25.0 25.0 30.0 30.0 75.0 75.0 patients independently as well as on adjusted analysis. Clinical stage is the most important prognostic factor of In conclussions, methylation status and expression of the cancer. Relative five years survival of ovarian cancer 75.0 75.0 46.8 46.8 56.3 56.3 51.1 51.1 BRCA1 were not to be prognosticator of EOC patients in all stages was 53%, for stage III and IV were 31 % 51.7 51.7 54.2 54.2 and they were not correlated to clinicopathological50.0 50.0 and for stage I and II were 95% (Landis et al., 1998). The 31.3 31.3 30.0 30.0 characteristics of the patients such as; age, menarche, present study found that clinical stage independently as parity, menopauze status, BMI, CA125 level, clinical prognostic factor of EOC in which the late stage (stage stage, histopathological type, grade, residual tumor. The II-IV) had HR 3.79 (p=0.01) compare with early stage 25.0 25.0 study found that menarche, CA125 level, clinical stage eventhough in the adjusted analysis had HR 3.15 (p=0.18). 38.0 38.0 33.1 33.1 31.3 31.3 31.3 31.3 30.0 30.0 27.6 27.6 and residual tumor were independently as prognosticator Residual tumor was demonstrated to be a prognostic 25.0 25.0 23.7 23.7 of EOC patients, eventhough in the multivariable analysis factor to determine survival in patients with EOC stage 0 0 statistically were not significant its due to the power of IV (Bristow et al., 2002; Winter et al., 2008). Elstrand et the study was low. al (2012) reported that among patients with EOC stage IV who underwent at least one surgical prosedure residual disease was an important prognostasicator for overall References survival. While in this study found residual tumor was Bai X, Fu, Y, Xue, H, et al (2014). BRCA1 promoter methylation as prognostic factor independently for survival of EOC in sporadic epithelal ovarian carsinoma: Association with patients. Grade of differentiation mainly in the early stages low expression of BRCA1, improved survival and co- of the disease is an important prognostic factor that affects expression of DNA methyltransferases. Oncol Letters, 7, treatment planning (Morgan et al., 2011 cited Hoffman 1088-96. et al., 2012), This study found that well differentiated, Bast Jr RC, Ravdin P, Hayes DF, et al (2000). Update of moderate differentiated and poor differentiated were recommendations for the use of tumor markers in breast clinically significant and likely to be a prognostic factor and colorectal cancer: clinical practice guidelines of the of EOC which the Hazard Ratio (HR) were 1.22 and American society of clinical oncology. J Clin Oncol, 19, 3.83 respectively even though statistically they were 1865-78. Bristow RE, Tomacruz RS, Armstrong DK, Trimble EL, Monntz not significant. Then menarche, CA125 level, clinical FJl (2002). Suvival effect of maximal cytoreductive surgery stage, residual tumor during surgery were independently for advanced ovarian carcinoma during the platinum era: a prognostic factors for survival of EOC, however, in the meta-analysis. J Clin Oncol, 20, 1248–59. multivariable analysis showed to be insignificant as Chan JK, Urban R, Cheung MK, et al (2006). Ovarian cancer in prognostic factors of survival, its seems that those results younger vs older women: a population-based analysis. Br J are due to the power of study being low. Cancer, 95, 1314–20. Age of EOC patients most commonly occured in Chiang JW, Karlan BY, Cass I, Baldwin RL (2006). BRCA1 age ≥40 years 73.9% and 26.1% aged <40 years, others promoter methylation predicts adverse ovarian cancer studied reported in Lahore India that the median age of prognosis. Gynecol Oncol, 101, 403-10. Duska LR, Chang YC, Flynn CE, et al (1999). Epithelial EOC was 47 years old (Saeed and Akram, 2012) and in ovarian carcinoma in the reproductive age group. Cancer, Sweden median age of ovarian cancer was 75 years old 85, 2623-9. (Segelman et al., 2010). The studies on the prognostic Elstrand MB, Sandstad B, Oksefjell H, Davidson B, Trope CG implication of age in ovarian cancer are inconclusive. (2012). Prognostic significance of residual tumor in patients Although most reports have shown that younger women with epithelial ovarian carcinoma stage IV in a 20 years with ovarian cancer have an improved outcome compared perspective. Acta Obstet Gynecol Scan, 91, 308-17. to older women due to they have lower stage and well Esteller M, Corn PG, Baylin, SB (2001). Herman JG. A gene differentiation tumors (Rodriguez et al., 1994: Chan et hypermethylation profile of human cancer. Cancer Res, al., 2006), others researchers have found that age was 61, 3225-9. Esteller M, Sanchez-Cespedes M, Rosell R, et al (1999). not an independent prognostic factor (Massi et al., 1996: Detection of aberrant promoter hypermethylation of tumor Duska et al., 1999). However, population-based studied suppressor genes in serum DNA from non-small cell lung found that across all stages of EOC very young women cancer patients. Cancer Res, 59, 67-70. (<30 years) had significant survival advantage over Hahn WC, Weinberg RA (2002). Rules for making human tumor young (30-60 years) and older (>60 years) group with 9484 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 Newly diagnosed without treatment Newly diagnosed without treatment Newly diagnosed with treatment Newly diagnosed with treatment Persistence or recurrence Persistence or recurrence R Remi emissi ssion on None None Chemotherapy Chemotherapy Radiotherapy Radiotherapy Concurr Concurrent ent chemor chemoradi adiati ation on DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9479 cells. N Engl J Med, 347, 1593-603. expression and tumor grade, stage or overall survival in Heo I, Joo C, Cho J (2008). Lin28 mediate terminal undylation platinum-tested epithelial ovarian cancer patients. Asian of let-7 precursur microRNA. Mol Cell, 32, 276-8. Pac J Cancer Prev, 15, 4275-9. Hilton JL, Geisler JP, Rathe JA, et al (2002). Inactivation of Thrall M, Gallion, HH, Kryscioz R, et al (2006). BRCA1 BRCA1 and BRCA2 in Ovarian Cancer. J Natl Cancer expression in a large series of sporadic ovarian carcinomas: Inst, 94, 1396-406. a Gynecologic Oncology Group study. Int J Gynecol Cancer, Hoffman BL, Schorge JO, Schaffer JL, et al (2012). Epithelial 16, 166-71. nd Ovarian Cancer. In Williams Gynecology, 2 Ed, Mc Graw Urban N (2003). Specific keynote: Ovarian cancer risk Hill Co, 853-78. assessment and the potential for early detection. Gynecol Hogdall EV, Christensen L, Kjaer SK, et al (2007). CA 125 Oncol, 88, 75-9. expression pattern, prognosis and correlation with serum Winter III WE, Maxwell GL, Tian C, et al (2008). Tumor residual CA 125 in ovarian tumor patients from the Danish “Malova” after surgical cytoreduction in prediction of clinical outcome ovarian cancer study. Gynecol Oncol, 104, 508-17. in stage IV epithelial ovarian cancer: a gynecologic oncology Issa J-P J, Kantarjian HM, Kirkpatrick P (2005). Fresh from the group study. J Clin Oncol, 26, 83-9. pipeline azacitidine. Nat Rev, 4, 275-6. Yang D, Khan S, Sun Y, et al (2011). Association of BRCA1 and Jacobs I, Bast RC (1989). The CA125 tumour-associated antigen: BRCA2 mutations with survival, chemotherapy sensitivity, a review of the literature. Human Reprod, 4, 1-12. and gene mutator phenotype in patients with ovarian cancer. Kawahara N, Nishikura K (2009). Regulation of the miRNA JAMA, 306, 1557-65. function by RNA editing. Tanpakushitsu Kakusan Koso, 54, 1133-40. Laird PW (2003). The power and the promise of DNA methylation markers. Nat Rev Cancer, 3, 253-66. Lan VTT, Thuan TB, Thu DM, et al (2013). Methylation profile of BRCA1, RASSF1A and ER in Vietnamese women with Ovarian Massi D, Susini T, Savino L, et al (1996). Epithelial ovarian tumors in the reproductive age group: age is not an independent prognostic factor. Cancer, 77, 1131-36. Cancer. Asian Pac J Cancer Prev, 14, 7713-8. Landis SH, Murray T, Bolden S, Wingo P (1998). Cancer statistics. CA Cancer J Clin, 48, 6-30. Lesnock JL, Darcy KM, Tian C, et al (2013). BRCA1 expression and improved survival in ovarian cancer patients treated with intaperitoneal cisplatin and paclitaxel: a gynecologic oncology group study. Br J Cancer, 108, 1231-37. Li C, Feng Y, Coukos G, Zhang L (2011). MicroRNAs in epithelial ovarian cancer. In William CS Cho Editor. in MicroRNAs in Cancer Translational Research. Springer Science, 309-42 Montavon C, Gloss BS, Warton K, et al (2012). Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer. Gynecol Oncol, 124, 582-88. Munkarah AR, Hallum AV, Morris M, et al (1997). Prognostic significance of residual disease in patients with stage IV epithelial ovarian cancer. Gynecol Oncol, 64, 13-7. Nagele F, Petru E, Medl M, et al (1995). Preoperative CA 125: an independent prognostic factor in patients with stage I epithelial ovarian cancer. Obstet Gynecol, 86, 259-64. Paramasivam S, Tripcony L, Crandon A, et al (2005). Prognostic importance of preoperative CA-125 in international federation of gynecology and obstetrics stage I epithelial ovarian ancer: an Australian multicenter study. J Clin Oncol, 23, 1-5. Petri AL, Hogdall EV, Christensen IBJ, et al (2006). Preoprative CA125 as a prognostic factor in stage I epithelial ovarian cancer. APMIS, 114, 358-63. Rodriguez M, Nguyen HN, Averette HE, et al (1994). National survey of ovarian carcinoma XII. Epithelial ovarian malignancies in women less than or equal to 25 years of age. Cancer, 73, 1245-50. Saeed S, Akram, M (2012). Epithelial ovarian cancer: epidemiology and clinicopathological features. Profesional Med J, 19, 1040-5. Segelman J, Floter-Radestad A, Hellborg H. Sjovall A, Martling A (2010). Epidemiology and prognosis of ovarian metastases in colorectal cancer. Br J Surg, 97, 1704-9. Shawky AEA, El-Hafez AA, El-Tantawy, Hamdy R (2014). No association between BRCA1 immunohistochemical Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9485 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Asian Pacific Journal of Cancer Prevention Unpaywall

Methylation Status and Immunohistochemistry of BRCA1 in Epithelial Ovarian Cancer

Asian Pacific Journal of Cancer PreventionNov 28, 2014

Loading next page...
 
/lp/unpaywall/methylation-status-and-immunohistochemistry-of-brca1-in-epithelial-q7X6wK90tL

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
1513-7368
DOI
10.7314/apjcp.2014.15.21.9479
Publisher site
See Article on Publisher Site

Abstract

Background: Cancer initiation and progression are controlled by genetic and epigenetic events. One epigenetic process which is widely known is DNA methylation, a cause of gene silencing. If a gene is silenced the protein which it encodes will not expressed. Objectives: 1. Identify the methylation status of BRCA1 in patients with epithelial ovarian cancer (EOC)and assess BRCA1 protein expression in tumor tissue. 2. Examine whether BRCA1 gene methylation and BRCA1 protein are associated with survival of epithelial ovarian cancer patients. Methods: The study design was a prospective-cohort study, conducted at Sardjito hospital, Yogyakarta, Indonesia. Results: A total of 69 cases were analyzed in this study. The data showed that the methylation status of BRCA1 in EOC was positive in 89.9%, with clear protein expression of BRCA1 in 31.9%. Methylation status and expression of BRCA1 were not prognosticators of EOC patients. Menarche, CA125 level, clinical stage and residual tumor were independent factors for prognosis. Keywords: EOC - methylation - BRCA1 - prognosis factors - survival Asian Pac J Cancer Prev, 15 (21), 9479-9485 of expression is said to be regulated by the transcriptional Introduction implications of methylation of CpG nucleotide promoter. Cancer develops through a multistep process in which Esteller et al. research in the methylation of some the genomes of the new cancer cells undergo mutations in suppressor genes in various types of cancer showed that some groups of the specific genes such as protooncogen, the methylation of BRCA1 in sporadic ovarian cancer tumor suppressor genes and other genes that directly was 19% (11/58) (Esteller et al., 2001), in Vietnam found or indirectly control a cell proliferation. Cancer cells the methylation status of BRCA1 in women with ovarian also have a genetic instability that allows cells to get cancer patients was 11/59 (18.6%) (Lan et al., 2013). other changes all the time. One of the characteristics of The examination of the profile of BRCA1 methylation in malignant tumors is its heterogeneity. Cancers are caused ovarian cancer is very crucial to understand the molecular by the accumulation of mutations of several categories pathology of ovarian cancer, which in the end it will be of genes, the initiation and the progression of cancer are very useful in the clinical management, due to methylation controlled by the genetic and epigenetic events. is a reversible procces. The most known epigenetic process is a DNA As it was known that the use of azacitidine (AZA), methylation. Methylation is adding four atoms on cytosine, an inhibitor of DNA methylation, has been approved by one of four DNA nucleotides. This additional atom blocks the FDA for the treatment of myelodysplastic syndrome the protein that transcribes genes. DNA methylation is (MDS), a precancerous condition of acute myeloid an epigenetic mechanism that becomes very clear in the leukemia (Issa et al., 2005). Different combinations of recent years that there is a synergy between the genetic genetic changes are found in genomes of more than 100 and epigenetic changes. different types of human cancers. Thus, each cancer The expression of BRCA1 protein is ubiquitous in may be unique and the spectrum of genetic changes that humans, located in the nucleus, whereas the highest levels initiates the incidence of cancer may have many variations. are obtained in the ovarian, testis and thymus. It is a There is no single rule that underlies the occurrence of tumor suppressor and the reduced expression is associated all cancer and cancer is a phenomenology of infinite with the transformation procedures and the etiology of complexity. Hahn and Weinberg made an alternative view sporadic breast cancer and ovarian cancer. The reduction that the pathogenesis of cancer in humans was governed 1 2 3 Department of Obstetric and Gynecology, Department of Histology and Biomolecular, Department of Pathology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia *For correspondence: pradjatmo@yahoo.com; herupradjatmo@ gmail.com Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9479 Heru Pradjatmo et al by a number of genetic and biochemical rules applied in (Figure 2). Tumor were catagorised as having aberrant most types of human cancers (Hahn and Weinberg, 2002). BRCA1 expression for very low to no staining (<10%); This study was conducted to determine the methylation 0 or 1 score) and normal BRCA1 expression for >10% status of BRCA1 gene in epithelial ovarian cancer and the BRCA1 staining (2-4 score). (Thrall et al., 2006; Lesnock expression of BRCA1 protein in the tumor whether the et al., 2013). proportion of expression appropriate with the proportion of methylation. Does the proportion of methylation Results status of BRCA1 genes and the proportion of expression of BRCA1 protein influence survival of patients with The cases of ovarian tumors had been collected epithelial ovarian cancer? found 69 cases were malignant epithelial ovarian cancer, which were then used for the study of this manuscript. The patient’s survival was followed up and the longest Materials and Methods follow-up was 54 months and the shortest was 12 months. The study design was a prospective cohort, it was Several clinicopathological characteristic which were conducted at Sardjito Hospital, Yogyakarta, Indonesia in associated with risk factors of ovarian tumors such as which patients with ovarian tumors when the results of patients age, menarche, parity, menopausal age, nutritional frozen section showed malignant ovarian neoplasm, then status (BMI), the CA125 level before operation, residual some of the tumors were stored at -70 C, then the tissues tumor during operation, histopathological type and grade were used as the sample of the study to examine the of differentiation were recorded and analyzed. Those methylation status by methylation specific PCR (MSP). clinicopathological characteristic of the patients were While the other tumor tissues were examined at pathology clasified in two or three group. Table 1 showed the results anatomy to determine the type of histopathology, the of the methylation of BRCA1 genes in the tissue of EOC degree of differentiation and examined the expression of was 62/69 cases (89.9%) without methylation 7/69 cases BRCA1 in the epithelial ovarian cancer. (10.1%). IHC staining of BRCA1 normal (positive) was 22/69 cases (31.9%) and aberrant (negative) was 47/69 BRCA1 methylation status examination (68.1%). The agreement between methylation status of DNA tissue extraction was performed with QIAamp the BRCA1 gene and the expression of BRCA1 protein in DNA Mini Kit (Qiagen, Germany) as appropriate with the the tumor was -0.019 its mean that there is no agreement manual kit. The conversian procedure of DNA bisulphite between methylation and expression of BRCA1. This used protocol of MethylEasy Xceed Rapid DNA Bisulphite result can be proved either in the electrophoresis seen modification Kit. For PCR reaction, 1μl DNA which was that although some cancer cells the BRCA1 gene was converted with sodium bisulphide add 18μl PCR mix found methylated some cells found unmethylated either which contain 15PCR buffer, optimum concentration and these cells still express BRCA1 protein (Figure. Mg2+ for each primer, 0.4μl primer and 0.1μl Taq 1). Distribution of methylation status and IHC BRCA1 polymerase. The primer length which were used for staining correlated with clinicopathologial factors were BRCA1_M75 bp, BRCA1_U76 bp, with a nukleotid base not statistically significant different with p>0.05 (Table sequence as follows: BRCA1_MF: 5’-TCG TGG TAA CGG AAA AGC GC-3’, BRCA1_MR: 5’-AAA TCT CAA CGA ACT CAC GCC G-3’, BRCA1_UF: 5’-TTG GTT TTT GTG GTA ATG GAA AAG TGT-3’, BRCA1_UR : 5’-AAA AAA TCT CAA CAA ACT CAC ACC A-3’, Immunohistochemistry (IHC) for BRCA1 expression and scoring. Paraffin block section 3-5µm thick of ovarian tumor tissue on glass slide was examined for IHC. The IHC assay for BRCA1 expression used the MS110 clone monoclonal antibody (Biocare Medical, LLC, 4040, Pike Lane Concord, CA 94520, LA, USA) that reacts with N-terminal portion of the BRCA1 protein. The possitive BRCA1 expression of breast cancer tissue were used for Figure 2. IHC Examination of BRCA1: (B1-P2) Show positive control, and negative control used the same tissue Positive 2 Staining, (B1-P3) Show Positive 3, (B1-P4) and the staining without BRCA1 antibody. The percentage Show Positive 4 of staining was determined by an independent pathologist Table 1. Kappa Statistic of the Methylation Status of he was blinded to the identity and clinical outcome of the BRCA1 Gene and Expression of BRCA1 Protein samples. The ascribes score was based on the number of cells with nuclear staining. The score was classified Methylation Status I H C p value Kappa as Thrall et al. used: slides was score as 0 if there was Aberrant<10%(-) Normal≥10%(+) no staining, 1 if there was scattered staining (<10%). 2 Methylated (+) 42 (60.9%) 20 (29.0%) 0.84 -0.019 if 10-50% of the cells were stained, 3 if 50-90% of cells Unmethylated (-) 5 (7.2%) 2 (2.9%) Total 47 (68.1%) 22 (31.9%) were stained and 4 if nearly all cells (>90%) were stained 9480 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 100.0 6.3 12.8 10.1 20.3 10.3 25.0 30.0 75.0 75.0 46.8 56.3 51.1 51.7 54.2 50.0 31.3 30.0 25.0 38.0 33.1 31.3 31.3 30.0 27.6 25.0 23.7 Newly diagnosed without treatment Newly diagnosed with treatment Persistence or recurrence Remission None Chemotherapy Radiotherapy Concurrent chemoradiation DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9479 2). In the survival analysis by bivariable analysis found was 19% (11/58) (Esteller et al., 2001), 10% of 49 patients that menarche, CA125 level, stage of disease and residual ovarian cancer (Rathi et al., 2001), 31% (Wang et al., tumor during operation were significantly influence the 2004), Lan et al (2013) in Vietnamese women suffer from survival of the EOC patients with HR 2.55 (p=0.03), 4.42 ovarian cancer was 18,6%. It was considered whether (p=0.01), 3.77 (p=0.01), 2,4 (p=0.04) respectively, but in there was a possibility of technical error, for checking multivariable analysis there were no factors influence the and proving the absence of errors in the examination of survival of the patients, even methylation status of BRCA1 this research, it had carried out an internal validation with gene and expression of BRCA1 protein as well (Table 3). positive and negative controls using positive and negative From the results of the present research, its were controls on each of the electrophoresis examination found that the methylation levels of BRCA1 was 89.9% (Figure 1). We also conducted an external validation in the compared with the results of other studies elsewhere was way of re-examining randomly samples of the study. We high. Methylation of BRCA1 in sporadic ovarian cancer also did the examination to some samples of other normal Table 2. Characteristic of Epithelial Ovarian Cancer and Comparibility of the Methylation Status of BRCA1 Gene and IHC Result of BRCA1 Protein Variable Methylation Status BRCA1 IHC BRCA1 protein Methylated (%) Unmethylated (%) p value Aberrant/- (%) Normal/+ (%) p value Age of patients: < 40 years 17 (24.6) 1 (1.4) 0.45 12 (17.4) 6 (8.7) 0.87 ≥ 40 years 45 (65.2) 6 (8.7) 35 (50.7) 16 (23.2) Age of menarche < 15 years 42 (60.9) 5 (7.2) 0.84 32 (46.4) 15 (21.7) 0.99 ≥ 15 years 20 (29.0) 2 (2.9) 15 (21.7) 7 (10.1) Parity: < 2 30 (43.5) 3 (4.3) 0.78 23 (33.3) 10 (14.5) 0.78 ≥ 2 32 (46.4) 4 (5.8) 24 (34.8) 12 (17.4) Menopauze: Not menopauze 33 (47.8) 2 (2.9) 0.21 22 (31.9) 13 (18.8) 0.34 Menopauze 29 (42.0) 5 (7.2) 25 (36.2) 9 (13.0) B M I: < 25 kg/m 47 (68.1) 5 (7.2) 0.79 35 (50.7) 17 (24.6) 0.8 ≥ 25 kg/m 15 (21.7) 2 (2.9) 12 (17.4) 5 (7.2) CA 125 level ≤ 70 IU/ml 21 (30.4) 3 (4.3) 0.63 17 (24.6) 7 (10.1) 0.72 > 70 IU/ml 41 (59.4) 4 (5.8) 30 (43.5) 15 (21.7) Clinical Stage: Early Stage 20 (29.0) 3 (4.3) 0.57 18 (26.1) 5 (7.2) 0.2 Late Stage 42 (60.9) 4 (5.8) 29 (42.0) 17 (24.6) Histophatological type: Serous 18 (26.1) 1 (1.4) 0.5 10 (14.5) 9 (13.0) 0.23 Musinous Ca. 30 (43.5) 5 (7.2) 26 (37.7) 9 (13.0) Others 14 (20.3) 1 (1.4) 11 (15.8) 4 (5.8) Grade of Differentiation Well differentiated 30 (43.5) 5 (7.2) 0.3 24 (34.8) 11 (15.9) 0.69 Moderate 15 (21.7) 0 (0.0) 9 (13.0) 6 (8.7) Poor differentiated 17 (24.6) 2 (2.9) 14 (20.3) 5 (7.2) Residual tumor: Optimal operation 36 (52.2) 4 (5.8) 0.96 29 (42.0) 11 (15.9) 0.35 Not optimal 26 (37.7) 3 (4.3) 18 (26.1) 11 (15.9) Table 3. Survival Analysis for Bivariable and Multivariable Analysis with Cox’s Regression Variable Bivariable analysis Multivariable analysis HR (95% CI) p-value HR (95% CI) p-value Age of patients: < 40 years Ref Ref. ≥ 40 years 1.54 (0.52-4.59) 0.43 1.38 (0.35-5.33) 0.63 Menarche < 15 years Ref. Ref. ≥ 15 years 2.55 (1.07-6.03) 0.03 2.64 (0.93-7.48) 0.06 Parity: < 2 Ref. Ref. ≥ 2 2.00 (0.80-4.96) 0.13 2.28 (0.91-7.79) 0.22 Menopauze: Not menopauze Ref. Ref. Menopauze 1.07 (0.45-2.53) 0.86 0.74 (0.26-2.15) 0.59 B M I: < 25 kg/m Ref. Ref. ≥ 25 kg/m 0.67 (0.22-2.01) 0.48 0.41 (0.11-1.49) 0.17 CA 125 ≤ 70 IU/ml Ref. Ref. >70 IU/ml 4.42 (1.29-15.10) 0.01 3.01 (0.74-12.19) 0.12 Clinical Stage: Early Stage Ref. Ref. Late Stage 3.77 (1.11-128.6) 0.01 3.15 (0.58-17.15) 0.18 Histophatological type: Serous 0.84 (0.25-2.78) 0.77 0.78 (0.20-2.98) 0.71 Musinous Ca. 0.82 (0.30-2.27) 0.71 1.75 (0.35-7.99) 0.46 Others (Clear cell+ Ref. Ref. Endometrioid) Grade of Differentiation Well differentiated Ref. Ref. Moderate 1.98 (0.66-5.92) 0.22 2.25 (0.52-9.63) 0.27 Poor differentiated 2.29 (0.82-6.36) 0.11 3.83 (0.71-20.69) 0.11 Residual tumor: Optimal operation Ref. Ref. Not optimal 2.45 (1.02-5.89) 0.04 0.81 (0.24-2.73) 0.74 Methylation status BRCA1 Methylated Ref. Ref. Unmethylated 1.02 (0.23-4.41) 0.97 2.11 (0.38-11.71) 0.39 IHC BRCA1 Protein Aberrant Ref. Ref. Normal 0.83 (0.32-2.15) 0.7 0.39 (0.11-1.36) 0.14 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9481 Heru Pradjatmo et al and would pause the mRNA translation process if the protein that was resulted from the translation of gene was no longer needed by binding to the mRNA so mRNA would be silent or undergo degradation. The protein expression of BRCA1 and others were regulated by various molecular devices within the cell. The setting was at the level of Figure 1. The Result Electrophoresis of BRCA1 transcription, translation and post translation. In the result Methylation of Some Sample of the Epithelial Ovarian of the latest research, it was reported that the causes and Cancer the early incidence of EOC were governed by a group of genes including microRNA genes, which could be cells, namely leukocytes cells of the normal people and modulated at four levels, namely the level of genomic, the result were same. The above validation checks proved transcriptional regulation, post-translation modification that the possibility of technical errors in the examination and regulatory processes of miRNA (Li et al., 2011). The could be ruled out or the results of the examination in the position of miRNA genes had been mapped to be scattered study were reliable. The results of this study might show in chromosome 1 to 22, and the X and Y sex chromosomes. characteristics of ovarian cancer patients in Indonesia, Apparently, around 16% miRNA was regulated at the particularly in areas of southern Central Java. level of post-translational. Changes in the pair of A-T base As it was known that the process of carcinogenesis became the pair of A-I base (inosine that acts on RNA was caused by various factors as described in the editing) caused a conversion of miRNA biogenesis, the literature. Likewise, promoter methylations of suppressor changes of miRNA product and the targeting changes of genes could be caused by various factors, namely, specific miRNA (Kawahara et al., 2009). environment, nutrition, chemicals, infections, and so on. It had also been reported that lin28, a repressor let7 For Indonesians, these factors were different to those in miRNA when a change of the base pairs occurred, caused Western countries or the other more developed countries. a Dicer enzyme that did not function and the degradation So, it would greatly affect the methylation status of the of pre-miRNA; consequently, the target protein of mRNA genes in these individuals in our population. had over-expression (Heo et al., 2008). This might explain why there was a difference of expression profile of BRCA1 and they had methylations, but the expression of the Discussion proteins in the tissue was high. As mentioned by Hilton et The data showed that frequency methylation status al. that the universal inactivation of BRCA1 had multiple of the patients were 89.9% and the expression of mechanisms that were not only because of mutation and BRCA1 were 31.9%. If all of the tumor cells underwent hypermethylation but there were still other factors or methylations in the BRCA1 genes, certainly, the tumor mechanisms (Hilton et al., 2002). These mechanisms cells would not express the BRCA1 protein but on the could include: first, the mutations of BRCA1 promoter, examination it obtained 22/69 cases (31.9%) that still so failing to identify the correct CpG island of BRCA1 showed the expression of BRCA1 protein and 47/69 promoter; second, the loss of the function of gene products case (68.1%) showed no expression of BRCA1. It meant required for the transcription of BRCA1, as well as no loss that not all ovarian tumor cells in a patient who on the of heterozygosity (LOH) as the third cause (Hilton et al., examination had positive methylation status had cells 2002). Similarly, not all cancer cells on these examination without methylation either. This was confirmed on the showed the negative expression of BRCA1 although the electrophoresis examination of any sample even it gained results of the examination of the methylation of BRCA1 a band that showed the positive methylation genes but on gene were positive. the other hand there was also a band which showed the There were no correlation among BRCA1 methylation presence of the genes not methylated (Figure 1). there and BRCA1 expression with several clinocopathological was almost no agreement between methylation status of variables such as; age, menarche, parity, menopauze BRCA1 genes and expression of BRCA1 protein in EOC status, BMI, CA125 level, clinical stage of disease, of this study as the result of Kappa statistic=0.01 (Table histopathological type, grade and residual tumor during 1). The methylation of BRCA1 promoter is important in surgery as seen in the distribution of methylation status of silencing BRCA1 in sporadic EOC. The loss of expression BRCA1 gene and expression of BRCA1 protein in EOC of tumor suppressor genes such as BRCA1 is known tissue statistically were not different with Chi square test occur through biallelic inactivation. The sugestion is that p>0.05 (Table 2). Lan et al (2013) found that association somatic mutation of BRCA1 is rare in sporadic EOC, thus, between methylation status of BRCA1, RASSF1A and the loss of BRCA1 expression is considered to be due to ER genes with the clinical and pathological parameters of a combination of allelic loss and methylation (Esteller women suffering from ovarian cancer in Vietnam. Other et al., 2001). Other authors suggested that the silent or studied find expression of BRCA1 protein in EOC was inactive BRCA1 gene on the tumor development were 40% and no association between BRCA1 expression with not only because of the epigenetic or genetic processes tumor grade, stage and overall survival of EOC patients (gene mutation), but also because of a variety of other (Shawky et al., 2014). As already were mentioned that mechanisms that might occur together, as the role of micro methylation of promoter gene or aberrant methylation RNA (miRNA) where miRNA was an RNA with a short was influenced by several environmental agents. Aberrant nucleotide chain length less than 22 bases of nucleotide DNA methylation currently is recognized as a common 9482 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9479 molecular abnormality in cancer and its become potential cancer. BRCA1 was a tumor suppressor that reduced the molecular marker for diagnosis, prognosis and treatment expression associated with the transformation process and (Laird, 2003). Previous studies regarding the association the etiology of breast cancer and sporadic ovarian cancers. between BRCA1 gene methylation and patient survival Reduced expression of BRCA1 was quite possible related remain controversial. Yang et al. and Montovan et al. to the presence of gene methylation. The present study of indicated that BRCA1 methylation was not assosiated with the examination of BRCA1 gene methylation obtained patient survival (Yang et al., 2011; Montovan et al., 2012). as much as 89.9%. Thus, 68.1% of epithelial ovarian Chiang et al. demonstrated survival disavantage in patients tumors without the BRCA1 protein expressed was very whose neoplasm were methylated at BRCA1 (Chiang likely because of methylation while the remaining 31.9% et al., 2006). However, reported significant association expressed with the positive levels (≥10%) or normal. between BRCA1 gene methylation and improved survival Therefore, it was very likely that the lack of BRCA1 rate in patients with advanced stage (stage III-IV) (Bai protein expression was because of the presence of down et al., 2014). The inconsistency of these results may be regulation due to the methylation process. due to the varying population and different environtment It was said that ovarian cancers caused by the germline condition that were involved in the studies. mutation had frequency of 10-15% while the rest were the In the present study demonstrated that there was sporadic very likely due to the role of lost BRCA1 gene no significant association between methylation status because of the presence of methylation on the promoter of BRCA1 with survival of the patients. Even though, to be inactive. Thrall et al (2006) found 84% of cases of the data from this studied show methylation status of EOC were still expressed positives, statistically there BRCA1 at EOC was high (89.1%) in this population. By was a significant correlation (p <0.001) between the acquiring a fact that the proportion of methylation BRCA1 expression of BRCA1 in the tumor tissue and the tumor gene was high, the population of patients with epithelial stage, where the expression seemed in all stage I and ovarian cancer in Indonesia especially in Central Java stage II, completely negative in 16% of tumor in stage would be used as the basic rationale for the presence of a III, or 65% of tumors in stage III had minimal expression strong indication of the use of gene targeted therapy with until completely negative (0-<10%) compared with 22% inhibitors of DNA methylation such as DMT inhibitors in stage I and 14% in stage II. So, overall there was a (azacitidine) and HDAC inhibitors in the therapy of significant decrease in BRCA1 protein expression with ovarian cancer in the future in Indonesia when the use of increasing stages of epithelial ovarian cancer (Bast et this therapy is already approved by authorized institutions al., 2000). The data studied found that early stage (stage for being used in ovarian cancer. As it was known that I) compare to late stage (stage II - IV) no significance the use of azacitidine (AZA), an inhibitor of DNA different between the two group p=0.20. Expression of methylation, has been approved by FDA since May 2004 BRCA1 protein in tumor cells seem to have advatages to for the therapy of myelodysplastic syndrome (MDS), a the survival of EOC patients independently or adjusted precancerous condition of acute myeloid leukemia. This even though it was not statistically significance, where was an example of drug pioneer in which the target was with expression were positive HR=0.83 (p=0.70) and HR “epigenetic gene silencing”, a mechanism that occurred 0.39 (p=0.14) respectively. in cancer cells to inhibit the expression of genes where The data of this studied also found that menarche, the effect inhibited a malignancy phenotype (Issa et al., CA125 level, clinical stage, residual tumor during surgery 2005). Because ovarian cancer was a highly heterogeneous were independently as prognostic factors for survival of cancer in which it has many types of the histopathology EOC with p=0.03, p=0.01, p=0.01, p=0.04 respectively. and malignancy with a progression ranging from slow to However, after adjusted for other variables they were not very fast and the patient could die quickly. Thus, using significant as a prognostic factors (p>0.05). As far we the molecular profiles of ovarian cancer to reduce the researched no study mentioned menarche corellated with heterogeneity will be important for the patient selection prognosis of EOC. Age of menarche usually corellated in determining the therapy. It also happened in the MDS with risk of ovarian cancer pathogenesis. However, in this (Myelo Dysplastic Syndrome), a heterogeneous disease study menarche was independently as prognostic factor group in which the outcomes differed greatly depending on for survival. The levels of CA125 in this studied was the profile of clinical pathology from chronic and slow to classified into two groups with the cut off point of 70 U/ aggressive with a short survival. The results of this study ml due to the level of CA125 was still normal is <35U/ml. got that the high frequency of BRCA1 methylation was a There are several conditions of non neoplastic conditions new fact for us in Indonesia. Hypermethylation in BRCA1 CA125 level are increase, nearly 6% of women without gene in the epithelial ovarian cancer had largely various ovarian cancer had CA125 levels more than 35mlU/ml levels of variation and it might likely be very different (Bast et al., 2000; Urban, 2003), then in this study of in each country because of differences in environmental malignancy condition espescially EOC the level of CA125 factors, nutrition, chemical exposures and polution factors, twice of normal limit used as the cut of high level. The as these greatly affected the occurrence of promoter data showed that CA125 was as independent prognostic methylation of suppressor genes and other genes. factor of the survival of EOC of all stage with HR 4.42 Without the BRCA1 protein expressed in ovarian and p=0.01, after adjusted to other clinicopathological tumors it showed the presence of dysfunction of BRCA1 factors HR 3.01 (95% CI 0.74-12.18) and p=0.12. Other gene due to either genetic or epigenetic changes that could studied found that CA125 were as prognostic factor for lead to the occurrence of the transformation of cells into EOC stage I (Nagele et al., 1995; Paramasivam et al., Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9483 Heru Pradjatmo et al 5-years survival estimates at 78.8% vs 58.8% and 35.3% 2005; Petri et al., 2006). A review of 15 studies showed respectively (p<0.001), even after adjusted for race, stage, that CA125 levels increased in 50% of patients with stage grade and surgical treatment the difference between the I of the disease, 90% in stage II, 92% in stage III, 94% age group persist (Chan et al,. 2006). In this studied in the stage IV of disease (Jacob and Bast, 1989). It was show that older women ≥ 40 years had lower survival also reported a positive correlation between the increased than younger women <40 years, but statistically not serum levels of CA125 and the expression levels of CA125 significant with HR 1.54 (p=0.43) and after adjusted for in the epithelial ovarian cancer tissues. However, in the other variables HR 0.93 (p=0.83). The present study show epithelial ovarian cancer of serous type the expression that parity and more than one child had less chances of was significantly more positive than the other types of survival compared to patients with no or one child as seen epithelial ovarian cancer. It was also found significantly independently as well as adjusted analysis HR more than shorter survival in the patients with ovarian cancer of stage 100.0 100.0 twice though statistically not significant, Other factor in III and IV without the expression of CA125 compared with 6.3 6.3 12.8 12.8 10.1 10.1 20.3 20.3 this study were histopathological type, menopauze status the patient of ovarian cancer in stage III and IV with the 10.3 10.3 apparently were not influential to the survival of EOC expression of CA125 in the tumor (Hogdall et al., 2007). 25.0 25.0 30.0 30.0 75.0 75.0 patients independently as well as on adjusted analysis. Clinical stage is the most important prognostic factor of In conclussions, methylation status and expression of the cancer. Relative five years survival of ovarian cancer 75.0 75.0 46.8 46.8 56.3 56.3 51.1 51.1 BRCA1 were not to be prognosticator of EOC patients in all stages was 53%, for stage III and IV were 31 % 51.7 51.7 54.2 54.2 and they were not correlated to clinicopathological50.0 50.0 and for stage I and II were 95% (Landis et al., 1998). The 31.3 31.3 30.0 30.0 characteristics of the patients such as; age, menarche, present study found that clinical stage independently as parity, menopauze status, BMI, CA125 level, clinical prognostic factor of EOC in which the late stage (stage stage, histopathological type, grade, residual tumor. The II-IV) had HR 3.79 (p=0.01) compare with early stage 25.0 25.0 study found that menarche, CA125 level, clinical stage eventhough in the adjusted analysis had HR 3.15 (p=0.18). 38.0 38.0 33.1 33.1 31.3 31.3 31.3 31.3 30.0 30.0 27.6 27.6 and residual tumor were independently as prognosticator Residual tumor was demonstrated to be a prognostic 25.0 25.0 23.7 23.7 of EOC patients, eventhough in the multivariable analysis factor to determine survival in patients with EOC stage 0 0 statistically were not significant its due to the power of IV (Bristow et al., 2002; Winter et al., 2008). Elstrand et the study was low. al (2012) reported that among patients with EOC stage IV who underwent at least one surgical prosedure residual disease was an important prognostasicator for overall References survival. While in this study found residual tumor was Bai X, Fu, Y, Xue, H, et al (2014). BRCA1 promoter methylation as prognostic factor independently for survival of EOC in sporadic epithelal ovarian carsinoma: Association with patients. Grade of differentiation mainly in the early stages low expression of BRCA1, improved survival and co- of the disease is an important prognostic factor that affects expression of DNA methyltransferases. Oncol Letters, 7, treatment planning (Morgan et al., 2011 cited Hoffman 1088-96. et al., 2012), This study found that well differentiated, Bast Jr RC, Ravdin P, Hayes DF, et al (2000). Update of moderate differentiated and poor differentiated were recommendations for the use of tumor markers in breast clinically significant and likely to be a prognostic factor and colorectal cancer: clinical practice guidelines of the of EOC which the Hazard Ratio (HR) were 1.22 and American society of clinical oncology. J Clin Oncol, 19, 3.83 respectively even though statistically they were 1865-78. Bristow RE, Tomacruz RS, Armstrong DK, Trimble EL, Monntz not significant. Then menarche, CA125 level, clinical FJl (2002). Suvival effect of maximal cytoreductive surgery stage, residual tumor during surgery were independently for advanced ovarian carcinoma during the platinum era: a prognostic factors for survival of EOC, however, in the meta-analysis. J Clin Oncol, 20, 1248–59. multivariable analysis showed to be insignificant as Chan JK, Urban R, Cheung MK, et al (2006). Ovarian cancer in prognostic factors of survival, its seems that those results younger vs older women: a population-based analysis. Br J are due to the power of study being low. Cancer, 95, 1314–20. Age of EOC patients most commonly occured in Chiang JW, Karlan BY, Cass I, Baldwin RL (2006). BRCA1 age ≥40 years 73.9% and 26.1% aged <40 years, others promoter methylation predicts adverse ovarian cancer studied reported in Lahore India that the median age of prognosis. Gynecol Oncol, 101, 403-10. Duska LR, Chang YC, Flynn CE, et al (1999). Epithelial EOC was 47 years old (Saeed and Akram, 2012) and in ovarian carcinoma in the reproductive age group. Cancer, Sweden median age of ovarian cancer was 75 years old 85, 2623-9. (Segelman et al., 2010). The studies on the prognostic Elstrand MB, Sandstad B, Oksefjell H, Davidson B, Trope CG implication of age in ovarian cancer are inconclusive. (2012). Prognostic significance of residual tumor in patients Although most reports have shown that younger women with epithelial ovarian carcinoma stage IV in a 20 years with ovarian cancer have an improved outcome compared perspective. Acta Obstet Gynecol Scan, 91, 308-17. to older women due to they have lower stage and well Esteller M, Corn PG, Baylin, SB (2001). Herman JG. A gene differentiation tumors (Rodriguez et al., 1994: Chan et hypermethylation profile of human cancer. Cancer Res, al., 2006), others researchers have found that age was 61, 3225-9. Esteller M, Sanchez-Cespedes M, Rosell R, et al (1999). not an independent prognostic factor (Massi et al., 1996: Detection of aberrant promoter hypermethylation of tumor Duska et al., 1999). However, population-based studied suppressor genes in serum DNA from non-small cell lung found that across all stages of EOC very young women cancer patients. Cancer Res, 59, 67-70. (<30 years) had significant survival advantage over Hahn WC, Weinberg RA (2002). Rules for making human tumor young (30-60 years) and older (>60 years) group with 9484 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 Newly diagnosed without treatment Newly diagnosed without treatment Newly diagnosed with treatment Newly diagnosed with treatment Persistence or recurrence Persistence or recurrence R Remi emissi ssion on None None Chemotherapy Chemotherapy Radiotherapy Radiotherapy Concurr Concurrent ent chemor chemoradi adiati ation on DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9479 cells. N Engl J Med, 347, 1593-603. expression and tumor grade, stage or overall survival in Heo I, Joo C, Cho J (2008). Lin28 mediate terminal undylation platinum-tested epithelial ovarian cancer patients. Asian of let-7 precursur microRNA. Mol Cell, 32, 276-8. Pac J Cancer Prev, 15, 4275-9. Hilton JL, Geisler JP, Rathe JA, et al (2002). Inactivation of Thrall M, Gallion, HH, Kryscioz R, et al (2006). BRCA1 BRCA1 and BRCA2 in Ovarian Cancer. J Natl Cancer expression in a large series of sporadic ovarian carcinomas: Inst, 94, 1396-406. a Gynecologic Oncology Group study. Int J Gynecol Cancer, Hoffman BL, Schorge JO, Schaffer JL, et al (2012). Epithelial 16, 166-71. nd Ovarian Cancer. In Williams Gynecology, 2 Ed, Mc Graw Urban N (2003). Specific keynote: Ovarian cancer risk Hill Co, 853-78. assessment and the potential for early detection. Gynecol Hogdall EV, Christensen L, Kjaer SK, et al (2007). CA 125 Oncol, 88, 75-9. expression pattern, prognosis and correlation with serum Winter III WE, Maxwell GL, Tian C, et al (2008). Tumor residual CA 125 in ovarian tumor patients from the Danish “Malova” after surgical cytoreduction in prediction of clinical outcome ovarian cancer study. Gynecol Oncol, 104, 508-17. in stage IV epithelial ovarian cancer: a gynecologic oncology Issa J-P J, Kantarjian HM, Kirkpatrick P (2005). Fresh from the group study. J Clin Oncol, 26, 83-9. pipeline azacitidine. Nat Rev, 4, 275-6. Yang D, Khan S, Sun Y, et al (2011). Association of BRCA1 and Jacobs I, Bast RC (1989). The CA125 tumour-associated antigen: BRCA2 mutations with survival, chemotherapy sensitivity, a review of the literature. Human Reprod, 4, 1-12. and gene mutator phenotype in patients with ovarian cancer. Kawahara N, Nishikura K (2009). Regulation of the miRNA JAMA, 306, 1557-65. function by RNA editing. Tanpakushitsu Kakusan Koso, 54, 1133-40. Laird PW (2003). The power and the promise of DNA methylation markers. Nat Rev Cancer, 3, 253-66. Lan VTT, Thuan TB, Thu DM, et al (2013). Methylation profile of BRCA1, RASSF1A and ER in Vietnamese women with Ovarian Massi D, Susini T, Savino L, et al (1996). Epithelial ovarian tumors in the reproductive age group: age is not an independent prognostic factor. Cancer, 77, 1131-36. Cancer. Asian Pac J Cancer Prev, 14, 7713-8. Landis SH, Murray T, Bolden S, Wingo P (1998). Cancer statistics. CA Cancer J Clin, 48, 6-30. Lesnock JL, Darcy KM, Tian C, et al (2013). BRCA1 expression and improved survival in ovarian cancer patients treated with intaperitoneal cisplatin and paclitaxel: a gynecologic oncology group study. Br J Cancer, 108, 1231-37. Li C, Feng Y, Coukos G, Zhang L (2011). MicroRNAs in epithelial ovarian cancer. In William CS Cho Editor. in MicroRNAs in Cancer Translational Research. Springer Science, 309-42 Montavon C, Gloss BS, Warton K, et al (2012). Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer. Gynecol Oncol, 124, 582-88. Munkarah AR, Hallum AV, Morris M, et al (1997). Prognostic significance of residual disease in patients with stage IV epithelial ovarian cancer. Gynecol Oncol, 64, 13-7. Nagele F, Petru E, Medl M, et al (1995). Preoperative CA 125: an independent prognostic factor in patients with stage I epithelial ovarian cancer. Obstet Gynecol, 86, 259-64. Paramasivam S, Tripcony L, Crandon A, et al (2005). Prognostic importance of preoperative CA-125 in international federation of gynecology and obstetrics stage I epithelial ovarian ancer: an Australian multicenter study. J Clin Oncol, 23, 1-5. Petri AL, Hogdall EV, Christensen IBJ, et al (2006). Preoprative CA125 as a prognostic factor in stage I epithelial ovarian cancer. APMIS, 114, 358-63. Rodriguez M, Nguyen HN, Averette HE, et al (1994). National survey of ovarian carcinoma XII. Epithelial ovarian malignancies in women less than or equal to 25 years of age. Cancer, 73, 1245-50. Saeed S, Akram, M (2012). Epithelial ovarian cancer: epidemiology and clinicopathological features. Profesional Med J, 19, 1040-5. Segelman J, Floter-Radestad A, Hellborg H. Sjovall A, Martling A (2010). Epidemiology and prognosis of ovarian metastases in colorectal cancer. Br J Surg, 97, 1704-9. Shawky AEA, El-Hafez AA, El-Tantawy, Hamdy R (2014). No association between BRCA1 immunohistochemical Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 9485

Journal

Asian Pacific Journal of Cancer PreventionUnpaywall

Published: Nov 28, 2014

There are no references for this article.