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Tlo13244 297.305

Volume 6 Number 3 pp. 297304 297 RASSF1A Promoter Methylation Viera Kajabova*, Bozena Smolkova*, Levels Positively Correlate with Iveta Zmetakova*, Katarina Sebova*,Tomas Krivulcik*, Vladimir Bella†, Karol Kajo‡, Estrogen Receptor Expression Katarina Machalekova‡ and Ivana Fridrichova* in Breast Cancer Patients1,2 *Laboratory of Cancer Genetics, Cancer ResearchInstitute of Slovak Academy of Sciences, Bratislava,Slovakia; †Department of Senology, St Elizabeth CancerInstitute, Bratislava, Slovakia; ‡Department of Pathology,Slovak Medical University and St Elizabeth CancerInstitute, Bratislava, Slovakia The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.
Translational Oncology (2013) 6, 297304 Address all correspondence to: Ivana Fridrichova, PhD, Laboratory of Cancer Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Breast cancer is the most common cancer in women worldwide.
Slovak Republic. E-mail: According to data published by the International Agency for Research 1This publication is the result of the implementation of the following projects: on Cancer, in 2008, 1,383,000 breast cancer patients were newly No. APVV-0076-10 supported by the Slovak Research and Development Agency(60%); No. 26240220058, Research and Development Operational Programme funded diagnosed and 458,000 breast cancer–related deaths occurred [1].
by the European Regional Development Fund (20%); Nos 2/0065/10 and 2/0120/13 More than 25% of breast cancer patients develop metastatic disease that funded by the Scientific Grant Agency of the Ministry of Education, Science, Research is mostly incurable and for which there are only palliative therapeutic and Sport of the Slovak Republic and the Slovak Academy of Sciences (20%). All authors options [2]. Clinicopathologic characteristics such as tumor size, lymph declare that they have no competing interests.
2This article refers to supplementary material, which is designated by Table W1 and is node (LN) status, invasion of vessels, and hormone receptor status available online at
play important roles in metastasis risk [3]. However, the results of a Received 27 February 2013; Revised 27 February 2013; Accepted 18 March 2013 recent multicenter study found differences in clinicopathologic fea- Copyright 2013 Neoplasia Press, Inc. All rights reserved 1944-7124/13/$25.00 tures between patients with and without primary metastases, and for DOI 10.1593/tlo.13244 RASSF1A Methylation and ER Expression Kajabova et al.
Translational Oncology Vol. 6, No. 3, 2013 metastasis risk, the lobular histology and luminal B positivity in T1 pri- with breast cancer clinicopathologic features to identify useful molec- mary metastatic breast cancer were determined [2].
ular markers indicating the metastatic potential of tumors and patient Similar to other cancer types, breast tumorigenesis is characterized response to hormonal therapy.
by the progressive accumulation of genetic and epigenetic changes inmany genes that regulate cell proliferation and differentiation. There- Materials and Methods fore, molecular characterization of tumor tissues allows determina-tion of novel cancer markers including those predicting metastatic potential and therapy response.
A total of 151 paraffin-embedded tumor tissue samples and Epigenetic abnormalities in neoplastic cells, such as hypermethyla- matched 151 peripheral blood samples from nonfamilial breast cancer tion and hypomethylation of DNA, altered patterns of histone modi- patients and blood samples of 50 healthy controls were obtained from fication, and remodeled chromatin structure, result in the modified the Department of Pathology and Department of Senology at hospitals expression of many essential genes. A well-categorized epigenetic in Bratislava, Slovakia. This study was approved by Ethics Committee change is hypermethylation of tumor-suppressor promoters that led of the University Hospital in Bratislava, and written informed consent to inappropriate transcription silencing of these genes [4]. The tumor was obtained from all patients and controls. Relevant clinical and suppressor gene RAS-association domain family member 1 (RASSF1A) pathologic data were retrieved from the patients' clinical records, and encodes a member of the group of RAS effectors that regulates cell tumors were characterized according to the primary tumor, regional proliferation, apoptosis, and microtubule stability. Hypermethylation lymph nodes, distant metastasis (TNM) classification. The age of pa- of RASSF1A was found in a substantial percentage of various primary tients ranged from 23 to 91 years (mean, 61.2 ± 10.8 years) at the time tumors [5]. Epigenetic inhibition of RASSF1A is considered to be an of breast cancer diagnosis. Typing was performed according to the cur- early cancer biomarker; however, this phenomenon is extended from rent World Health Organization (WHO) classification for breast neo- primary to metastatic tumors during tumor progression [6]. Moreover, plasms (Table 1). No preoperative radiotherapy or chemotherapy had in invasive breast cancers, significantly higher RASSF1A methylation been performed in any of the cases. Controls included 25 individuals of levels were shown compared with in situ carcinomas [7]. These results <50 years and 25 individuals of >50 years who had no signs and symp- indicate the possible association of RASSF1A silencing with metastasis.
toms of cancer or other serious diseases.
Other studies reported higher frequencies of methylation in RASSF1Aalone or in combination with HIN-1 in estrogen receptor (ER)–positive DNA Extraction and Sodium Bisulfite Modification cases compared with ER-negative cases [8,9]. Moreover, a recent Blood samples of patients and controls were collected in EDTA-treated in vitro study revealed that RASSF1A inhibits ERα expression and tubes and centrifuged at 1000g for 10 minutes at room temperature function [product of estrogen receptor 1 (ESR1) gene]; thereby, it plays within 2 hours of venepuncture. Then, supernatants were collected a key role in suppressing transformation of mammary epithelial cells and centrifuged at 1000g for 10 minutes at room temperature to prevent and ERα-positive breast cancer initiation [10]. In addition to the cellular DNA contamination. Plasma samples were stored at −70°C un- potential RASSF1A-mediated epigenetic regulation of ESR1, mild or til further processing. Cell-free DNA from plasma samples was isolated moderate DNA methylation of the ESR1 promoter alone was observed using a QIAamp DSP Virus Kit (Qiagen, Hilden, Germany), DNA in breast tumorigenesis, indicating the possible influence of epigenetic from paraffin-embedded tumor tissues was isolated by the MagneSil processes on hormonal therapy response [11,12]. In tumorigenesis, Genomic, Fixed Tissue System (Promega, Madison, WI), and genomic there are numerous changes in the cadherin-catenin adhesion com- DNA from peripheral blood was obtained using a FlexiGene DNA Kit plexes, including the cell adhesion protein E-cadherin encoded by (Qiagen) according to the manual instructions. DNA concentrations cadherin 1, type 1, E-cadherin (CDH1). In primary breast cancer, the were measured using a NanoDrop 1000 spectrophotometer (Thermo heterogeneous loss of E-cadherin expression corresponding with vari- Fisher Scientific, Bremen, Germany). Tumor DNA (2 μg), cell-free able patterns of promoter methylation was observed in the early stages DNA (2 μg), and genomic DNA (1 μg) were modified by sodium before cell invasion [13]. CDH1 hypermethylation with loss of protein bisulfite treatment according to the protocols of the EpiTect Bisulfite expression was found in both ductal and lobular breast carcinomas; Kit (Qiagen) and CpGenome DNA Modification Kit (Chemicon, however, no significant correlation was observed between E-cadherin Billerica, MA), respectively. DNA was stored at −18°C until use.
expression and the CDH1 promoter methylation profile [14]. Thetissue inhibitors of metalloproteinase (TIMPs) prevent degradation of Quantitative Multiplex Methylation-Specific Polymerase the extracellular matrix by the metalloproteinases. TIMP metallopepti- Chain Reaction Analysis dase inhibitor 3 (TIMP3) is a matrix-bound protein regulating matrix For quantitative evaluation of promoter methylation, the two-color composition that affects tumor growth, angiogenesis, invasion, and modification of quantitative multiplex methylation-specific polymerase metastasis. TIMP3 promoter methylation was observed in 21% to chain reaction (QM-MSP) technology was used [19]. QM-MSP was 27% of breast cancer patients and in invasive ductal carcinomas that performed in two sequential polymerase chain reaction (PCR) reac- were associated with high tumor grading and LN metastasis [15,16].
tions. In the first step, co-amplification of three and two gene loci The spleen tyrosine kinase (SYK) is an intracellular receptor protein (RASSF1A, CDH1, SYK and ESR1, TIMP3) was performed using kinase involved in cell proliferation, differentiation, and phagocytosis three and two pairs of methylation-independent external primers, re- and plays a suppressive function in breast cancer progression and spectively. Multiplex PCRs were performed in 30-μl volumes contain- metastasis [17]. The frequencies of SYK promoter hypermethylation ing 30 to 60 ng of modified DNA, 15 μl of 2× QIAGEN Multiplex at different stages of breast cancer indicate its occurrence shortly before PCR Master Mix (Qiagen), and aliquots of six/four primers at a final the development of the invasion phenotype [18]. The objective of concentration of 0.2 μM. PCR conditions were 95°C for 15 minutes, the present study was to determine the association of the promoter 35 cycles at 94°C for 30 seconds, 62/56°C for 60 seconds, hybridiza- methylation profiles of five genes related to invasion and metastasis tion at 72°C for 90 seconds, and final extension at 72°C for 10 minutes.
Translational Oncology Vol. 6, No. 3, 2013 RASSF1A Methylation and ER Expression Kajabova et al.
Table 1. RASSF1A Methylation Levels in Different Clinical and Histopathologic Categories in Breast Cancer Patients.
RASSF1A Methylation in Tumor Samples RASSF1A Methylation in Plasma Samples Histologic grading ER− PR− HER2+ ER− PR− HER2− DIC indicates ductal invasive carcinomas; LIC, lobular invasive carcinomas; Others, tubular, micropapillar invasive, cribriform invasive, or mucinous breast carcinomas; LN status, lymph node status;ER status, estrogen receptor status; PR status, progesterone receptor status; IHC subtypes, immunohistochemical subtypes.
P < .05 was regarded as statistically significant (in bold). LN status was categorized according to the number of cancer cell–positive nodes as 0, 1, 2, and 3 with none, 1 to 3, 4 to 10, and >10 of positiveLNs, respectively. ER or PR status was considered as positive in cases with ≥1% of positively responding cells. HER2 expression was regarded as positive, if the intensity of IHC reaction was 3+ in 30%of tumor cells or with fluorescence in situ hybridization proven HER2 gene amplification in cases with ambiguous IHC positive at 2+ intensity reaction. According to ER, PR, and HER2 expression, fourIHC subtypes were recognized, luminal A and B (ER+ and/or PR+ HER− and ER+ and/or PR+ HER2+), HER2 overexpression positive (ER− PR− HER2+), and triple negative (ER− PR− HER2−).
Tumor sizes are shown in millimeters.
In the second step (quantitative real-time PCR), 1 μl of the first re- 100. Concentrations of methylated (M) and unmethylated (U ) por- action PCR product was used at a dilution of up to 1:102 in a duplex tions were determined from simultaneously amplified standard curves reaction with both pairs of primers and specific TaqMan probes for for each gene. Methylation levels up to 0.5% were considered to be methylated and unmethylated DNA substrates for each gene. PCRs the background of this sensitive quantitative method. The cumulative were performed in 15-μl volumes containing 7.5 μl of Maxima Probe methylation index (CMI) was calculated as the sum of percentage meth- qPCR Master Mix (2×; Fermentas, Amherst, NY), methylation- and ylation for all evaluated genes. For all five genes, CMI of 500 was the unmethylation-specific primers for RASSF1A, ESR1, CDH1, TIMP3, maximum value of methylation. Primers and TaqMan probes are or SYK gene at a final concentration of 0.3 μM, and methylation- and summarized in the supplementary material (Table W1) [21].
unmethylation-specific TaqMan probes at concentrations rangingfrom 0.1 to 0.27 μM. The reaction conditions were 50°C for 2 min- Statistical Analysis utes, 95°C for 10 minutes, followed by 35 cycles at 95°C for 15 seconds For statistical analyses, SPSS statistics 15.0 was applied, with P < .05 and 60°C for 30 to 90 seconds with final extension at 72°C for 30 sec- regarded as statistically significant. Normally distributed data were onds. For quantitative PCR, a CFX96TM Real-Time PCR System tested by Pearson correlations, Student's t tests, or analysis of variance (Bio-Rad, Hercules, CA) was used. Standard curve establishment and with Bonferroni or Tamhane tests for multiple comparisons, depend- evaluation of quantitative analysis of DNA methylation were performed ing on homogeneity of variance. For non-normally distributed data, as previously described [20]. The relative amount of methylation (%) Spearman correlations, nonparametric Mann-Whitney U or Kruskal- was calculated in each sample according to the formula [M/(U + M)] × Wallis H tests were used. Normality of distribution was assessed by RASSF1A Methylation and ER Expression Kajabova et al.
Translational Oncology Vol. 6, No. 3, 2013 Figure 1. Methylation levels of five genes evaluated in tumor and plasma samples of breast cancer patients.
Kolmogorov-Smirnoff tests. All tests were two-tailed. Categorical data mined in individual patients. Of 129 patients with any methylation were tested by Chi square.
in tumor tissues, 91, 30, and 7 samples were methylated in one,two, and three evaluated genes, respectively. One patient manifestedpromoter methylation in all five genes; however, the CMI was only 77.14. Of 49 patients with methylation in plasma, 37, 7, and 5 sampleswere methylated in one, two, and three genes, respectively (Figure 2).
DNA Methylation in Tumor and Plasma Samples The cumulative methylation levels for the five evaluated genes were sig- Quantitative analyses of DNA methylation were performed in nificantly higher in the tumors and plasma than in the genomic DNA of paraffin-embedded tumor tissues, blood cells, and plasma samples the same patients. In the methylation-positive tumor and plasma sam- from 151 breast cancer patients. Of these, 129 patients (85.4%) dem- ples, the mean CMIs were 50.52 and 32.96, respectively, compared with onstrated different levels of methylation in at least one of the evaluated no methylation in genomic DNA except for four patients with TIMP3 promoters in their tumors: in 124, 19, 15, 15, and 6 patients, mean methylation. However, a similar range of cumulative methylation in methylation levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81% tumor and plasma DNA (0.74–156.57 and 0.51–151.62) was observed.
were found in RASSF1A, ESR1, CDH1, TIMP3, and SYK, respectively.
In the majority of tumors, the substantial portion of CMI was repre- In plasma samples, RASSF1A methylation was observed at a markedly sented by RASSF1A methylation when compared with more frequent lower frequency of 22.54% in 25 patients. ESR1, CDH1, TIMP3, and methylation of other genes in plasma samples.
SYK were methylated in 10, 15, 17, and 3 patients at 17.20%, 22.87%,31.93%, and 27.42%, respectively. DNA methylation levels of five RASSF1A Methylation Levels and evaluated genes in tumor and plasma samples are graphically depicted in Figure 1. Simultaneously methylated promoters in both tumor and Statistical analysis of the correlation between methylation levels and plasma samples were found in 25, 1, 2, 4, and 1 patients in RASSF1A, clinicopathologic features of 151 breast cancer patients was performed ESR1, CDH1, TIMP3, and SYK genes, respectively (Table 2). Low for the highly and frequently methylated RASSF1A gene. The evalu- levels of TIMP3 methylation (0.53–5.15%) were detected in the ated categories were age, histologic type, tumor size, histologic grading, genomic DNA of four breast cancer patients. Of 50 healthy controls, LN status, TNM staging, ER status, progesterone receptor (PR) status, methylation levels of >0.5% in the CDH1 promoter was observed in human epidermal growth factor receptor 2 (HER2) expression, and the genomic DNA of one person alone. However, in the control plasma immunohistochemical (IHC) subtypes. In plasma samples, RASSF1A samples, rare methylation events in CDH1 were found in one person methylation ranged from 0% to 15.8%, with no significant differences (1.67%), TIMP3 in two (16.52% and 20.40%), and ESR1 in two between the subgroups of each clinicopathologic category. The tumor (7.12% and 16.72%). The number of methylated genes was deter- samples of these patients exhibited visibly higher levels of RASSF1A Table 2. Frequencies of Breast Cancer Patients with DNA Methylation in Tumor and Plasma Samples.
Promoter Methylation in Tumor, N (%) Promoter Methylation in Plasma, N (%) Promoter Methylation in Both Tumor and Plasma, N (%) Translational Oncology Vol. 6, No. 3, 2013 RASSF1A Methylation and ER Expression Kajabova et al.
methylation, and statistically significant differences were observed of DNA methylation in ER silencing [11,23]. In the present study, between patients with various tumor size (P = .029), ER-negative 9.9% of patients exhibited CDH1 promoter methylation levels of and ER-positive status (P = .000), PR-negative and PR-positive status up to 14.56% in tumor tissues, similar to other groups of patients (P = .004), and different IHC subtypes (P = .000; Table 1 and Figure 3).
where low levels of CDH1 methylation frequencies from 5.8% to Moreover, there was a positive correlation between RASSF1A methyla- 22.5% were observed [20,22,24]. Variable TIMP3 methylation levels tion levels and the percentage of cancer cells with ER (r = 0.251, of 3% to 42% were identified in the samples of nine patients [25], in P = .002) or PR expression (r = 0.200, P = .014).
accordance with our findings; however, to our knowledge, quantitativeevaluation of SYK methylation has not yet been performed. Compar-ison of studies in European, American, and Saudi Arabian females re- vealed that the highest methylation levels were in RASSF1A similar Quantification of DNA methylation levels in cancer-associated genes to our study, confirming the important role of RASSF1A epigenetic contributes to the more complex molecular characterization of tumors silencing in breast cancer regardless of ethnicity [21,23,24].
required for the development of new diagnostic and therapeutic strat- Circulating cell-free DNA from plasma, serum, or other body fluids egies for cancer patients. High methodical diversity of DNA methyla- seems to be an appropriate biologic material for qualitative or quanti- tion status evaluation was found in the current literature; therefore, we tative testing of tumor-specific molecular alterations including DNA compared our results with these studies using quantitative methods methylation. The serum of patients with invasive ductal carcinomas based on real-time technology. In tumor samples, we found RASSF1A showed significantly higher RASSF1A methylation frequencies com- methylation in 82.1% of evaluated breast cancer patients, with a mean pared with control persons [26], as in our study. We found significantly level of 48.45% in methylated cases. In other studies, analogous fre- lower frequencies of RASSF1A methylation in plasma samples (16.6%) quencies (68% and 82.5%) but lower means of methylation levels than in tumors (82.1%); however, all 25 patients with positive find- (18.5 ± 4.7% and more than 10%) were found in the majority of ings in plasma had simultaneous methylation in their tumor samples.
patients [21,22]. Similar to our results, previous studies recorded For ESR1, CDH1, TIMP3, and SYK, rare incidences were observed low incidences of ESR1 methylation in breast cancers and weak cor- in both tumor and plasma samples. ESR1 was evaluated in the serum relation with low ERα expression levels, indicating a sporadic role of healthy controls and disease-free breast cancer and metastatic breast Figure 2. Cumulative DNA methylation levels in breast cancer patients. The results from tumor tissues of 129 patients (A) and plasmasamples of 49 patients (B) are shown. The CMI is the sum of percentage methylation for five evaluated genes.
RASSF1A Methylation and ER Expression Kajabova et al.
Translational Oncology Vol. 6, No. 3, 2013 Figure 3. Distribution of RASSF1A methylation levels in four clinical and histopathologic categories of breast cancer patients. Box plotsshow the significant differences in the subgroups of patients with different tumor sizes (A), negative and positive estrogen or progesteronestatus (B and C), and different IHC subtypes (D). The length of the boxes is the interquartile range (IQR) that represents values betweenthe 75th and 25th percentiles. Values more than three IQRs from the end of a box are labeled as extreme (*). Values more than 1.5 IQRsbut less than 3 IQRs from the end of the box are labeled as outliers (O). The median is depicted by a horizontal line.
cancer patients and revealed no differences in the low levels of ESR1 In our previous study, no relationship between tumor size and methylation between these three groups [27]. Our results oppose the RASSF1A methylation levels was observed [20], but in the presented above-mentioned hypothesis describing the possible influence of ESR1 group of patients we found significantly higher levels in four cases with epigenetic silencing alone in the strategy of breast cancer therapy. Many breast tumors larger than 50 mm, three of which were at an advanced researchers have focused on the identification of useful sets of methyl- stage of disease. The accumulation of DNA methylation changes could ated genes to improve diagnosis, prognosis, or therapeutic strategy; be associated with aggressive phenotype rather than larger size of tumor, therefore, CMI appears to be a useful parameter. The high incidence because small cancers can also invade and metastasize as a result of of RASSF1A methylation in CMI shows the value of this silenced gene higher numbers of molecular changes compared with early-stage in tumor development in our patients. However, in 49 plasma sam- cancer. Most importantly, we found a relationship between RASSF1A ples, we found a different spectrum in CMI, with visibly higher occur- methylation levels and expression of hormonal receptors. Previous rences of TIMP3 and CDH1 methylation. These results indicate that studies showed higher frequency of RASSF1A methylation in breast cell-free DNA could be derived from a degraded cell subpopulation, cancers with ER+ and PR+ status than in ER− and PR− cases which is active in invasive and metastatic processes, for example, circu- [24,30]; however, we observed a positive correlation between RASSF1A lating tumor cells, rather than from products of apoptosis and necrosis methylation levels in tumor tissues and number of cancer cells with in heterogeneous tumor masses [28]; therefore, these DNA samples positive expression in both ER and PR. Analyses of RASSF1A methyla- could be used for metastatic potential testing. However, after the critical tion in four different IHC subtypes showed very low levels in ER− evaluation of methodical diversity, variability of results, and limited PR− HER2− but not in ER− PR− HER2+. Moreover, in cancers with diagnostic sensitivity and specificity of cell-free DNA alterations in HER2 overexpression, higher but statistically insignificant differences many published studies, we agree that clinical utilization of such DNA in methylation levels were observed compared with HER2-negative requires further studies to assess sample collection, processing, analysis, cancers. These results indicate the possible influence of HER2 on and measurement of results [29].
DNA methylation processes.
Translational Oncology Vol. 6, No. 3, 2013 RASSF1A Methylation and ER Expression Kajabova et al.
In normal human breast epithelium, ERα expression is fairly consis- tent over time, and women with ER overexpression in the normal We thank Gabriela Gasajova for excellent technical assistance.
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Table W1. Primers and TaqMan Probes.
Oligonucleotide Sequence (5′–3′) Product Size (bp) RASSF1A Met R Probe RASSF1A Umet R Probe CDH1 Umet F Probe ESR1 Umet F Probe TIMP3 Met F Probe TIMP3 Umet F Probe


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