Abstract
Background: The use of cell-free DNA (cfDNA) as a non-invasive biomarker has been evaluated in many types of cancer. This study investigated the prognostic significance of cfDNA level for ovarian cancer. Materials and Methods: Preoperative sera of 36 patients with ovarian cancer and of 16 with benign tumors were analyzed using commercially available copy number assay kits to measure the cfDNA level of genes including beta-2-microglobulin (B2M), member RAS oncogene family (RAB25), claudin 4 (CLDN4) and ATP-binding cassette subfamily F member 2 (ABCF2). Cox regression analysis was used to calculate hazard ratios (HR) and 95% confidence intervals (CI). Results: cfDNA level of these genes had no association with other prognostic factors of ovarian cancer. In particular, in patients with advanced stage disease, a low RAB25 level was an independent prognostic factor for disease-free survival (HR=18.2, 95% CI=2.0-170.0) and overall survival (HR=33.6, 95% CI=1.8-634.8). Conclusion: Our findings suggest that the preoperative serum cfDNA level of RAB25 could be a useful biomarker predicting survival outcomes in patients with advanced ovarian cancer.
Ovarian cancer is the most lethal gynecological malignancy among all types of gynecological cancer (1). The majority of patients are diagnosed with disease at advanced stages due to the absence of specific symptoms and screening biomarkers. The prognosis of patients with advanced disease is still poor despite the use of extensive surgery and adjuvant chemotherapy. Although the serum cancer antigen 125 (CA125) level has been used as a biomarker to detect recurrence during follow-up, the role of CA125 in screening is controversial because in only 50% of stage I ovarian cancer cases is CA125 elevated (2). In addition, an elevated CA125 level is frequently associated with benign gynecological disease and non-gynecological malignancies, causing a significant false-positive rate. Accordingly, there is a clear need to develop new biomarkers for ovarian cancer.
Cell-free DNA (cfDNA) is extracellular DNA found in serum or plasma that can be detected by sophisticated techniques such as polymerase chain reaction (PCR) (3). The elevation of cfDNA has been reported in various inflammatory diseases, including acute pancreatitis, cardiomyopathy and rheumatoid arthritis (4-7). It is known that elevation of cfDNA can also be found in patients with various types of cancer, and these cfDNA are believed to be derived from the tumor itself through active metabolism or tumor cell death (8). The use of cfDNA as a screening method has been evaluated in many types of cancer including renal cell carcinoma, lung cancer, testicular germ cell tumor, colorectal cancer, and breast cancer, suggesting that it could be used as a non-invasive biomarker for a wide variety of cancer types (9-11). It was also reported that the detection of a high level of cfDNA has a role in the prediction of prognosis in patients with bladder cancer (12).
In ovarian cancer, a few studies have evaluated the role of cfDNA as a biomarker for ovarian cancer. In studies with patients, total cfDNA levels were higher in patients than in normal controls (13, 14). An animal study with female nude mice demonstrated a correlation of tumor burden with the level of cfDNA (15). It remains unclear whether cfDNA levels have an association with the prognosis of ovarian cancer.
More than 20% of ovarian carcinomas are associated with a copy number abnormality at various chromosomal locations (16). Recent studies using comparative genomic hybridization have revealed specific copy number changes and gene expression level of different genes including RAS oncogene family (RAB25), claudin 4 (CLDN4) and ATP-binding cassette subfamily F member 2 (ABCF2) (17, 18). Although the cfDNA is assumed to originate from tumor, it remains unclear whether it could be useful in detecting the change in copy number for a particular gene.
Median expression of selected genes in serum cell-free DNA level of patients with epithelial ovarian cancer and those with benign cyst.
The purpose of this study was to investigate the prognostic significance of serum levels of total cfDNA and cancer-specific genes in patients with ovarian cancer.
Materials and Methods
Patients. This study included 36 patients with primary epithelial ovarian cancer and 16 patients with benign ovarian tumors, including five cases of serous cystadenoma, three of mucinous cystadenoma, six of endometriotic cyst, and four of mature cystic teratoma. All patients underwent surgery between 2005 and 2009 at Seoul National University Bundang Hospital. The mean age of the ovarian cancer group was 51.8 years; for the benign group, this value was 36.5 years. Patients with ovarian cancer underwent platinum-based adjuvant chemotherapy if indicated. The median duration of follow-up was 30.7 months. Data regarding surgical stage, the presence of residual tumor after cytoreductive surgery, and histologic type were obtained from medical records retrospectively. Stages were determined according to the International Federation of Gynecology and Obstetrics (FIGO) criteria (17), and the four major groups were used for analysis. The FIGO stage distribution was as follows: stage I, 10 patients (19.2%); stage II, 2 patients (3.8%); stage III, 20 patients (38.5%); stage IV, 4 patients (7.7%).
DNA extraction and quantitative real-time PCR. Venous blood samples were obtained preoperatively from the patients. After extracting serum from whole blood, the serum was stored in a deep freezer until it was used for analysis. DNA was isolated from 1 ml of serum using the QIAamp DNA mini Kit (Qiagen Inc, Valencia, CA, USA) according to the manufacturer's instruction. Extracted DNA was resuspended in 30 μl of deionized distilled water. Target genes were amplified, and the amplicons were analyzed with the Topo-TA Cloning kit (Invitrogen, Eugene, OR, USA). These plasmids were used for target gene quantification. Serial dilutions of plasmid DNA were used to determine the dynamic range of quantification.
Commercially available copy number assay kits were used for DNA quantification. TaqMan primers and probes for beta-2-microglobulin (B2M: assay ID: Hs00119644_cn), ABCF2 (assay ID: Hs03001077_cn), RAB25 (assay ID: Hs02686900_cn) and CLDN4 (assay ID: Hs07520413_cn) genes used in this study were purchased from Applied Biosystems (Carlsbad, CA, USA) and confirmed using the Genbank database. A master mix for PCR was prepared with 2.5 μl of RNase DNase free water, 1.25 μl primers (9 μM) and probe (2.5 μM), 12.5 μl TaqMan Master PCR 2× master mixture (Applied Biosystems). DNA in 5 μl was added as the PCR template. Quantitative real-time PCR was performed on 96-well optical plates using the above reagents; the results were analyzed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). The following PCR conditions were used. After initial activation of uracyl-N-glycosylase at 50°C for 2 minutes, AmpliTaq Gold was activated at 95°C for 10 min. The subsequent PCR condition consisted of 45 cycles of denaturation at 95°C for 15 seconds and annealing extension at 60°C for 1 minute per cycle. During the PCR amplification, the amplified products were measured continuously by determination of the fluorescence emission. A standard curve consisting of known DNA concentration was created by serial dilution to quantify the copy number of specific gene.
Statistical analysis. SPSS for Windows version 12.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. The mean values of serum DNA level were compared using Mann-Whitney U-tests. The relationships between categorical data were evaluated using the chi-square test or Fisher's exact test. Pearson's correlation coefficient was employed to investigate the correlation. The differences in disease-free survival (DFS) and overall survival (OS) between positive and negative groups were analyzed by means of log-rank test and Cox regression analysis. Cut-off points for each gene were determined by median level of patients with ovarian cancer. A p-values less than 0.05 was considered as being statistically significant.
Results
cfDNA level in benign and malignant ovarian tumors. To investigate the role of cfDNA as a screening marker for ovarian cancer, we compared the cfDNA levels in patients with benign tumors and those with ovarian cancer (Table I). The serum B2M gene level in cfDNA in ovarian cancer was similar to that of those with benign ovarian tumors. There was no significant difference in cfDNA level according to the histologic types of benign tumors.
Association of cell-free DNA level according to prognostic factors of ovarian cancer.
Survival analysis according to serum cell-free DNA level in patients with advanced-stage ovarian cancer.
To test whether cfDNA has a role for predicting the prognosis of ovarian cancer, we investigated the association of prognostic factors with the evel of different genes in cfDNA (Table II). Compared to other histological types, patients with serous ovarian cancer had lower levels of B2M, RAB25 and CLDN4 in cfDNA. Other prognostic factors including age>50 years, advanced stage and presence of residual tumor had no association with the cfDNA level. No correlation between cfDNA and CA125 or CA19-9 was found.
Survival analysis according to cfDNA level. In multivariate survival analysis using Cox regression analysis, advanced stage was an independent prognostic factor for poor DFS. Interestingly, a low level of RAB25 was also an independent prognostic factor for poor DFS (hazard ratio, HR=8.6, 95% confidence interval, CI=1.3-57.1). According to a subsequent subgroup analysis on patients with advanced stages of disease, RAB25 was an independent prognostic factor for DFS (HR=18.2, 95% CI=2.0-170.0) and OS (HR=33.6, 95% CI=1.8-634.8) (Table III).
Discussion
It has recently been reported that circulating cfDNA could be used as a non-invasive biomarker for a wide variety of cancer types. In this study, we investigated the role of cfDNA as a screening and prognostic biomarker for ovarian cancer. Our findings showed that there was no statistical difference in the levels of B2M, RAB25, CLDN4 and ABCF2 genes in serum cfDNA between patients with ovarian cancer and patients with benign ovarian tumors. Our finding is not consistent with a previous study showing that cfDNA levels in patients with ovarian cancer were higher than that of 12 age-matched normal controls (13). However, another study showed that plasma cfDNA in patients with ovarian cancer was lower compared to patients with benign tumors (14). These findings are consistent with previous studies showing that the discriminatory power of cfDNA between benign and malignant disease has been found to be modest for various types of cancer (3). One explanation for these results is the elevation of cfDNA by other causes, including variation in the source. Circulating cfDNA in serum is reported to be higher than that in plasma because the release of DNA from leukocytes during blood clotting leads to nonspecific elevation of total cfDNA (20). The use of serum in this study might have limited the detection of differences in cfDNA levels between benign and malignant tumors. Further research into better understanding the role of cfDNA in screening of ovarian cancer is warranted.
Next we investigated whether cfDNA could predict the prognosis of patient with ovarian cancer. Our finding showed that B2M gene measured in cfDNA had no association with prognosis of ovarian cancer. This result is similar to that of a previous study showing that the serum cfDNA level had no association with prognostic factors in patients with ovarian cancer (14).
Recently, researchers have sought to find specific alterations in cfDNA, such as cfDNA strand integrity, mutation of tumor suppressor genes, methylation alterations, or microsatellite alterations. In ovarian cancer, one study showed that tumor-specific polymorphic microsatellite loci were detected in the cfDNA of 85% of patients with ovarian cancer. Another study using a microarray-based methylation assay showed that differential methylation of promoters in Ras association domain family member 1A (RASSF1A), calcitonin-related polypeptide alpha (CALCA) and E1A binding protein p300 (EP300) in cfDNA may be a useful biomarker to differentiate between certain benign and malignant ovarian tumors (21). Thus, we investigated whether copy number variance of a specific gene in cfDNA could predict the prognosis of patients with ovarian cancer. Interestingly, we found that a low serum cfDNA level of RAB25 was associated with increased risk of death from ovarian cancer. A previous study using array comparative genomic hybridization showed an increase in DNA copy number of the RAB25 gene on chromosome 1q22 in 54% in ovarian cancer (18). In that study, RAB25 copy number was directly correlated with the expression level, and the increased expression of RAB25 in tumor tissue was associated with poor DFS and OS in patients with ovarian cancer. It has been suggested that RAB25 could play key roles in cell proliferation and in the prevention of apoptosis by reducing the expression of proapoptotic molecules (22). The association of poor survival with low RAB25 in serum cfDNA might be explained by reduced apoptosis of tumor cells and lower release of RAB25 gene in serum in our study population (18, 22). Recently, several studies have shown the tumor suppressor function of RAB25 in breast and colon cancer (23, 24). These results also could explain the impact of RAB25 on the survival in our study.
In conclusion, our study suggests that the detection of RAB25 gene copy number variation could be useful as a prognostic biomarker in patients with advanced ovarian cancer, although measuring total cfDNA levels has limited value as a screening marker for ovarian cancer.
Acknowledgements
This work was supported by grant number 11-2009-032 from Seoul National University Bundang Hospital Research Fund.
- Received March 19, 2012.
- Revision received April 24, 2012.
- Accepted April 25, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
