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Cell-Free DNA Level As a Prognostic Biomarker for Epithelial Ovarian Cancer

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Cell-Free DNA Level As a Prognostic Biomarker for Epithelial Ovarian Cancer ANTICANCER RESEARCH 32: 3467-3472 (2012) Cell-free DNA Level as a Prognostic Biomarker for Epithelial Ovarian Cancer JAE HONG NO1, KIDONG KIM1, KI HO PARK2 and YONG-BEOM KIM1 1Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Gyunggi, Republic of Korea; 2Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea Abstract. Background: The use of cell-free DNA (cfDNA) is controversial because in only 50% of stage I ovarian as a non-invasive biomarker has been evaluated in many cancer cases is CA125 elevated (2). In addition, an elevated types of cancer. This study investigated the prognostic CA125 level is frequently associated with benign significance of cfDNA level for ovarian cancer. Materials gynecological disease and non-gynecological malignancies, and Methods: Preoperative sera of 36 patients with ovarian causing a significant false-positive rate. Accordingly, there is cancer and of 16 with benign tumors were analyzed using a clear need to develop new biomarkers for ovarian cancer. commercially available copy number assay kits to measure Cell-free DNA (cfDNA) is extracellular DNA found in the cfDNA level of genes including beta-2-microglobulin serum or plasma that can be detected by sophisticated (B2M), member RAS oncogene family (RAB25), claudin 4 techniques such as polymerase chain reaction (PCR) (3). The (CLDN4) and ATP-binding cassette subfamily F member 2 elevation of cfDNA has been reported in various (ABCF2). Cox regression analysis was used to calculate inflammatory diseases, including acute pancreatitis, hazard ratios (HR) and 95% confidence intervals (CI). cardiomyopathy and rheumatoid arthritis (4-7). It is known Results: cfDNA level of these genes had no association with that elevation of cfDNA can also be found in patients with other prognostic factors of ovarian cancer. In particular, in various types of cancer, and these cfDNA are believed to be patients with advanced stage disease, a low RAB25 level was derived from the tumor itself through active metabolism or an independent prognostic factor for disease-free survival tumor cell death (8). The use of cfDNA as a screening (HR=18.2, 95% CI=2.0-170.0) and overall survival method has been evaluated in many types of cancer including (HR=33.6, 95% CI=1.8-634.8). Conclusion: Our findings renal cell carcinoma, lung cancer, testicular germ cell tumor, suggest that the preoperative serum cfDNA level of RAB25 colorectal cancer, and breast cancer, suggesting that it could could be a useful biomarker predicting survival outcomes in be used as a non-invasive biomarker for a wide variety of patients with advanced ovarian cancer. cancer types (9-11). It was also reported that the detection of a high level of cfDNA has a role in the prediction of Ovarian cancer is the most lethal gynecological malignancy prognosis in patients with bladder cancer (12). among all types of gynecological cancer (1). The majority of In ovarian cancer, a few studies have evaluated the role of patients are diagnosed with disease at advanced stages due cfDNA as a biomarker for ovarian cancer. In studies with to the absence of specific symptoms and screening patients, total cfDNA levels were higher in patients than in biomarkers. The prognosis of patients with advanced disease normal controls (13, 14). An animal study with female nude is still poor despite the use of extensive surgery and adjuvant mice demonstrated a correlation of tumor burden with the chemotherapy. Although the serum cancer antigen 125 level of cfDNA (15). It remains unclear whether cfDNA (CA125) level has been used as a biomarker to detect levels have an association with the prognosis of ovarian recurrence during follow-up, the role of CA125 in screening cancer. More than 20% of ovarian carcinomas are associated with a copy number abnormality at various chromosomal locations (16). Recent studies using comparative genomic Correspondence to: Yong-Beom Kim, MD, Ph.D., Department of hybridization have revealed specific copy number changes Obstetrics and Gynecology, Seoul National University Bundang and gene expression level of different genes including RAS Hospital, Gumi-ro 300, Seongnam-si, Gyunggi-do, Republic of Korea. Tel: +82 317877253, Fax: +82 317874054, e-mail: [email protected] oncogene family (RAB25), claudin 4 (CLDN4) and ATP- binding cassette subfamily F member 2 (ABCF2) (17, 18). Key Words: Cell-free DNA, ovarian cancer, biomarker. Although the cfDNA is assumed to originate from tumor, it 0250-7005/2012 $2.00+.40 3467 ANTICANCER RESEARCH 32: 3467-3472 (2012) Table Ⅰ. Median expression of selected genes in serum cell-free DNA level of patients with epithelial ovarian cancer and those with benign cyst. Expression (copies/ml) Gene Benign (n=16) Epithelial ovarian cancer (n=36) p-Value* Median (IQR) Median (IQR) B2M 450.5 (280.1-560.0) 292.7 (121.8-551.2) 0.127 RAB25 69.2 (46.2-80.3) 61.3 (20.5-80.2) 0.276 CLDN4 77.2 (57.3-106.1) 55.2 (16.2-92.7) 0.085 ABCF2 1.2 (1.0-2.4) 2.0 (1.1-2.6) 0.968 *Mann-Whitney U-test. IQR: interquartile range; B2M: beta-2-microglobulin; RAB25: member RAS oncogene family; CLDN4: claudin 4; ABCF2: ATP-binding cassette subfamily F member 2. remains unclear whether it could be useful in detecting the (assay ID: Hs07520413_cn) genes used in this study were purchased change in copy number for a particular gene. from Applied Biosystems (Carlsbad, CA, USA) and confirmed using The purpose of this study was to investigate the prognostic 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 significance of serum levels of total cfDNA and cancer- (2.5 μM), 12.5 μl TaqMan Master PCR 2× master mixture (Applied specific genes in patients with ovarian cancer. Biosystems). DNA in 5 μl was added as the PCR template. Quantitative real-time PCR was performed on 96-well optical plates Materials and Methods using the above reagents; the results were analyzed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Carlsbad, Patients. This study included 36 patients with primary epithelial CA, USA). The following PCR conditions were used. After initial ovarian cancer and 16 patients with benign ovarian tumors, activation of uracyl-N-glycosylase at 50˚C for 2 minutes, AmpliTaq including five cases of serous cystadenoma, three of mucinous Gold was activated at 95˚C for 10 min. The subsequent PCR cystadenoma, six of endometriotic cyst, and four of mature cystic condition consisted of 45 cycles of denaturation at 95˚C for 15 teratoma. All patients underwent surgery between 2005 and 2009 at seconds and annealing extension at 60˚C for 1 minute per cycle. Seoul National University Bundang Hospital. The mean age of the During the PCR amplification, the amplified products were measured ovarian cancer group was 51.8 years; for the benign group, this continuously by determination of the fluorescence emission. A value was 36.5 years. Patients with ovarian cancer underwent standard curve consisting of known DNA concentration was created platinum-based adjuvant chemotherapy if indicated. The median by serial dilution to quantify the copy number of specific gene. duration of follow-up was 30.7 months. Data regarding surgical stage, the presence of residual tumor after cytoreductive surgery, and Statistical analysis. SPSS for Windows version 12.0 (SPSS Inc., histologic type were obtained from medical records retrospectively. Chicago, IL, USA) was used for data analysis. The mean values of Stages were determined according to the International Federation of serum DNA level were compared using Mann-Whitney U-tests. The Gynecology and Obstetrics (FIGO) criteria (17), and the four major relationships between categorical data were evaluated using the chi- groups were used for analysis. The FIGO stage distribution was as square test or Fisher’s exact test. Pearson’s correlation coefficient follows: stage I, 10 patients (19.2%); stage II, 2 patients (3.8%); was employed to investigate the correlation. The differences in stage III, 20 patients (38.5%); stage IV, 4 patients (7.7%). disease-free survival (DFS) and overall survival (OS) between positive and negative groups were analyzed by means of log-rank DNA extraction and quantitative real-time PCR. Venous blood test and Cox regression analysis. Cut-off points for each gene were samples were obtained preoperatively from the patients. After determined by median level of patients with ovarian cancer. A p- extracting serum from whole blood, the serum was stored in a deep values less than 0.05 was considered as being statistically significant. freezer until it was used for analysis. DNA was isolated from 1 ml of serum using the QIAamp DNA mini Kit (Qiagen Inc, Valencia, Results 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 cfDNA level in benign and malignant ovarian tumors. To Topo-TA Cloning kit (Invitrogen, Eugene, OR, USA). These investigate the role of cfDNA as a screening marker for plasmids were used for target gene quantification. Serial dilutions ovarian cancer, we compared the cfDNA levels in patients of plasmid DNA were used to determine the dynamic range of with benign tumors and those with ovarian cancer (Table Ⅰ). quantification. Commercially available copy number assay kits were used for The serum B2M gene level in cfDNA in ovarian cancer was DNA quantification. TaqMan primers and probes for beta-2- similar to that of those with benign ovarian tumors. There microglobulin (B2M: assay ID: Hs00119644_cn), ABCF2 (assay ID: was no significant difference in cfDNA level according to Hs03001077_cn), RAB25 (assay ID: Hs02686900_cn) and CLDN4 the histologic types of benign tumors.
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