“Liquid biopsy” in which body fluids are screened for biomarkers like circulating tumor cells (CTCs), circulating free DNA, or exosomes are gathering substantial research and support. CTCs are shed into bloodstream from primary tumor, metastases or recurrences, and display tumor specific characteristics, rendering them good candidates for liquid biopsy. The feasibility of CTCs in epithelial ovarian cancer (EOC) has been investigated through various methods and varying detection rates between 12-90% have been reported.
To evaluate the role of CTCs as predictive markers for chemoresistance and overall survival (OS), we enrolled 62 histologically confirmed EOC subjects. Magnetic powder labeled monoclonal antibodies for epithelial cell adhesion molecule (EpCAM), Mucin 1 cell surface associated ( MUC1) ,mucin 16, cell surface associated MUC16 or CA125 were prepared according to the manufacturer’s protocol. Expression of three ovarian cancer related genes were detected in CTCs using multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR). The test was considered positive if a PCR fragment of at least one tumor associated transcript was clearly detected or amplicon concentration was >=0.15ng/µl. Patients showing recurrence within 6 months of completion of treatment were considered chemotherapy resistant.
Prediction of resistance to chemotherapy, PFS, OS
Only 34 out of 62 EOC patients responded for complete follow up of more than 6 months (5, 2, 16,11 patients of stages I,II,III and IV respectively). 28 patients had opted for non-neoadjuvant chemotherapy, and rest 6 had neoadjuvant chemotherapy. Of these, 4 patients showed resistance to chemotherapy and the rest were chemo-sensitive. CTCs were detected at primary diagnosis in 100% (4/4) of these chemotherapy resistant patients, and only in 23.3% (7/30) of those who were chemotherapy respondent (p=0.007). In univariable analysis, presence of CTC at primary diagnosis correlated with platinum resistance (OR, 3.00; 95% CI, 1.12-7.95; p=0.028)
Statistically significant increased levels of EpCam mRNA (EpCAM+ CTC) were detected in chemotherapy therapy resistant patients, as determined by univariable analysis (OR, 5.78; 95% CI, 1.39-23.95; p=0.016). No significant differences were observed in the mRNA levels of MUC1(p=0.188) and MUC16 (p=0.139), and serum concentrations of CA125 (p=0.414) between the chemotherapy resistant and sensitive groups.
During the follow up 5 sufferers died. CTCs at primary diagnosis were present in 5/5 (100%) patients who died, whereas in only 6/29 (20.7%) of those who survived. Presence of CTC significantly correlated with decreased OS (HR, 2.15; 95% CI, 1.21-3.85; p=0.009), but not with PFS (HR, 1.49; 95% CI, 0.80-2.78; p=0.198) on univariable analysis.
Significantly higher levels of only EpCAM mRNA were detected in those who perished than those who survived (p=0.019). Multivariable analysis revealed the presence of EpCAM+CTC at primary diagnosis to be an independent predictor of a poor PFS (HR,3.3; 95% CI, 1.3-8.4; p=0.010), OS (HR, 2.6; 95% CI,1.1-5.6; p=0.019) and resistance to chemotherapy (OR 8.6; 95% CI, 1.8-43.7; p=0.009). None of the other genes studied (MUC1, MUC16) showed any significant correlation with PFS and OS. No significant differences were realized between serum CA125 concentrations and PFS or OS.
To conclude, expression of EpCAM in CTC positive patients predicts chemotherapy resistance and prognosis, could aid clinicians in individualizing therapy, saving the patient from unnecessary side effects and systemic toxicity of platinum-based chemotherapy. EpCAM+CTC may be distinguished by their amplified capacity to break DNA-platinum adducts, thus bypassing cisplatinum-mediated cytotoxicity and making way to the well-known molecular phenotype of “on-target” platinum resistance. Also, EpCAM expression can be a strong basis for further investigating anti-EpCAM targeted therapies in ovarian cancer.