Impact of Cyclophosphamide to Modulates the Genetic Signature of Epithelial Mesenchymal Transition Markers CK19 & Vimentin in Circulating Tumor Cells Population and DNA Copy Number Variations in Breast Cancer Patients

Abstract

Circulating tumor cells are the frontier area of medicine for early prognosis and diagnosis during progression of disease like cancer. Etiopathology of breast cancer is complex due to heterogeneous group of cell population and epithelial-mesenchymal transition markers Sox4, EpCAM, CK19 and vimentin play significant role during onset of tumorigenesis after invasion to the blood vessels and to reach neighboring targeted tissue .Cyclophosphamide, an important antineoplastic drug by the clinicians and also used for auto-immune disease. Present study has been designed to explore the sensitivity (frequency) of cytokeratin and vimentin in circulating tumor cells population and compare after in- vitro   exposure with cyclophosphamide at different time intervals using flowcytometric analysis using specific antibodies and compare with controls. The same study further extended to evaluate DNA copy number variations to evaluate the tissue specific genetic susceptibility in breast cancer patients. : Flowcytometer, a highly sensitive tool for the qualitative and quantitative analysis of  circulating tumor cells using specific monoclonal antibodies of  cytokeratin 19 and vimentin, conjugated by PE (phycoerythrin) after gating. The sensitivity of epithelial-mesenchymal transition markers (CK19 and Vimentin) in circulating tumor cells population were further evaluated after in-vitro exposure with cyclophosphamide (1.0 ugm/ml) at three different time interval i.e. 24, 48 and 72 hours in lymphocytes cultures. Further, the tissue specific genetic susceptibility were evaluated by DNA copy number variations. Findings, reveals the mean values of circulating tumor cells population in control group was +vemAbCK19 (3243.667) followed by decreasing trend were observed after exposure at 24 hours (2753.50), 48 hours (2720.00) and 72 hours (1945.50). Statistical analysis showing significant differences (p<0.001) with respect to controls using chi square-test. Similarly, vimentin showing decreasing trends at first 24 hours (4565.00) and 72 hours (2997.50) with significant (p<0.001) differences. PCR based study using specific - primers of CK19 and vimentin on agarose gel (1.5 %) and individual bands density were evaluated for DNA copy number variations. The sensitivity of vimentin (323bp) its isoform (225 bp), and CK19 (573bp) showing decreasing trend significantly (p<0.05) with respect to controls. Data was further analyzed on basis of different age - groups of the breast cancer patients i.e  an  early (25-35 years) age-group that showing highest (42.85%) frequency followed by decreasing trend (28.50 %) were observed in older age groups ( 65-75 years).

Conclusion: Present study reveals that - 1) discordance in the frequency of circulating tumor cells population may be either due to different pathological stages or tissue specific sensitivity after  drug exposure at different time intervals mediated toxicity significantly at an early age-groups patients. 2) long-term exposure of the drug showing positive approach to the clinicians for  better  management, 3) variations in the  frequency of  DNA copy number significantly support the above findings of cell population that confirm drug mediated genetic susceptibility, and 4) vimentin isoforms are highly  relevant to increase dug binding capacity to  the antigen of sub-population of circulating tumor cells population during metastasis. Hence, present study may consider as prognostic, diagnostic biomarker for early detection of breast cancer patients.