Amr Mohamed Hussein
Immunology and Allergy Department, Medical Research Institute, Alexandria University, Alexandria, 21516
Mohammed Samy Afifi
Immunology and Allergy Department, Medical Research Institute, Alexandria University, Alexandria, 21516
Yasser Ahmed
Experimental and Clinical Surgery Department, Medical Research Institute, Alexandria University, Alexandria, 21516, Egypt
Asmaa Ahmed Hashiem
Zoology Department, Faculty of Science, Alexandria University, Alexandria 21516, Egypt
Shymaa Abdullah Mohamed
Molecular Biology Unit, Medical Technology Center & Applied Medical Chemistry Department, Medical Research Institute, Alexandria University, Alexandria, 21516, Egypt
ABSTRACT
Background. Circulating tumor cells (CTCs) Originating from the primary tumor and disseminating via the circulatory system. The abundance of circulating tumor cells is positively linked to metastatic progression, and this increase is inversely Correlated with the survival outcomes of breast cancer patients and provides more accurate information about patient-specific diagnoses. Therefore, Detection of CTCs with high sensitivity in peripheral blood can function as a reliable tool for cancer diagnosis in the context of liquid biopsy. Objective. This study developed an accurate technique for detecting CTCs in a single milliliter of peripheral blood. Materials and Methods. Eighty women with different clinicopathological data and 20 matched healthy volunteers were included. To identify CTCs via a qRT-PCR assay, cytokeratin-14 and 19 and vimentin were selected as target genes. A FACS assay was performed with antibodies against cytokeratin-14, 16, 19, vimentin, and CD45. For the spiking experiment, MDA-MB-231 cells and HCT 116 cells were used to validate both methods. The results. Vimentin is a crucial marker for the identification of CTCs via either “qPCR-RT” or “FCAS”, followed by cytokeratins. A statistical Assessment of the qRT-PCR data showed A notable positive relationship between cytokeratin-14 and vimentin expression. The clinical stage of disease was significantly negatively correlated with cytokeratin-19 and positively correlated with vimentin expression. The statistical analysis of the FACS data revealed A meaningful positive linkage between clinical stage and either VIM+CTCs or VIM+CK+CTCs. Conclusion. The qRT-PCR assay was more accurate and sensitive than the FACS assay for Identification of CTCs within peripheral blood samples with high performance. Due to the heterogeneity and variability of breast carcinoma, the study recommended using different cytokeratins and vimentin antibodies to assess the disease, especially in the early stages.
Keywords: Breast Cancer, Circulation Tumor Cells, qRT‒PCR, Flow Cytometry, Cytokeratins, Vimentin.
