Bulk-lysis protocols as a sensitive method for investigation of circulating CK19 cells in the peripheral blood of patients with breast cancer by flow cytometry

Abstract

Invasive Breast Carcinoma (IBC) is a heterogeneous group of tumours with varying responses to treatment. Proteins such as Cytokeratin 19 (CK19) play a crucial role in the prognostic evaluation of patients, demonstrating significant roles in breast tumours and metastatic progression, and are described as biomarkers used for the detection of disseminated cells in lymph nodes or distant organs in clinically metastasis-free patients (cM0). Thus, the objective of this study was to establish a methodology for investigating circulating CK19+ breast cancer cells (CTCs) in peripheral blood to detect the risk of metastasis early. The study included a total of 62 patients who were treated at the University Hospital of Santa Catarina. The patients were divided into two groups based on the radiological diagnosis of BI-RADS®. The control group (n = 15) included patients with BI-RADS® 3 and 4a, whose malignancy risk was equal to or less than 10%. The IBC group (n = 46) comprised patients with BI-RADS® 4b, 4c and 5, whose malignancy risk was greater than 10%. The sample preparation was carried out using the bulk-lysis technique to concentrate the peripheral blood sample. For the evaluation of CTCs, the panel was defined using the expression of CD45, a pan-leukocyte antigen not expressed in mammary cells, along with two markers present only in circulating metastatic cells and not in leukocytes, CK19 and Pan-CK. The samples were acquired using a FACSCanto II flow cytometer, equipped with FACSDiva 8.0 software, until tube drying was complete. Data analysis was conducted using Infinicyt 2.0 software. The results obtained in this study show that out of the total cases of IBC analyzed (n = 47), 6.38% (n = 3) presented CTCs detected by flow cytometry, correlating with the presence of metastases identified by imaging methods (cM1), indicating the efficiency of flow cytometry for the research of neoplastic emboli in peripheral blood. Thus, the development and standardization of flow cytometry tests can offer faster, more sensitive, and precise results, complementing the diagnosis and monitoring of IBC patients.