Detection of Circulating Tumor Cells in the Prognostic Significance of Patients With Breast Cancer: A Retrospective Study

Abstract

Background: Breast cancer (BC) is an aggressive tumor. Circulating tumor cells (CTCs) are a potential biomarker for the prognosis of cancer patients. This study aimed to explore the prognostic significance of CTCs in patients with BC.

Methods: Retrospectively, 108 BC patients were collected between January 2011 and December 2019, while 10 patients with benign nodules were included as controls. CTCs with different phenotypes of patients were isolated using CanPatrol and tricolor RNA in situ hybridization (RNA-ISH) methods. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) levels were measured by immunohistochemistry (IHC). The progression-free survival (PFS) was calculated using the Kaplan-Meier method. Independent risk factors for BC recurrence were determined by Cox proportional risk regression analysis.

Results: The higher the cancer stage (p = 0.00), the higher the ki-67 expression level (p < 0.01), and the lower the histologic grade (p < 0.01), the higher the number of CTCs. The PFS of patients with high CTCs was shorter than that of patients with low CTCs (p < 0.05). Total CTCs (≥ 6) and positive mesenchymal CTCs (MCTCs) were also associated with recurrence and metastasis.

Conclusions: Total CTCs in BC patients have an independent influence on PFS reduction. Higher total CTCs and MCTCs in peripheral blood are biomarkers for predicting the prognosis of BC patients. HER-2 high expression is also associated with the prognosis of the disease.

Keywords: breast cancer; circulating tumor cells; metastasis; progression‐free survival; recurrence; triple negative breast cancer.

FIGURE 1

Images of CTCs in breast cancer by CanPatrol and the RNA in situ hybridization technique. (A) Epithelial CTCs: EpCAM and CK8/18/19 genes were labeled with AF594 immunofluroresence dye conjugation (red dots). (B) Mesenchymal CTC: Vimentin and Twist genes were labeled with AF488 immunofluroresence dye conjugation (green dots). (C) Mixed CTCs: the cellular nucleus was stained with 6‐diamidino‐2‐phenylindole (DAPI). All pictures were taken by an immunofluroscence microscope at 100× magnification. CTCs, circulating tumor cells.

FIGURE 2

ROC curve determination of the CTC cutoff value for differentiating clinical significance. This graph shows the ROC curve of total CTCs in 108 BC patients and 10 patients with benign nodules. It determined sensitivity and specificity of CTCs at 6.0/5 mL of blood as a cutoff. AUC, area under the ROC curve; CTC, circulating tumor cells; ROC, receiver operating characteristic.

FIGURE 3

Comparison of progression‐free survival (PFS) in patients with total CTCs, epithelial CTCs, Mixed CTCs, and mesenchymal CTCs (MCTCs) by Kaplan–Meier curves. (A) Total CTCs, (B) epithelial CTCs, and (C, D) mixed MCTCs and MCTCs. CTC, circulating tumor cell; MCTC, mesenchymal CTC.

FIGURE 4

PFS comparison of patients with hormone receptor expression by Kaplan–Meier curves. (A) Estrogen receptor (ER), (B) progesterone receptor‐PR, (C) ER, PR, and HER2 triple positive (TR+), and (D) triple negative breast cancer (TNBC). PFS, progression‐free survival.