Identification of the plasma total cfDNA level before and after chemotherapy as an indicator of the neoadjuvant chemotherapy response in locally advanced breast cancer

Abstract

This study aimed to retrospectively evaluate the circulating free DNA (cfDNA) level in patients with locally advanced breast cancer (LABC) having different neoadjuvant chemotherapy (NCT) responses and to investigate whether dynamic changes in cfDNA level could predict the effectiveness of NCT in patients with LABC. Data on 61 patients with LABC were included. NCT responses were evaluated using the response evaluation criteria. Blood samples were collected for cfDNA detection before treatment and after the first and eighth courses of chemotherapy. The Alu 111-bp and 260-bp fragment levels were evaluated by polymerase chain reaction, and the predictive value of the cfDNA level in the NCT response was determined. In vitro, the MCF-7 and MCF-7/ADR cell lines were applied to simulate the phenomenon of drug resistance and explain the underlying mechanism. The Alu 111-bp level increased after the first NCT course (P = .014) and then remained high after NCT in the high-R group (P = .047), but it remained steady in the low-R group during NCT. A similar tendency in the Alu 260-bp level was revealed in different groups. The ∆∆Ct value of Alu 260-bp had good diagnostic efficiency in assessing predictive ability. The area under the curve for the ∆∆Ct1 and ∆∆Ct2 of Alu 260-bp was 0.697 and 0.647, respectively. The cfDNA level was closely related to epirubicin-induced apoptosis and changes in the Ki-67 index in vitro. The elevation of cfDNA after one chemotherapy cycle was mediated by the apoptosis of tumor cells and related to the improved chemotherapy response.

Keywords: apoptosis; biomarkers; breast cancer; neoadjuvant chemotherapy.

Figure 1

Correlation between cfDNA levels and patients with different Miller‐Payne grades. A, The Alu 111‐bp fragment levels increased after the first NCT in patients with Miller‐Payne 3‐5 grades. The Alu 111‐bp fragment levels remained stable during NCT in the low‐R group compared with the high‐R group. The Alu 260‐bp fragment levels increased after the NCT in both groups. CFDI remained at a constant level. B, No significant difference in the levels of both Alus and CFDI was found between different groups during the whole NCT

Figure 2

Correlation between cfDNA levels and patients with different changes in the Ki‐67 index. A, The levels of both Alus increased after the first and the eighth NCT in the high‐R group. The cfDNA level remained stable during NCT in the low‐R group. No significant difference was observed during NCT in any of the groups. B, No significant difference in both Alus and CFDI was found between different NCT response groups during NCT

Figure 3

Receiver operating characteristic (ROC) curves for cfDNA for discriminating the low‐response group from the high‐response group. AUC, Area under the curve. A, Relative levels of both Alus and a combined result before NCT. B, Relative levels of both Alus and a combined result after the first NCT. C, Relative levels of both Alus and a combined result after NCT. D, The ∆∆Ct1 (the difference between the ∆Ct at the first two points) of both Alus. E, The ∆∆Ct2 (the difference between the ∆Ct at the beginning and the end time points). F, Combined results of the ∆∆Ct1. G, Combined results of the ∆∆Ct2

Figure 4

The cfDNA levels in the supernatant after the treatment of epirubicin. A, A decrease and then increase in the Alu 111‐bp fragment levels in the supernatant was observed in both the MCF‐7 and MCF‐7/ADR cell lines. The Alu 111‐bp fragment levels decreased at the EPI concentration less than 1 μg/mL in the MCF‐7 cell line. The critical value was 4 μg/mL in the MCF‐7/ADR cell line. B, The Alu 260‐bp fragment levels decreased at the EPI concentration less than 2 μg/mL and then increased in the MCF‐7 cell line. No obvious change in the Alu 260‐bp fragment levels at different EPI concentrations was observed in the MCF‐7/ADR cell line

Figure 5

A, The cell apoptosis rate increased with the increasing concentration of EPI in the supernatant in the MCF‐7 cell line. The apoptosis rate was steady at the low concentration gradient of EPI and increased obviously until the concentration was 8 μg/mL in the MCF‐7/ADR cell line. B, The Ki‐67 index decreased with the increase in the EPI concentration in the MCF‐7 cell line. The Ki‐67 index showed minimal change in the MCF‐7/ADR cell line