Neutrophil Biomarkers Can Predict Cardiotoxicity of Anthracyclines in Breast Cancer

Abstract

Doxorubicin (DOX), a commonly used anticancer agent, causes cardiotoxicity that begins with the first dose and may progress to heart failure years after treatment. An inflammatory response associated with neutrophil recruitment has been recognized as a mechanism of DOX-induced cardiotoxicity. This study aimed to validate mRNA expression of the previously identified biomarkers of DOX-induced cardiotoxicity, PGLYRP1, CAMP, MMP9, and CEACAM8, and to assay their protein expression in the peripheral blood of breast cancer patients. Blood samples from 40 breast cancer patients treated with DOX-based chemotherapy were collected before and after the first chemotherapy cycle and > 2 years after treatment. The protein and gene expression of PGLYRP1/Tag7, CAMP/LL37, MMP9/gelatinase B, and CEACAM8/CD66b were determined using ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each candidate biomarker. Patients with cardiotoxicity (n = 20) had significantly elevated levels of PGLYRP1, CAMP, MMP9, and CEACAM8 at baseline, after the first dose of DOX-based chemotherapy, and at > 2 years after treatment relative to patients without cardiotoxicity (n = 20). The first dose of DOX induced significantly higher levels of all examined biomarkers in both groups of patients. At > 2 years post treatment, the levels of all but MMP9 dropped below the baseline. There was a good correlation between the expression of mRNA and the target proteins. We demonstrate that circulating levels of PGLYRP1, CAMP, MMP9, and CEACAM8 can predict the cardiotoxicity of DOX. This novel finding may be of value in the early identification of patients at risk for cardiotoxicity.

Keywords: ELISA; RT-qPCR; anthracyclines; cardiotoxicity; innate immunity; neutrophils.

Figure 1

PGLYRP1, CAMP, MMP9, and CEACAM8 mRNA expression levels are increased in the blood of breast cancer patients treated with DOX-based chemotherapy after the 1st cycle (T1) of DOX-based chemotherapy in comparison with the baseline (T0). (A) Histogram showing the ΔCt values of PGLYRP1, CAMP, MMP9, and CEACAM8 in PBMCs of patients with cardiotoxicity (n = 16) and patients without cardiotoxicity (n = 16). Each dot represents one sample, and the mean ± SD for each group is indicated. (B) Fold change (2^-ΔΔCt) for each of the examined genes comparing T1 and T2, considering the T0 as a calibrator. (C) Receiver operating characteristic (ROC) curves for the ability of blood levels of the four mRNAs to predict DOX-induced cardiotoxicity in breast cancer. NCTX, non-cardiotoxicity; CTX, cardiotoxicity; PGLYRP1, peptidoglycan recognition protein 1; CAMP, cathelicidin antimicrobial peptide; MMP9, matrix metallopeptidase; CEACAM8, carcinoembryonic antigen-related cell adhesion molecule 8. p-value, * < 0.001, ** < 0.01, *** > 0.1.

Figure 2

Protein levels of PGLYRP1, CAMP, MMP9, and CEACAM8 are increased in the plasma of breast cancer patients after the 1st chemotherapy cycle and at >1 year after chemotherapy in comparison with the baseline. (A) Scatter plots demonstrating the comparison of the protein concentrations of the examined proteins in the plasma of breast cancer patients with cardiotoxicity (CTX) and non-cardiotoxicity (NCTX) at baseline (T0), after the first chemotherapy dose (T1), and at >1 year post treatment (T2). Each dot represents one sample, and the mean and SD for each group is indicated. (B) Protein expression levels of the examined markers in comparison with the cutoff values. (C) Receiver operating characteristic (ROC) curves for all examined neutrophil markers at CTX_T1 versus HC + NCTX_T0 + CT_T0. NCTX, non-cardiotoxicity; CTX, cardiotoxicity; PGLYRP1, peptidoglycan recognition protein 1; CAMP, cathelicidin antimicrobial peptide; MMP9, matrix metallopeptidase; CEACAM8 carcinoembryonic antigen-related cell adhesion molecule 8. AUC = area under the curve. p-Value, * < 0.05, ** > 0.01.

Figure 3

Pairwise Pearson correlations between plasma markers among the study samples by cardiotoxicity and by time points. CTX, cardiotoxicity; NCTX, non-cardiotoxicity. (A) NCTX at T0; (B) NCTX at T1; (C) NCTX at T2; (D) CTX at T0; (E) CTX at T1; (F) CTX at T2.