The prognostic and therapeutic implications of circulating tumor cell phenotype detection based on epithelial-mesenchymal transition markers in the first-line chemotherapy of HER2-negative metastatic breast cancer

Abstract

Background: Epithelial-mesenchymal transition (EMT) is implicated in the metastatic process and presents a challenge to epithelial cell adhesion molecule-based detection of circulating tumor cells (CTCs), which have been demonstrated to be a prognostic indicator in metastatic breast cancer. Although evidence has indicated that heterogeneity of CTCs based on EMT markers is associated with disease progression, no standard recommendations have been established for clinical practice. This study aimed to evaluate the prognostic significance of dynamic CTC detection based on EMT for metastatic breast cancer patients.

Methods: We enrolled 108 human epidermal growth factor receptor 2-negative metastatic breast cancer patients from the prospective phase III CAMELLIA study and applied the CanPatrol CTC enrichment technique to identify CTC phenotypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) in peripheral blood samples. Receiver operating characteristic curve analyses of total CTC count and the proportion of mesenchymal CTCs for predicting the 1-year progression-free survival (PFS) rate were conducted to determine the optimal cut-off values, and Kaplan-Meier analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic value of the cut-off values of both total CTC count and the proportion of mesenchymal CTCs in combination.

Results: For predicting the 1-year PFS rate, the optimal cut-off value of total CTC count was 9.5 (Area under the curve [AUC] = 0.538, 95% confidence interval [CI] = 0.418-0.657), and that of the proportion of mesenchymal CTCs was 10.7% (AUC = 0.581, 95% CI = 0.463-0.699). We used the two cut-off values in combination to forecast PFS in which the total CTC count was equaled to or exceeded 10/5 mL with the proportion of mesenchymal CTCs surpassed 10.7%. Patients who met the combined criteria had significantly shorter median PFS than did those who did not meet the criteria (6.2 vs. 9.9 months, P =0.010). A nomogram was constructed based on the criteria and significant clinicopathological characteristics with a C-index of 0.613 (P = 0.010).

Conclusions: The criteria, which combine the total CTC count and the proportion of mesenchymal CTCs, may be used to monitor therapeutic resistance and predict prognosis in patients with metastatic breast cancer. Trial registration ClinicalTrials.gov. NCT01917279. Registered on 19 July 2013, https://clinicaltrials.gov/ct2/show/NCT01917279?term=NCT01917279&rank=1 .

Keywords: Breast cancer; Circulating tumor cells; Epithelial–mesenchymal transition; Prognosis; Therapeutic implication.

Fig. 1

Receiver operating characteristic curves of total CTC count and the proportion of M + CTCs for predicting 1-year PFS rate. CTC circulating tumor cell, M + CTCs mesenchymal CTCs, PFS progression-free survival

Fig. 2

Kaplan-Meier analysis-based estimation of PFS probabilities of metastatic breast cancer patients in the EMT and non-EMT groups. Patients in the EMT group met the combined criteria with a total CTC count equaled to or exceeded 10 per 5 mL peripheral blood and a proportion of M + CTCs surpassed 10.7%; Patients in the non-EMT group did not meet the criteria. PFS progression-free survival

Fig. 3

A prognostic nomogram for predicting PFS of patients with metastatic breast cancer. PFS progression-free survival, HR hormone receptor

Fig. 4

Comparison of total CTC count, CTC phenotype, serum tumor markers, and radiographic examination in identifying disease progression. a For patient No. 01058, the M + CTC count and their proportion in total CTCs manifested a continuous declining tendency and the total CTC count was fluctuated when the patient responded to chemotherapy. Moreover, when disease progression presented in the liver and pleura, the count and proportion of M + CTCs increased significantly compared with the prior measurement, while the total CTC count and the level of CA153 continued to descend. b For patient No. 01131, the total CTC count and M + CTC count were obviously increased 3 months earlier than the imaging evidence of disease progression. CTC circulating tumor cell; E + CTCs epithelial CTCs, E +/M + CTCs biphenotypic epithelial/mesenchymal CTCs, M + CTCs mesenchymal CTCs, CA153 carbohydrate antigen 153, CEA carcinoembryonic antigen, PR partial response, SD stable disease, PD progressive disease. The arrows and circles on the radiographic image were used to indicate the lesions