Epithelial/Mesenchymal Characteristics and PD-L1 Co-Expression in CTCs of Metastatic Breast Cancer Patients Treated with Eribulin: Correlation with Clinical Outcome

SIMPLE SUMMARY: Circulating tumor cells (CTCs) detected in cancer patients as single cells or in clusters are of prognostic value. There is currently no information concerning the co-expression of mesenchymal markers (important for metastasis) and PD-L1 (immune system suppressor) in CTCs of metastatic breast cancer patients. In this study, we aimed to evaluate the co-expression of these markers in single and cluster CTCs and to determine if there is any relationship with patients’ outcome after eribulin treatment. CTCs were detected and phenotypically analyzed by quantifying keratins (epithelial marker), vimentin (mesenchymal marker) and PD-L1 using immunofluorescence and confocal microscopy. We found, for the first time, distinct CTC subpopulations, such as cluster and single PD-L1(+) mesenchymal CTCs, at baseline and eight days after eribulin administration, respectively, that were associated with worse prognosis. Our study supports the role of CTC analysis in the investigation of mechanisms of resistance and disease progression in real time. ABSTRACT: We aimed to evaluate the co-expression of PD-L1 and epithelial-mesenchymal markers in CTCs from metastatic breast cancer (MBC) patients and to determine if there is any relationship with patients’ outcome after eribulin treatment. Using cytospin preparations of peripheral blood mononuclear cells (PBMCs) from MBC patients treated with eribulin and a combination of immunocytochemistry and immunofluorescence, we quantified PD-L1, keratins and vimentin in single and cluster CTCs on days 1 and 8 of the first-treatment cycle. CTCs (n = 173) were found in 31 out of 38 patients. At baseline, the presence of cluster CTCs (p = 0.048), cluster mesenchymal CTCs (mCTCs) (p = 0.0003) or cluster PD-L1(+)mCTCs (p = 0.006) was associated with shorter overall survival (OS). In multivariate cox regression analysis, the detection of cluster mCTCs was the only parameter associated with increased risk of death (p = 0.024). On day 8 post-eribulin administration, PD-L1(+)mCTCs and especially single PD-L1(+)mCTCs decreased in 75% and 89% of patients, respectively. The detection of single PD-L1(+)mCTCs after eribulin treatment was correlated with shorter PFS (p = 0.047) and OS (p = 0.020). In conclusion, our study identified for the first time that cluster and single PD-L1(+)mCTCs subpopulations are of clinical significance in patients with MBC and highlighted the importance of CTC phenotyping during treatment with eribulin.