Multi-Marker Immunofluorescent Staining and PD-L1 Detection on Circulating Tumour Cells from Ovarian Cancer Patients.

Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the Parsortix™ system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.007) and mesenchymal (p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1.

PD-L1 Expression on Circulating Tumor Cells May Be Predictive of Response to Pembrolizumab in Advanced Melanoma: Results from a Pilot Study.

BACKGROUND: PD-1 inhibitors are routinely used for the treatment of advanced melanoma. This study sought to determine whether PD-L1 expression on circulating tumor cells (CTCs) can serve as a predictive biomarker of clinical benefit and response to treatment with the PD-1 inhibitor pembrolizumab. METHODS: Blood samples were collected from patients with metastatic melanoma receiving pembrolizumab, prior to treatment and 6-12 weeks after initiation of therapy. Multiparametric flow cytometry was used to identify CTCs and evaluate the expression of PD-L1. RESULTS: CTCs were detected in 25 of 40 patients (63%). Patients with detectable PD-L1+ CTCs (14/25, 64%) had significantly longer progression-free survival (PFS) compared with patients with PD-L1- CTCs (26.6 months vs. 5.5 months; p = .018). The 12-month PFS rates were 76% versus 22% in the PD-L1+ versus PD-L1- CTCs groups (p = .012), respectively. A multivariate linear regression analysis confirmed that PD-L1+ CTC is an independent predictive biomarker of PFS (hazard ratio, 0.229; 95% confidence interval, 0.052-1.012; p = .026). CONCLUSION: Our results reveal the potential of CTCs as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with melanoma. PD-L1 expression on CTCs may be predictive of response to pembrolizumab and longer PFS. IMPLICATIONS FOR PRACTICE: The present data suggest that PD-L1 expression on circulating tumor cells may predict response to pembrolizumab in advanced melanoma. This needs further validation in a larger trial and, if proven, might be a useful liquid biopsy tool that could be used to stratify patients into groups more likely to respond to immunotherapy, hence leading to health cost savings.

Is the Blood an Alternative for Programmed Cell Death Ligand 1 Assessment in Non-Small Cell Lung Cancer?

Anti-programmed cell death (PD)-1/PD-ligand 1 (L1) therapies have significantly improved the outcomes for non-small cell lung cancer (NSCLC) patients in recent years. These therapies work by reactivating the immune system and enabling it to target cancer cells once more. There is a general agreement that expression of PD-L1 on tumour cells predicts the therapeutic response to PD-1/PD-L1 inhibitors in NSCLC. Hence, immunohistochemical staining of tumour tissue biopsies from NSCLC patients with PD-L1 antibodies is the current standard used to aid selection of patients for treatment with anti-PD-1 as first line therapy. However, issues of small tissue samples, tissue heterogeneity, the emergence of new metastatic sites, and dynamic changes in the expression of PD-L1 may influence PD-L1 status during disease evolution. Re-biopsy would expose patients to the risk of complications and tardy results. Analysis of PD-L1 expression on circulating tumour cells (CTCs) may provide an accessible and non-invasive means to select patients for anti-PD-1 therapies. Additionally, CTCs could potentially provide a useful biomarker in their own right. Several published studies have assessed PD-L1 expression on CTCs from NSCLC patients. Overall, analysis of PD-L1 on CTCs is feasible and could be detected prior to and after frontline therapy. However, there is no evidence on whether PD-L1 expression on CTCs could predict the response to anti-PD-1/PD-L1 treatment. This review examines the challenges that need to be addressed to demonstrate the clinical validity of PD-L1 analysis in CTCs as a biomarker capable of predicting the response to immune checkpoint blockade.