Background

Small molecular inhibitors and immunotherapy have emerged as a novel alternative treatment regime for a variety of epithelial cancers. A large number of clinical trials are in progress worldwide to gauge the efficacy of tyrosine kinase inhibitors (TKI) or immune checkpoint inhibitors (ICI) against actionable targets such as receptor tyrosine kinases (RTKs) and program death ligand 1 (PD-L1). Although highly effective, the outcome of PD-L1 based ICI or TKI against RTKs is vitally contingent on the presence of PD-L1 or RTK expression on cancer cells. Determining druggable targets on the basis of solid biopsy could be often misleading, especially if a patient has progressed in spite of chemotherapy. This could be due to the intrinsic heterogeneity of tumor cells or therapeutic selection pressure or both, leading to alteration in the expression profile of the targets. Additionally, the immunohistochemistry result depends on a multitude of quality controls such as age and integrity of a biopsy sample, lab-to-lab variations in tissue block preparation, and degradation of targets due to chemical fixation methods. This unmet need of reliable detection and monitoring of actionable target expression could be addressed by circulating tumor cells (CTCs) based liquid biopsy tests. To this end, we have developed a CTC-based liquid biopsy assay to detect PD-L1, HER2, and EGFR expression in different epithelial cancers.

Methods

We retrospectively evaluated peripheral blood samples from a total of 134 carcinoma patients for the presence of CTCs expressing PD-L1, HER2, or EGFR markers respectively. Among these, 45% of patients had lung cancer, while 25% and 20% presented with breast, GI, and colorectal (CRC) malignancies. The remaining were gall bladder, ovarian, prostate, and head and neck cancer (HNC) patients. All lung cancer patients were analyzed for CTCs expressing PD-L1. CTCs were isolated from DCGI-approved OncoDiscover technology based on immunomagnetic targeting of epithelial cell surface molecules (EpCAM). EpCAM-targeted, magnetically isolated cells were considered CTCs on the basis of expression of cytokeratins, absence of CD45, and prominent presence of DAPI-stained nuclei. The presence or absence of aforesaid markers was determined using automated fluorescence imaging. Expression of PD-L1, HER2, or EGFR was detected by fluorescence microscopy using fluorescently labeled anti-PD-L1, HER2, or EGFR antibodies respectively. Based on fluorescence intensity, CTCs were binned as PD-L1, HER2, or EGFR negative for no detectable fluorescence signal, or weakly or strongly positive based on low or high fluorescence signal.

Results

Among the evaluated cohort, 51% of all CTCs showed the presence of PD-L1 expression, while 63% showed HER2-positive CTCs (all from breast cancer patients). 20% from the PD-L1 positive population showed strong PD-L1 expression. 78% of CTCs from lung cancer patients showed the presence of a detectable PD-L1 signal, while 66% of breast, GI, and CRC patients showed CTCs with PD-L1 expression. CTCs from HNC and gall bladder cancer patients showed the least PD-L1 expression (25% and 50% respectively). Among CTCs originating from different cancer types, breast cancer CTCs showed higher mean expression of PD-L1 compared to CTCs from colorectal cancer patients. A clear subset of CTCs for PD-L1 and HER2 expression was observed in lung and breast cancer patients respectively, suggesting heterogeneity in expression or the presence of different subclones within the same tumor type. Among all CTCs evaluated for EGFR expression, 50% showed the presence of detectable EGFR compared to the cut-off value.

Conclusions

CTCs isolated from cancers of epithelial origin showed the presence of PD-L1. Similarly, CTCs obtained from breast and lung cancer patients showed HER2 and EGFR expression respectively. Our data suggest that CTCs can be used as a real-time surrogate for molecular profiling of PD-L1, HER2, and EGFR expression, besides being a prognostic marker. Detection of PD-L1, HER2, and EGFR in CTCs offers a potential and viable alternative for immunotherapy or targeted therapy decisions in a vast majority of epithelial cancers.