Background Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. Results Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33C100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. Conclusions Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the specificity of CTC identification and the accuracy of biomarker evaluation. Introduction Circulating tumor cells (CTCs) are rare cells that can be detected in the blood of patients with solid tumors and have been explored as a form of liquid biopsy [1C3]. Enumeration of CTCs following epithelial cell adhesion molecule (EpCAM) based capture serves as a prognostic and predictive biomarker in different malignancies such as prostate  and breast cancer , as well as non-small cell lung cancer (NSCLC) . However, the limited biological readout of enumeration DAPT does not inform on the wide range of therapeutic targets for patients with advanced cancer. Given the variety of new treatments in early phase and advanced clinical trials, there is a critical need to develop new biomarkers that may predict the benefits of these treatments and identify early signs of therapeutic resistance. Lung cancer is one of the DAPT most common and lethal types of cancer, with a 5-year survival rate of ~15% when diagnosed with metastatic disease . Immunotherapy has revolutionized the treatment of advanced lung cancer, leading to durable responses and improved survival benefit in a subset of patients with NSCLC [8C10]. Therefore, recent therapeutic advances in checkpoint inhibition have given rise to interest in developing predictive biomarkers. Programmed death ligand-1 (PD-L1) expression is quantified on tumor biopsies prior to PD-L1 based treatment of patients with NSCLC, DAPT yet the invasive nature of biopsy often leads clinicians to test archived biopsies that may not be representative of the disease after exposure to chemotherapy or targeted therapies. Thus, evaluating CTCs for PD-L1 expression could be useful as a surrogate to tumor biopsies. The standard molecular definition of a CTC is based on positive detection of cells with an intact nucleus that express cytokeratin (CK) but are negative for CD45, a white blood cell (WBC) marker . While FDA cleared, this limited definition of a CTC does not account for the diversity of WBCs in circulation with low or absent expression of CD45 such as neutrophils , myeloid-derived suppressor cells (MDSCs) , or other immature blasting myeloid populations . Confounding the issue to an even greater extent is the increase in neutrophil number in patients with progressive cancer , as well as evidence that neutrophils stain positive for CK , raising concerns over the specificity of Rabbit Polyclonal to MMP-2 traditional CTC identification approaches. These factors may be even more significant when evaluating CTC biomarkers amidst phenomena such as epithelial-mesenchymal transition (EMT) given the expression of EMT-related proteins such as Vimentin  and CD44  on neutrophils. However, EMT-based CTC capture may be required in NSCLC as traditional capture with EpCAM was shown to be effective in only DAPT 20% of samples from patients with metastatic NSCLC , as opposed to 57% from those with prostate cancer . Furthermore, PD-L1 can be expressed to varying degrees by neutrophils  and myeloid subsets , complicating blood sample analysis, and confounding any assay purporting to evaluate CTC immune biomarkers. These issues are particularly challenging for antibody-independent methods of CTC capture (i.e., filter.