Analysis of potential immunological markers associated with anti-PD-1 therapy in non- small cell lung cancer patients

Background: Immune checkpoint blockade represents a major breakthrough in advanced non-small cell lung cancer (NSCLC) therapy. However, success is limited to a subset of patients and identification of robust biomarkers associated with clinical response is strongly requested. In this study, we assessed whether pre-existing immunological features, as well as immune parameters measured during treatment, might result informative in providing clinical guidance. Methods: Starting from a cohort of advanced NSCLC patients (n=74) treated with nivolumab, we investigated blood samples collected at baseline and during treatment. Frequency of various circulating lymphocyte subsets was analyzed by flow cytometry. Potential blood cellular biomarkers were then correlated with overall survival (OS) as well as clinical response, assessed by RECIST criteria, by which patients have been stratified in partial response (PR), stable disease (SD) and progressive disease (PD). Soluble factors in plasma samples were evaluated using MILLIPLEX® MAP Kits (Luminex Technology) in 10 CD and 10 PD patients at different time points. Results: Patients characterized by longer OS displayed higher frequency of CD3+ T cells (p =0.048) but lower amounts of NK cells (p = 0.002), at baseline. Moreover, they displayed a statistically significant lower expression of PD-1 on both CD3+ and CD8+ T cells (p=0.013 and p=0.033, respectively). With regard to pretreatment level of exhausted T cells, patients grouped as controlled disease (CD), including PR and SD patients, showed a significant lower frequency than PD group (p=0.046). Notably, in CD patients, the frequency of exhausted CD8+ T cells further decreased during treatment (p=<0.0001, p=0.0032, and p=0.0239, respectively). With regard to plasma samples, we found an increase of IP-10 and MCP-1 levels during nivolumab therapy in CD patients. Conclusions: Our results suggest that the distribution of lymphocyte subsets and expression of PD-1 on T cells before treatment could indicate the outcome of anti-PD-1 treatment in NSCLC patients. In addition, assessing the initial amount of exhausted T cells as well as their decrease upon treatment could also predict response and clinical outcome. Furthermore, the modulation of IP-10 and MCP-1 soluble factors in NSCLC patients during nivolumab therapy may represent novel circulating biomarkers of controlled or uncontrolled disease.