ABSTRACT: Abstract Introduction: PD-(L)1 immunotherapy (IO) is a mainstay first-line (1L) treatment for advanced non-small-cell lung cancer without actionable alterations (aNSCLC), but reliable biomarkers are lacking. This study explored the potential utility of longitudinal blood gene expression profiling in this setting. Methods: 165 patients with aNSCLC and 1LIO as monotherapy (n=60), or combined with chemotherapy (CHTIO, n=71) were prospectively analyzed along with patients receiving 2LIO (n=34) and age-matched controls (n=21). PAXgene blood RNA was analyzed by quantitative PCR (qPCR) and Affymetrix U133 Plus 2.0 microarrays, including median fold changes [FC] and odds ratio (OR) between groups, with control of the false discovery rate (FDR). Progression-free survival (PFS) was verified through review of radiological images based on RECIST v1.1. Routine blood cell counts and PD L1 tumor proportion score (TPS) were available from the patients’ records. Results: Significantly higher neutrophil (FC 1.66, p<0.001) and lower lymphocyte counts (FC 0.65, p<0.001) in the blood of untreated patients vs. age-matched controls were significantly associated with shorter PFS (hazard ratio [HR] 1.94 for neutrophil/lymphocyte ratio [NLR] ≥5, p<0.001) and reduced expression of GATA3, FOXP3 and other T-cell specific genes (p<0.01). After 4 cycles of 1L treatment, blood neutrophil counts normalized (FC 0.59 vs. baseline, p<0.001), but lymphopenia persisted. Furthermore, IO monotherapy, but not CHTIO, was accompanied by an upregulation of T cell specific genes, like PD1, GATA3 and FOXP3, whose higher expression after 4 cycles showed stronger associations with longer PFS (HR 0.28-0.31, p<0.05) and long-term responses (LTR) >1year (OR 12-19x, p<0.01) than NLR and PD L1 TPS. Affymetrix profiling revealed profound deregulation of many immunologic pathways in the patients’ blood before and during treatment, whose prognostic value was linked to IO monotherapy, but not CHTIO, and more pronounced after 4 treatment cycles (n=19285 genes associated with LTR, p<0.05, FDR <0.1) compared to baseline (n=610 genes). Gene expression changes, NLR and PD-L1 TPS were independent IO biomarkers in multivariable analyses. Conclusions: Transcriptomic perturbations are pronounced and readily detectable by qPCR in the blood of NSCLC patients. While the prognostic utility of immunologic gene expression alterations at baseline in the blood was limited by the patients’ initial leucocytosis, an upregulation of PD1, GATA3, FOXP3 and other T-cell related genes specifically under IO monotherapy was strongly associated with clinical benefit and could serve as a novel IO biomarker independent of blood cell counts and tumor PD L1 expression.
