CTCs were isolated in 17/33 (51.5%) of non-small-cell lung cancer (NSCLC) patients. CTCs were determined to be PD-L1-positive in 11/17 (64.7%) NSCLC cases. 3D chromosomal DNA FISH for ALK and EGFR molecular targets showed better resolution than in 2D when imaging CTCs. PFS was not found to be associated with CTCs prior to therapy ([HR]:2.246; 95% [CI]:0.9565–5.273; p = 0.0632), nor the presence of PD-L1 expression ([HR]:1.646; 95% [CI]:0.5128–5.283; p = 0.4023) in NSCLC patients. The gene discussed is EGFR; the disease is non-small cell lung carcinoma.