Background <p>Programmed death-ligand 1 (PD-L1) immunohistochemistry is a predictive biomarker for anti-PD-(L)1 therapy in non-small cell lung cancer (NSCLC). It is not a reliable predictor of clinical benefit with non-invasive imaging providing a potential solution. We present the PECan study, the aim of which to assess the relationship of [^99mTc]-labeled anti-PD-L1 single-domain antibody (NM-01) single-photon emission computed tomography (SPECT)/CT with metabolic response to anti-PD-(L)1.</p> Methods <p>PD-L1 tumour proportion score (TPS) measured using SP263 assay. [^99mTc]NM-01 SPECT/CT and [¹⁸F]FDG PET/CT performed before and 9-weeks following pembrolizumab with/without chemotherapy in patients with advanced NSCLC. Tumor (T) to blood pool (BP) maximum region of interest (ROI_max) measurements performed in primary and metastatic lesions using SPECT/CT images.</p> Results <p>Fifteen patients were included (median age 63 years, 9 male). Intertumoural heterogeneity evident in 10(67%) patients. Mean [^99mTc]NM-01 T:BP demonstrated moderate correlation with PD-L1 TPS (<i>r</i> = 0.45, <i>p</i> &lt; 0.05). Depth of [¹⁸F]FDG PET/CT metabolic response at 9-weeks (<i>n</i> = 13), correlated strongly with baseline [^99mTc]NM-01 T:BP (<i>r</i> = −0.73, <i>p</i> &lt; 0.05), but only moderately with PD-L1 TPS (<i>r</i> = −0.46, <i>p</i> = 0.06).</p> Conclusion <p>[^99mTc]NM-01 SPECT/CT allows non-invasive quantification of PD-L1 in primary tumour and metastases in NSCLC. [^99mTc]NM-01 uptake moderately correlates with PD-L1 immunohistochemistry, determines heterogeneity, and is associated with early metabolic response to anti-PD-1 pembrolizumab.</p> Clinical trials registration <p>PD-L1 Expression in Cancer (PECan) study (NCT04436406), registered 18 June 2020 <a href="https://clinicaltrials.gov/ct2/show/NCT04436406">https://clinicaltrials.gov/ct2/show/NCT04436406</a></p>