BackgroundMyocardial programmed death-ligand 1 (PD-L1) expression is implicated in immune checkpoint inhibitor (ICI)-associated myocarditis. Measurement of myocardial PD-L1 expression may have potential use as a mechanistic and predictive biomarker. The aim of this study was to determine non-invasive assessment of myocardial PD-L1 expression using [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT.MethodsThoracic [99mTc]NM-01 SPECT/CT was performed in lung cancer patients (n= 10) at baseline and 9-weeks following anti-programmed cell death protein 1 (PD-1) therapy. Baseline and 9-week left ventricular and right ventricular to blood pool ratios (LVmax:BP) and (RVmax:BP) were measured. LVmaxwas compared to background skeletal muscle (musclemax). Intra-rater reliability was determined by intraclass correlation coefficient (ICC) and Bland–Altman analysis.ResultsMean LVmax:BP values were 2.76 ± 0.67 at baseline vs 2.55 ± 0.77 at 9 weeks (p= 0.42). Mean RVmax:BP was 1.82 ± 0.32 at baseline vs 1.76 ± 0.45 at 9 weeks (p= 0.67). Myocardial PD-L1 expression was at least threefold greater than skeletal muscle at baseline for the LV (LVmaxto musclemax3.71 ± 0.77 vs 0.98 ± 0.20 (p< 0.001)) and at least twofold for the RV (LVmaxto musclemax2.49 ± 0.63 vs 0.98 ± 0.20 (p< 0.001)). There was excellent intra-rater reliability for LVmax:BP with ICC 0.99 (95% confidence interval 0.94–0.99,p< 0.001), mean bias -0.05 ± 0.14 (95% limits of agreement -0.32 to 0.21). There were no major adverse cardiovascular events or myocarditis during follow-up.ConclusionThis study is the first to report PD-L1 expression of the heart that can be quantified non-invasively without invasive myocardial biopsy, with high reliability and specificity. This technique can be applied to investigate myocardial PD-L1 expression in ICI-associated myocarditis and cardiomyopathies.Clinical trial registrationPD-L1 Expression in Cancer (PECan) study (NCT04436406).https://clinicaltrials.gov/ct2/show/NCT04436406June 18th, 2020.