Background The ectonucleotidases CD39 and CD73 act sequentially to convert ATP into highly immunosuppressive adenosine that can accumulate in the extracellular milieu and suppress NK/T-cell anti-tumor responses. Blockade of CD73 in combination with other immunostimulatory antibodies can induce anti-tumor responses in subset of patients with non-small cell lung cancer (NSCLC). The levels, spatial distribution, clinicopathologic associations and biomarker potential of the CD39/CD73 pathway in NSCLC, as well as the impact of chemoradiotherapy, remain poorly understood.

Methods Using multiplexed quantitative immunofluorescence (mQIF), we simultaneously measured the levels of CD39, CD73 protein, CD8+ T-cells and cytokeratin (CK)-expressing cancer-cells in five retrospective NSCLC cohorts represented in tissue microarrays. The mQIF analysis included compartment-based measurements based on fluorescence co-localization as well as single-cell segmentation/phenotyping and spatial analysis. The first three cohorts included baseline tumor samples from patients with stages I-IV NSCLC treated with non-immunotherapy regimens (Cohort #1, n=179; Cohort #2, n=172; Cohort #3, n=267). The fourth cohort (Cohort #4, n= 68) included baseline tumor samples from patients treated with immunotherapy. The fifth cohort (Cohort #5, n=23 pairs) included paired biopsies before and after chemotherapy with or without radiotherapy. Associations between the markers with clinicopathologic variables and outcomes were studied.

Results 90% of NSCLCs showed detectable concurrent expression of both CD39 and CD73, but the levels were highly variable across cases. CD39+ cell density was higher in nonmalignant CK- stromal cell areas and CD73+ cell density was higher within the CK+ cancer cell nests. The levels of the markers were positively associated across the cohorts and high expression of CD39 or CD73 were consistently associated with higher local CD8+ T-cell infiltration and adenocarcinoma histology. CD8+ T-cells were more distant from CD73+ cancer cells than CD73- cancer cells. After chemoradiotherapy, CD73+ cells are significantly depleted. Stromal CD39+ cells and tumor CD73+ cells were associated with better outcomes after immunotherapy.

Conclusions The CD39/CD73 pathway is expressed in the majority of NSCLCs with a distinct tumor/stromal cell distribution. This pathway is associated with local adaptive anti-tumor immune responses, adenocarcinoma histology and better outcome after immunotherapy. Chemo-radiotherapy reduces the density of CD73+ expressing cells in the tumor microenvironment without significantly altering the spatial distribution of these cells relative to CD8+ TILs.

Acknowledgements Funding Source: AstraZeneca