Clinical and molecular correlates of digitally quantified anthracosis in lung tissue slides: utility of an automated quantitative workflow.

Air pollution, such as particulate matter with a diameter of ≤2.5 μm (PM2.5) is a major contributor to lung cancer in the never-smoking population. Anthracotic pigments (black deposits in the lungs) are physical evidence of environmental exposures. While links between anthracosis and lung disease have been established, anthracosis has not been routinely used as a measure of environmental exposure in research, because there is neither a standard quantitative measure of anthracosis nor an efficient method for quantifying it. We developed 'Slide-based methods for High-throughput Anthracosis Detection and Estimation' (SHADE). SHADE is an automated workflow that quantifies anthracotic pigments on scanned images of whole H&E slides. SHADE was optimized to identify anthracosis while avoiding detection of artefacts in images. SHADE scores and manual pathologist rankings of 10-image patches demonstrated high concordance (R = 0.864). Application of SHADE to background lung sections from 140 never-smoking lung cancer patients demonstrated significant associations between high anthracosis and older age, male sex, 30-year residential PM2.5 estimates, and birthplace in Asia (p < 0.05). Low anthracosis was associated with less aggressive adenocarcinomas (p = 0.052). No significant relationship was identified between anthracosis and 3-year residential PM2.5 estimates, lobe sampled, ethnicity, EGFR mutation status or pathologic tumor stage. Anthracosis levels in the never-smoking cohort were similar to that of background lung sections from 59 ever-smoking patients. Exploratory analysis of background lung sections of 15 individuals who had bronchoalveolar lavage specimen gene expression data available, SHADE scores were associated with the expression of several immunoregulatory genes (CEACAM6, CXCL6, IL5RA, LCN2, SPA17), the activation of inflammatory response and cytokine gene sets, and suppression of the antigen processing and presentation gene set (false discovery rate < 0.1). SHADE provides an automated workflow for anthracosis quantification and has demonstrated utility in uncovering insight into anthracosis-associated molecular changes in lung cancer.