Non-Smokers

Tumor slides from the internal training cohort were reviewed by 2 pathologists (K.K. and W.D.T.) who were blinded to patient clinical outcomes; they used an Olympus BX51 microscope (Olympus Optical Co. Ltd., Tokyo, Japan) with a standard 22-mm diameter eyepiece.
Tumor STAS was defined as tumor cells within air spaces in the lung parenchyma beyond the edge of the main tumor (Figure 1A and 1D) and was composed of 3 morphological patterns: 1) micropapillary structures consisting of papillary structures without central fibrovascular cores (Figure 1A and 1B),^{15 (link), 16 (link)} which occasionally form ring-like structures within air spaces (Figure 1C); 2) solid nests or tumor islands consisting of solid collections of tumor cells filling air spaces (Figure 1D and 1E)^{17 (link)}; and 3) single cells consisting of scattered discohesive single cells (Figure 1F). The edge of the main tumor was defined as the smooth surface of the tumor which is easily recognizable at gross or at low-power field examination as highlighted with the dotted line in Figure 1A. Tumor STAS was considered present when tumor STAS, as defined above, was identified beyond the edge of the main tumor even if it existed only in the first alveolar layer from the tumor edge. Lesions of STAS consist of tumor cells which morphologically appear to be situated within air spaces as micropapillary clusters, solid nests or single cells that are detached from alveolar walls. This differs from lepidic growth where tumor cells grow in a linear fashion along the surface of alveolar walls. Extent of air space filling by tumor cells varied from abundant cellular infiltrates to very inconspicuous single cells or micropapillary clusters that were sometimes difficult to distinguish from alveolar macrophages. In addition, distance between tumor surface and farthest STAS from tumor edge was measured by a ruler. Since lung specimens were not consistently inflated during processing, in order to account for artifactual atelectasis, we also measured according to the number of alveolar spaces.
Tumor cells of STAS were distinguished from alveolar macrophages using the following methods. Macrophages in smokers typically have cytoplasm containing faint brown pigment and black carbon granules while in nonsmokers the pigment is lacking and cytoplasm is sometimes foamy. Nuclei are small, uniform, and regular, without atypia. Nuclear folds are frequent and nucleoli are inconspicuous or absent. In contrast, tumor cells of STAS typically lack cytoplasmic pigment or foamy cytoplasm. They often grow in cohesive clusters and nuclei are atypical with hyperchromasia and frequent nucleoli. The distinction of STAS from artifacts was done in the following way. Tumor floaters were favored, by the presence of clusters of cells often randomly scattered over tissue and at the edges of the tissue section. Presence of jagged edges of tumor cell clusters suggested tumor fragmentation or edges of a knife cut during specimen processing rather than STAS. Linear strips of cells that were lifted off of alveolar walls also favored the presence of artifact. Identification of tumor cells distant from the main tumor was regarded as an artifact unless intraalveolar tumor cells could be demonstrated in a continuum of airspaces containing intraalveolar tumor cells back to the tumor edge.
According to the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society histological classification, the percentage of each histologic pattern—lepidic, acinar, papillary, solid, and micropapillary—was recorded in 5% increments and tumors were classified by their predominant pattern.^{1 (link)} Each histologic pattern was considered present in the tumor when it comprised ≥5% of the overall tumor.^{7 (link)} Presence of visceral pleural, lymphatic, and vascular invasion was also recorded.