Figure 1. CD117 expression in normal lung (NL) and NSCLC.

Photomicrographs: Expression of CD117 and Ki-67 in NSC lung cancer and normal lung. The left columns show sections stained with CD117 (red), cytokeratin (green) and DAPI (blue). Sections in the center column show CD117 (red) and DAPI (blue) only, in order to reveal CD117 staining obscured by bright cytokeratin expression. Sections in the right column shows CD117 (red), the proliferation marker Ki67 (green) and DAPI (blue). Tumors were classified as KIT+ or negative on the basis of CD117 immunofluorescent staining of FFPE. In KIT+ tumors (top photomicrographs) CD117 (red stain, center and right panels) was expressed in virtually all cytokeratin+ tumor cells (green stain, left panels). Ki-67+ proliferating cells (green stain, right panels) were frequently seen among CD117+ tumor cells. In KIT negative tumors (center row of photomicrographs), only solitary CD117+ mast cells were detected (red stain, center and right panels). Proliferating Ki-67+ cells were frequent among cytokeratin+ CD117 negative tumor cells. Normal tumor-adjacent lung also appeared to lack CD117 expression among cytokeratin+ airway cells (bottom photomicrographs). Proliferating Ki-67+ cells were infrequent and confined to the basal layer of airway epithelium. When all NSCLC tumors are considered together, flow cytometry revealed bimodal CD117 expression (center panels A-C). Cells were gated on hematopoietic lineage negative singlet events with DNA content ≥2N, as detailed in supplementary figure 1. The percent of CD117+ cells, among cytokeratin positive cells, plotted on a linear scale, allowed the distinction between CD117low (≤20% positive) and CD117high (>21%). An example of CD117 detection on a tumor single cell suspension is given for a KIT negative tumor (panel A) and a KIT+ tumor (panel C). Arrows indicate the corresponding data points (open circles). The percent CD117+ cells among cytokeratin+ cells, shown on a log scale, reveals the presence of detectable CD117+/cytokeratin+ cells in normal lung, KIT “negative” tumors, and malignant pleural effusions (MPE, panel D). The proportion of cells expressing CD117 in KIT+ tumors was statistically significant from that of normal lung (NL, p = 0.004), KIT negative tumor (p = 0.008) and MPE (p = 0.001). Panel E shows the relative intensity of CD117 expression on CD117+ cells, as measured by CD117 mean fluorescence intensity (MFI). In KIT+ tumors, CD117 expression on CD117+ cells was significantly greater than that of normal lung (NL, p = 0.002), KIT negative tumor (p = 0.026) and malignant pleural effusion (MPE, p = 0.002). The filled red circle indicates that the sole outlier in CD117 expression in the KIT negative group, is at the top of the range of percent CD117+. Box plots (panels B, D, E) indicate the sample median and quartiles, the whiskers indicate the sample range, exclusive of outliers.