Figure 2

Identification of unique EV and circulating miRNA profiles Lung-RADS4 cancer patients. (a-l) Next Generation Sequencing (NGS) of blood EV or circulating miRNA identified Let-7b-5p, miR-184, and miR-22-3p as differentially expressed in NSCLC patients compared to controls. Let-7b-5p (a,b) and mir-184 (c,d) are deregulated in EV and mir-22-3p in plasma (i,j). The relative abundance of the indicated miRNA in screening controls (blue) versus confirmed diseased patients only (green) or versus all lung-RADS2 plus false-positive lung-RADS4 (magenta) are shown in (a–d) and (i,j), respectively. P values were Benjamini–Hochberg adjusted. *p < 0.1, **p < 0.05, ***p < 0.01, ****p < 0.001. Note that 2–3 patient samples with undetectably low reads were excluded from the analysis. Corresponding Reads per million (RPM) were used to plot miRNA levels in confirmed cancer patients versus disease-free individuals and to perform ROC analysis shown in (e–h,k,l). EdgeR generalized linear models (GLM) were used to assess significance of miRNA regulations. Multiple logistic regression analysis was performed to determine the combined classification performance of the three miRNA biomarker candidates (m). Statistical significance of miRNA regulations was determined by EdgeR GLM, as indicated above. (n–p) Quantitative Polymerase Chain Reaction (qPCR) data showing expression fold changes (means) of let-7b-5p (n) or miRNA-184 (o) or miRNA-21-5p (n) or miRNA-22-3p (p) in EV samples obtained from confirmed cancer patients (IV +) versus screening controls (II). RNA samples were pooled from 14 cancer patients and 14 randomly selected screening individuals. Expression was normalized to GAPDH. Error bars denote SD values. P values are derived from student t-test analyses.