K., Shiba K., Siljander P. leading cause by 2030 (= 2 stage III and = 2 stage IV). We found that the KRASG12D and P53mut marker panel Briciclib disodium salt fully separated these four samples from healthy control samples (= 5) and had, on average, greater than fivefold increased percentage of EV staining positive for the marker panel consisting of MUC1, EGFR, and FG-P4OH (Fig. 5C). To establish the correlation between the EV onco/tumor suppressor protein analysis and next-generation sequencing (NGS) of surgical samples, we analyzed samples where matching information was available (i.e., KRASG12D, = 13; KRAS wild type, = 8; P53mut, = 8; P53 wild type, = 15). KRASG12D could be detected in Briciclib disodium salt this cohort with 100% specificity and sensitivity (= 0.0002), and P53mut was detected at 100% specificity and 87.5% sensitivity (Fig. 5C). Open in a separate window Fig. 5. EV-based KRASG12D detection in clinical plasma sample.(A) Representative images of a stage 4 PDAC, demonstrating superb colocalization of KRASG12D staining with TFP-labeled EV (green). Note that the bulk EV analysis of this sample failed to detect KRASmut. (B) Larger FOV of the same plasma sample. In this image, the TFP-ROI mask of all labeled EV is depicted by gray outlines, while the KRASG12D staining is shown in red. Of 1618 EVs imaged (only a fraction is shown for better visibility), 1.24% were positive for KRASG12D. (C) Group analysis of EV. The top graphs show quantitative analysis of late-stage PDAC samples (= 4) against controls (= 5). Note the high levels of EV positivity for KRASmut/P53mut as well as other PDAC biomarkers. The bottom graphs show the correlation of EV positivity for KRASmut/P53mut against the known mutational status derived from sequencing of surgical tumor samples (= 24 samples). values were indicated by significance: 0.0332 (*), 0.0021 (**), 0.0002 (***), and 0.0001 (****). Note the excellent correlation between EV analysis and NGS results. See text for details. sEVA of plasma samples enables early cancer detection We next expanded the measurements to plasma samples from stage 1 pancreatic cancer patients (= 16), although this is a rare clinical scenario as most PDACs will present at Briciclib disodium salt a later stage. Nevertheless, early PDAC detection has high clinical value in high-risk groups (= 16) and in healthy controls (= 5). Note that virtually all stage 1 PDAC plasma samples contained EV positive for either KRASmut Briciclib disodium salt (row 1) or P53mut (row 2). This marker positivity is higher than for PDACEV alone (row 3), as only two samples were above background. We also determined the fraction of EV double positive for both KRASmut and P53mut (row 4) in an effort to increase specificity, but this was a rare event. When all markers were combined, 15 of the 16 samples had positive EV (row 5). The bottom graph shows the fraction of KRASmut- or P53mut-positive EV in plasma samples. When analyzing the stage 1 PDAC plasma samples, 15 of the 16 EV samples were positive for KRASmut and/or P53mut. All five samples with known KRAS mutation were positively identified, and another five samples with known P53 mutation were all positively identified. Only 1 1 of the 16 samples had dually positive EV staining for KRASmut and P53mut. For nine samples, the mutational status was not known, but five of these samples (56%) had EV positive for P53mut and seven samples (78%) had EV positive for KRASG12D, in line with the expected rate of these mutations in pancreatic cancer (= 25 patient samples were studied including healthy controls (= 5), surgically proven stage 1 PDAC (= 16), and biopsy-proven stage 2/4 PDAC (= 4; table S3). Of the stage 1 PDAC, sequencing information was available in seven patients; the remaining patient samples predated routine sequencing, or sequencing costs were not covered by the health care provider. Blood collection was optimized for plasma EV analysis, and all samples were deidentified and analyzed in blinded fashion. Briefly, whole blood was collected in one 10-ml purple-top EDTA tube and was inverted 10 times to mix. Whole blood was stored upright at 4C and processed within 1 hour of collection. To process blood for plasma isolation, the tube was Itgb1 centrifuged for 10 min at 400(4C). The plasma layer was collected in a 15-ml tube using a.