Phyllis A. Gimotty

Supplemental Figure 2. Box-and-whisker plots with data points in jittered overlay show the mRNA expression levels of PIGR and ANPEP in tumor samples from across the TCGA cohort “pan-cancer analysis of whole genomes” (ICGC/TCGA, Nature 2020)

Supplemental Table 2: LFQ MS Significant Protein Output from WUSTL

Supplemental Table 1: Plasma Pool Composition

Supplemental Table 6.1: Univariate Biomarker Analysis of Penn Phase 2 Cohort – Disease Control Reference Supplemental Table 6.2: Two-variable Biomarker Analysis of Penn Phase 2 Cohort – Disease Control Reference Supplemental Table 6.3: Multivariable Analysis of Penn Phase 2 Cohort – Disease Control Reference

TMT-labeled MS Significant Protein Output from Wistar

Supplemental Table 7: Biomarker concentration cutoff points based on percentiles of distribution in Mayo Phase 2 control plasma samples (n = 140).

Supplemental Table 8.1: PIGR values by sex Supplemental Table 8.2: ANPEP values by sex Supplemental Table 8.3: PIGR values by diabetes mellitus status Supplemental Table 8.4: ANPEP values by diabetes mellitus status Supplemental Table 8.5: Spearman correlation analysis of age and ANPEP or PIGR values

Purpose:Pancreatic ductal adenocarcinoma (PDAC) is typically detected too late for useful therapeutic interventions; hence, we sought blood biomarkers to detect early-stage disease.Experimental Design:Using mass spectrometry and ELISA on plasma pools from the University of Pennsylvania (Penn) and the Mayo Clinic (Mayo), we identified aminopeptidase N (ANPEP) and polymeric immunoglobulin receptor (PIGR) as increased in early-stage (stage I/II) PDAC plasma compared with controls. We tested ANPEP and PIGR, along with prior data for thrombospondin 2 (THBS2) and carbohydrate antigen 19-9 (CA19-9), in retrospective phase II studies using separate cohorts of PDAC plasmas at different stages versus healthy or nonmalignant disease controls (DC) from Penn (n = 135) and Mayo (n = 537).Results:Comparing healthy controls with stage I/II PDAC, we obtained area under the receiver-operating characteristic curves (AUC) of 0.78 [95% confidence interval (CI), 0.68–0.86]/0.80 (95% CI, 0.74–0.85; ANPEP) and 0.81 (95% CI, 0.70–0.88)/0.86 (95% CI, 0.82–0.90; PIGR) for the Penn/Mayo phase II studies, respectively. In multivariable models, CA19-9/THBS2/ANPEP, CA19-9/THBS2/PIGR, and CA19-9/THBS2/ANPEP/PIGR elicited AUC of 0.94 to 0.96 for Penn and 0.97 for Mayo. Notably, the four-marker panel elicited AUC of 0.87 for the Mayo stage I/II versus DC and 0.91 for stages I to IV versus DC. At a specificity of 95%, a plasma biomarker panel composed of CA19-9 (≥35 U/mL), THBS2 (≥42 ng/mL), ANPEP (≥2,995 ng/mL), and PIGR (≥1,800 ng/mL) yielded a sensitivity of 91.9% for PDAC stages I to IV and 87.5% for PDAC stage I/II.Conclusions:Adding ANPEP and PIGR to a plasma biomarker panel of CA19-9 and THBS2 enhances the detection of early-stage PDAC when comparing cancer versus healthy or nonmalignant DC. Given the concordance of our data in two retrospective phase II studies, assessments in prediagnostic cases are warranted.

Supplemental Table 5: Demographic and clinical characteristics of patients whose samples were used in Mayo Phase 2