Fig 1: High protein levels of ISG15 in cancerous tissues and serum of PA patients. (A) Protein expressions of ISG15 in cancerous and adjacent noncancerous tissues based on the IHC score (staining percentage × intensity). Typical images of IHC staining for ISG15 were magnified 100-fold, 200-fold, and 400-fold in the TMA (scale bars: 200 µm, 100 µm, and 50 µm from left to right). ISG15 expression was classified as follows: samples with IHC scores < 6 were defined as low expression, and scores = 6 were defined as high expression. The percentage of cells staining positive was scored as follows: < 10% (score = 0), 10%–25% (score = 1), 26%–50% (score = 2), 51%–75% (score = 3), and 76%–100% (score = 4). (B) Representative images of PDAC tissues with no staining/weak/moderate/strong ISG15 expression magnified 200-fold. Scale bars: 100 µm. The staining intensity was scored as follows: no staining (score = 0), weak (score = 1), moderate (score = 2), and strong (score = 3). (C) The proportion of cancerous (Figure 7C, left) and adjacent noncancerous (Figure 7C, right) tissues with ISG15 protein levels based on IHC scores (0 to 12). (D) Correlation between the protein level of ISG15 and histological type. IHC scores = 6 were considered high levels of ISG15. A P-value < 0.05 was considered to be statistically significant. (E) An ROC curve of ISG15 protein levels [high levels of ISG15 (IHC scores = 6) and low levels of ISG15 (IHC score < 6)] was used to differentiate cancerous tissues (n = 35) from adjacent noncancerous tissues (n = 22) (P < 0.001). (F) High protein levels of serum ISG15 in PA patients (n = 40) compared with those in healthy controls (n = 38) (P < 0.001). (G) A ROC curve of the serum ISG15 levels was used to differentiate PA patients from healthy controls. PDAC, pancreatic ductal adenocarcinoma; IHC, immunohistochemistry; TMA, tissue microarray; n, number, ROC, receiver operating characteristic; AUC, area under the ROC curve; CI, confidence interval.
Fig 2: Analyses of ISG15 mRNA expression in PA patients vs. normal controls for diagnosis. (A) Analysis of ISG15 mRNA expression in PA vs. normal pancreatic tissues from TCGA and GTEx datasets. The following settings were used for the analysis: “expression on box plots”, “gene = ISG15”, “|log2(FC)| cut-off = 1”, “P-value cut-off = 0.01”, “datasets = PAAD”, “log scale = log2(TPM + 1)”, “jitter size = 0.4”, and “matched TCGA normal and GTEx data”. (B) Heat map of ISG15 mRNA expression in PA vs. normal tissues with a P-value < 0.05 from 6 independent datasets from Oncomine. The 6 datasets included the Badea dataset29, Grutzmann dataset41, Iacobuzio-Donahue dataset42, Logsdon dataset43, Pei dataset30, and Segara dataset44. (C) ROC curve analysis of ISG15 mRNA expression with a P-value < 0.05 was used to diagnose PA from the 6 independent datasets from Oncomine. PA, pancreatic adenocarcinoma; T, tumor; N, normal; *P-value < 0.01; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression; FC, fold change; PAAD, pancreatic adenocarcinoma; TPM, transcripts per million; ROC, receiver operating characteristic; AUC, area under the ROC curve; CI, confidence interval.
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