Peptidase inhibitor 15 as a novel blood diagnostic marker for cholangiocarcinoma

Abstract

Background: We aimed to screen a specific secretory protein that could serve as blood diagnostic marker for cholangiocarcinoma (CCA).

Methods: Starting with the analysis of gene expression profiles in tumor tissues and matched normal tissues from cases with CCA and hepatocellular carcinoma (HCC), we identified peptidase inhibitor 15 (PI15) was a potential diagnostic marker for CCA. We demonstrated PI15 expression levels in CCA, HCC, and normal liver tissues. Furthermore, quantitative enzyme-linked immunosorbent assay (ELISA) assessed plasma PI15 levels in CCA (n = 61), HCC (n = 72), benign liver disease (n = 28), chronic hepatitis B (CHB) patients (n = 45), and healthy individuals (n = 45). The diagnostic value of PI15 was estimated by the area under the receiver operating characteristic (ROC) curve (AUC).

Findings: The positive rate of PI15 expression was 70% in CCA and only 9.1% in HCC; PI15 was not detected in normal liver tissue. High levels of plasma PI15 were evident in CCA patients, whereas only low levels were observed in cases involving HCC, benign liver disease, CHB patients, and healthy individuals. Plasma PI15 levels in CCA patients were obviously reduced (p = .0014) after surgery. The AUC of plasma PI15 for discriminating between CCA and HCC was 0.735. Furthermore, with a specificity of 94.44%, the combination of CA19-9 (>98.5 U/ml) and PI15 (>13 ng/ml) yielded a sensitivity of 80.39% for CCA and HCC.

Interpretation: PI15 exhibits promise as a novel marker for predicting the diagnosis and follow-up of CCA patients. FUND: Natural Science Research Foundation of Anhui Province and Natural Science Foundation of China.

Keywords: Biomarker; Blood diagnosis; Cholangiocarcinoma; PI15.

Fig. 1

Discovery of a candidate marker for CCA. (a) Gene expression profile assay of CCA tumor tissues and matched normal tissues for screening candidate diagnostic markers. Heat map shown differentially expressed secretory proteins and tumor markers in CCA. The left heat map was based on the gene expression profile of CCA tumor tissues and matched normal tissues. The right heat map was based on CCA (CHOL, n = 36) RNA-seq data from the TCGA database. (b) Gene expression profile assay of HCC tumor tissues and matched normal tissues. Heat map shown selected secretory proteins and tumor markers in HCC. The left heat map was based on microarray data of HCC tumor tissues and matched normal tissues. The right heat map was based on HCC (LIHC, n = 374) RNA-seq data from the TCGA database. Each column depicts an individual sample. Blue squares represent normal tissues, yellow squares represent tumor tissues. RNA-seq data were normalized with MultiExperiment Viewer. (c) The positive rate of PI15 expression in various tumors. (d) The fold change of PI15 expression in various tumors. CCA (CHOL), cholangiocarcinoma (n = 36); HCC (LIHC), hepatocellular carcinoma (n = 374); PAAD, pancreatic adenocarcinoma (n = 178); STAD, stomach adenocarcinoma (n = 375); COAD, colon adenocarcinoma (n = 480); READ, rectum adenocarcinoma (n = 167); LUSC, lung squamous cell carcinoma (n = 502); LUAD, lung adenocarcinoma (n = 535); KIRP, kidney renal papillary cell carcinoma (n = 289); KIRC, kidney renal clear cell carcinoma (n = 539); KICH, kidney chromophobe (n = 65); BRCA, breast invasive carcinoma (n = 312); and PRAD, prostate adenocarcinoma (n = 499). Tumor samples are from the TCGA database. Red columns indicate positive expression of PI15, blue columns indicate negative expression of PI15.

Fig. 2

Expression of secretory protein PI15 was significantly upregulated in CCA. (a) The expression of PI15 and tumor markers (AFP, CEA, CA125, PSA, and GH) were determined by reverse transcription PCR (RT-PCR) in CCA tumor tissues (n = 10) and matched normal tissues (n = 10). Each band represents a different patient sample. (b) The expression of PI15 and tumor markers (AFP, CEA, CA125, PSA, and GH) were determined by RT-PCR in normal liver tissues (n = 5), HCC tumor tissues (n = 11), and matched normal tissues (n = 11). Each band represents a different patient sample. (c-d) ROC curve analysis of the expression of PI15 and tumor markers (assessed by RNA-seq) for discriminating tumor tissue from normal tissue in CCA (CHOL, n = 36) and HCC (LIHC, n = 374). The genes that encode the tumor markers (AFP, CEA, CA125, PSA, and GH) were AFP, CEACAM5, MUC16, KLK3, and GH1, respectively. PI15–1 and PI15–2 were the two primers used to amplify PI15. CCA/CHOL, cholangiocarcinoma; HCC/LIHC, hepatocellular carcinoma; Tumor adjacent, matched normal tissue; Normal liver, normal liver tissue.

Fig. 3

PI15 as a potential diagnostic marker for CCA. (a) Plasma PI15 levels were measured by quantitative ELISA in CCA (HBV-) patients (n = 51), CCA (HBV+) patients (n = 10), HCC patients (n = 72), benign liver disease patients (n = 28), CHB patients (n = 45), and healthy individuals (n = 45). (b) Plasma PI15 levels in iCCA (n = 26), pCCA (n = 12), and dCCA (n = 13) patients. (c) ROC curves for PI15 levels in plasma samples from patients with CCA (HBV-) patients (n = 51) versus HCC patients (n = 72), benign liver disease patients (n = 28), CHB patients (n = 45), and healthy individuals (n = 45). HBV-, HBV negative; HBV+, HBV positive; Benign, benign liver disease; CHB, chronic hepatitis B; Normal, healthy individuals; iCCA, intrahepatic cholangiocarcinoma; pCCA, perihilar cholangiocarcinoma; dCCA, distal cholangiocarcinoma. Unpaired t-test; Data are presented as mean ± SEM.

Fig. 4

The combination of PI15 and CA19–9 improves diagnostic performance for CCA. (a) ROC curves for PI15, CA19–9, and PI15 + CA19–9 levels in patients with CCA (HBV-) patients (n = 51) versus HCC patients (n = 72), benign liver disease patients (n = 28), CHB patients (n = 45), and healthy individuals (n = 45). (b) ROC curves for PI15, CA19–9, and PI15 + CA19–9 levels in patients with iCCA (HBV-) patients (n = 26) versus HCC patients (n = 72), benign liver disease patients (n = 28), CHB patients (n = 45), and healthy individuals (n = 45). HBV-, HBV negative; CHB, chronic hepatitis B; Normal, healthy individuals; iCCA, intrahepatic cholangiocarcinoma.

Fig. 5

Determination of plasma PI15 level for the prediction of postoperative recovery in CCA patients. (a) The levels of plasma PI15 and serum CA19–9 in preoperative (day “-1”) and postoperative (days “4” and “7”) CCA patients (n = 27) were measured by quantitative ELISA and electrochemiluminescence respectively. The PI15 and CA19–9 concentration of representative CCA cases were shown. (b) Plasma PI15 levels in preoperative (day “-1”) and postoperative (days “4” and “7”) HCC patients (n = 30) were measured by quantitative ELISA. The PI15 concentration of representative HCC cases were shown. (c) Plasma PI15 levels in preoperative (day “-1”) and postoperative (days “4” and “7”) benign liver disease patients (n = 20) were measured by quantitative ELISA. The PI15 concentration of representative benign liver disease cases were shown. The abscissa of the coordinate axis is the number of days after surgery (postoperative days), and “-1” refers to the preoperative day. Before, before surgery; After, after surgery. Paired t-test; Data are presented as mean ± SEM.