Serum ITIH5 as a novel diagnostic biomarker in cholangiocarcinoma

Abstract

Cholangiocarcinoma often remains undetected until advanced stages due to the lack of reliable diagnostic markers. Our goal was to identify a unique secretory protein for cholangiocarcinoma diagnosis and differentiation from other malignancies, benign hepatobiliary diseases, and chronic liver conditions. We conducted bulk RNA-seq analysis to identify genes specifically upregulated in cholangiocarcinoma but not in most other cancers, benign hepatobiliary diseases, and chronic liver diseases focusing on exocrine protein-encoding genes. Single-cell RNA sequencing examined subcellular distribution. Immunohistochemistry and enzyme-linked immunosorbent assays assessed tissue and serum expression. Diagnostic performance was evaluated via receiver-operating characteristic (ROC) analysis. Inter-alpha-trypsin inhibitor heavy chain family member five (ITIH5), a gene encoding an extracellular protein, is notably upregulated in cholangiocarcinoma. This elevation is not observed in most other cancer types, benign hepatobiliary diseases, or chronic liver disorders. It is specifically expressed by malignant cholangiocytes. ITIH5 expression in cholangiocarcinoma tissues exceeded that in nontumorous bile duct, hepatocellular carcinoma, and nontumorous hepatic tissues. Serum ITIH5 levels were elevated in cholangiocarcinoma compared with controls (hepatocellular carcinoma, benign diseases, chronic hepatitis B, and healthy individuals). ITIH5 yielded areas under the ROC curve (AUCs) from 0.839 to 0.851 distinguishing cholangiocarcinoma from controls. Combining ITIH5 with carbohydrate antigen 19-9 (CA19-9) enhanced CA19-9's diagnostic effectiveness. In conclusion, serum ITIH5 may serve as a novel noninvasive cholangiocarcinoma diagnostic marker.

Keywords: ELISA; ITIH5; cholangiocarcinoma; diagnosis; serum marker.

FIGURE 1

Experimental analysis flowchart. COAD, colon adenocarcinoma; CHOL, bile duct cancer; EP‐upDEGs, exocrine protein‐upregulated differentially expressed genes; ESCA, esophageal cancer; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic cancer; READ, rectum adenocarcinoma; scRNA‐seq, single‐cell RNA sequencing; STAD, stomach cancer.

FIGURE 2

Identification of a potential cholangiocarcinoma (CCA) marker. (A) Venn diagram of intersecting genes of upregulated differential expression genes in TCGA‐CHOL and genes encoding secreted proteins. (B) The volcano plot of upregulated differentially expressed genes in TCGA‐CHOL. The genes in the white box represent 39 exocrine protein‐upregulated differentially expressed genes (EP‐upDEGs), among which ITIH5 is the most prominently elevated gene. (C) Expression of ITIH5 across various cancer types. Red dotted boxes represent ITIH5 expression in CCA. (D) The tumor group displayed significantly higher ITIH5 expression than the normal CCA group. (E) There was no significant difference in ITIH5 expression between tumor and nontumor groups in hepatocellular carcinoma (HCC). CHOL, bile duct cancer.

FIGURE 3

Single‐cell RNA sequencing (scRNA‐seq) analysis. (A) The Uniform Manifold Approximation and Projection (UMAP) visualization revealed 23 distinct cell clusters. (B) The distribution of ITIH5 expression in the adjacent samples. (C) The distribution of ITIH5 expression in the tumor samples. (D) The dot plot illustrated the expression of cell‐type‐specific marker genes for clusters 1, 3, 5, and 6.

FIGURE 4

The diagnostic value of ITIH5 at the mRNA level for cholangiocarcinoma (CCA). (A) The positive rate of ITIH5 in the various cancer types. (B) Fold change in ITIH5 expression in the various cancer types. (C) Heatmap illustrating the expression level of ITIH5 and the tumor markers in TCGA‐CHOL. (D) Heatmap illustrating the expression level of ITIH5 and the tumor markers in TCGA‐LIHC. (E) ROC curve illustrating the diagnostic significance of ITIH5 and the tumor markers in CCA. (F) ROC curve illustrating the diagnostic significance of ITIH5 in distinguishing between CCA and hepatocellular carcinoma (HCC). CHOL, bile duct cancer; LIHC, liver hepatocellular carcinoma.

FIGURE 5

The expression of ITIH5 in benign hepatobiliary diseases and chronic hepatic diseases. (A–F) The expression levels of ITIH5 between the control and case groups in IHCA (GSE11819), liver angioma (GSE62029), PSC (GSE159676), NASH (GSE159676), and other liver diseases (GSE159676), respectively. CLD, chronic liver diseases; IHCA, inflammatory hepatocellular tumor; NASH, nonalcoholic steatohepatitis; PSC, primary sclerosing cholangitis.

FIGURE 6

ITIH5 immunohistochemistry analysis. (A) Cholangiocarcinoma (CCA) tissues exhibited positive cytoplasm and membrane staining for ITIH5. The right panel displays an enlarged view of the area highlighted by the black box in the left panel. (B–D) ITIH5 expression in nontumorous bile duct, hepatocellular carcinoma (HCC), and nontumorous hepatic tissues, respectively. (E) The immunohistochemistry (IHC) score of ITIH5 expression levels in CCA, nontumorous bile duct, HCC, and nontumorous hepatic tissues. ***p < 0.001. Scale, 50 μm.

FIGURE 7

Serum ITIH5 levels and diagnostic accuracy assessment. (A) Serum ITIH5 levels in CCA, HCC, Benign, CHB, and Healthy. (B) Serum ITIH5 levels in iCCA, pCCA, and dCCA. (C–G) The ROC analysis results for ITIH5, CA19‐9, and the ITIH5/CA19‐9 panel in discriminating CCA patients from those with HCC, Benign, CHB, Healthy and NCCA, respectively. (H–L) The ROC analysis results for ITIH5, CA19‐9, and the ITIH5/CA19‐9 panel in discriminating iCCA patients from those with HCC, Benign, CHB, Healthy, and NCCA, respectively. ***p < 0.001. ns, no statistical differences. Benign, benign hepatobiliary diseases; CHB, chronic hepatitis B; CCA, cholangiocarcinoma; dCCA, distal cholangiocarcinoma; Healthy, healthy individuals; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; pCCA, perihilar cholangiocarcinoma; NCCA, the total patients without CCA.