Analysis of N-linked Glycan Alterations in Tissue and Serum Reveals Promising Biomarkers for Intrahepatic Cholangiocarcinoma

Abstract

There is an urgent need for the identification of reliable prognostic biomarkers for patients with intrahepatic cholangiocarcinoma (iCCA) and alterations in N-glycosylation have demonstrated an immense potential to be used as diagnostic strategies for many cancers, including hepatocellular carcinoma (HCC). N-glycosylation is one of the most common post-translational modifications known to be altered based on the status of the cell. N-glycan structures on glycoproteins can be modified based on the addition or removal of specific N-glycan residues, some of which have been linked to liver diseases. However, little is known concerning the N-glycan alterations that are associated with iCCA. We characterized the N-glycan modifications quantitatively and qualitatively in three cohorts, consisting of two tissue cohorts: a discovery cohort (n = 104 cases) and a validation cohort (n = 75), and one independent serum cohort consisting of patients with iCCA, HCC, or benign chronic liver disease (n = 67). N-glycan analysis in situ was correlated to tumor regions annotated on histopathology and revealed that bisected fucosylated N-glycan structures were specific to iCCA tumor regions. These same N-glycan modifications were significantly upregulated in iCCA tissue and serum relative to HCC and bile duct disease, including primary sclerosing cholangitis (PSC) (P < 0.0001). N-glycan modifications identified in iCCA tissue and serum were used to generate an algorithm that could be used as a biomarker of iCCA. We demonstrate that this biomarker algorithm quadrupled the sensitivity (at 90% specificity) of iCCA detection as compared with carbohydrate antigen 19-9, the current "gold standard" biomarker of CCA.

Significance: This work elucidates the N-glycan alterations that occur directly in iCCA tissue and utilizes this information to discover serum biomarkers that can be used for the noninvasive detection of iCCA.

FIGURE 1

Bisected and biantennary fucosylated structures are highly expressed in iCCA tumor. Representative images of the relative intensity of biantennary fucosylated N-glycan (1809.646 m/z); (A) bisected fucosylated N-glycan (2012.717 m/z); (B) tetraantennary branched N-glycan (2393.840 m/z); (C) high mannose N-glycan (1905.630 m/z); (D) proposed N-glycan structures at the bottom correspond to the respective m/z value (observed mass). (E) H&E staining. Tumor regions are outlined in red, normal areas are outlined in black, and fibrotic regions are outlined in blue. Intrahepatic Cholangiocarcinoma (iCCA), and Hepatocellular Carcinoma (HCC). For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.

FIGURE 2

Bisected fucosylated N-glycan alterations are specific to patients with iCCA in tissue—analysis in a discovery and validation tissue cohort. Representative images of the relative intensity of bisected/triantennary single fucosylated N-glycan (1850.667 m/z); (A) double fucosylated N-glycan (1996.720 m/z); (B) double fucosylated with a galactose N-glycan (2158.791 m/z); (C) tetraantennary branched N-glycan (2393.824 m/z) N-glycan (D) and high mannose N-glycan (1905.644 m/z) in TMA 1 (E). This TMA includes two cores per patient: 50 HCC, 20 iCCA, and 6 normal hepatic tissue. OLD: other liver diseases. (F–J) same N-glycans as in A–E but analyzed in a second independent validation tissue cohort (TMA 2). This TMA includes one core per patient: 45 HCC, 23 iCCA, and 5 normal hepatic tissue. (K–O) relative intensity quantification of both TMAs comparing iCCA (n = 43) v non iCCA (n = 108). Each point in box plots represents a patient. The mass defect used for each m/z value is based on TMA 1 run. The asterisk indicates statistical difference (Mann–Whitney, P < 0.001) and error bars represent the SD. Darker red colors represent a higher intensity for the specific glycan while more blue tones represent less intensity. For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.

FIGURE 3

Bisected core fucosylated N-glycan alterations are specific to patients with iCCA in tissue. (A) Cartoon description of Endo F3 (including mass shift) and PNGase F cleavage sites, and the respective FUTs that catalyze the addition of the fucose residue. Representative images of core fucosylated N-glycans after Endo F3 treatment on TMA2, bisected core fucosylated N-glycan (B) bisected core, and outer-arm fucosylation N-glycan (C) bisected core, and outer-arm fucosylation with two galactose residues N-glycan (D) and tetraantennary core fucosylated N-glycan (E). For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.

FIGURE 4

Fucosylated N-glycans are significantly altered in iCCA serum. (A) Serum N-glycan imaging workflow. Relative intensity quantification of bisected double fucosylated N-glycan (2158.791 m/z); (B) triantennary fucosylated N-glycan (2174.779 m/z); (C) triantennary double fucosylated N-glycan (2320.808 m/z); (D) and tetraantennary double fucosylated N-glycan (2361.849 m/z); (E) biantennary N-glycan (1339.463 m/z); (F) Each point in box plots represents a patient. The asterisk indicates statistical difference (Mann–Whitney, P < 0.001) and error bars represent the SD. Non iCCA n = 62 and iCCA n = 30. The mass defect used for each m/z value is based on TMA 1 run. For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.

FIGURE 5

Profile data clustering reveals N-glycan grouping based on the number of fucose residues in iCCA serum and tissue. Dendrograms representing clustering for 12 N-glycans (A) and 6 N-glycans (B) in serum and TMAs. Glycan (G), G1: 1217, G2: 1339, G3:1501, G4:2158, G5:2174, G6:2320, G7:2631, G8:2465, G9:2487, G10:2539, G11:2685, G12:2852. PCA and their dimension (Dim) of serum (C) and TMAs (D) showing N-glycan clustering based on the number of fucose residues. 2Na (doubly sodiated N-glycan). For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.

FIGURE 6

N-glycans from serum and TMA as promising biomarkers to differentiate iCCA from other liver diseases. (A) ROC curve for serum. Non iCCA n = 62 and iCCA n = 30. (B) ROC curve for TMA, non iCCA n = 108 and iCCA n = 43. For ROC curve labeling: N-glycan 1339 m/z (green-dashed line), N-glycan 2158 m/z (blue-dashed line), and a combination of N-glycans 1339 m/z and 2158 m/z (red-solid line). (C) Relative intensity quantification boxplots of N-glycan 1339 m/z and 2158 m/z in serum, non iCCA n = 62, and iCCA n = 30. (D) Representative images of TMA 1 showing the N-glycan intensity for 1339 m/z and 2158 m/z. The asterisk indicates statistical difference (Mann–Whitney, P < 0.001) and error bars represent the SD. For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.

FIGURE 7

N-glycan combinations as promising biomarkers to differentiate iCCA from PSC. (A) ROC curve classification of a combination of 1339 m/z, 2158 m/z, and 1257 m/z. (B) ROC curve of combination of 1339 m/z, 2158 m/z, 1257 m/z, and CA19-9. For ROC curves: iCCA (n = 30) and PSC (n = 17). For N-glycans, red triangle, fucose; blue square, N-acetylglucosamine; green circles, mannose; yellow circles, galactose.