Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis

Abstract

Objective: A detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options.

Design: We analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling.

Results: Patients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but ROBO2 mutations occurred exclusively in invasive CCA and CTNNB1 mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin.

Conclusion: Integrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.

Keywords: bilary duct carcinoma; cholangiocarcinoma; gene mutation.

Conflict of interest statement

Competing interests: PS: Grant, boards and presentations from Novartis, and boards from Incyte. CP: Advisory board honoraria from BioMedX. BG: Advisory board from Novartis.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Study design, sample and patient characterisation. (A) A total of 54 patients with IPNB or ITPN neoplasms of the biliary tree were included in this study. The heatmap indicates the available DNA sequencing and DNA methylation/CNA data for IPNB/ITPN precursor lesions, invasive CCA and normal tissue. For 45 precursors lesions all three analyses, DNA sequencing, DNA methylation and CNA data were obtained. (B) For DNA sequencing, precursor lesions of 48 patients and 26 corresponding invasive CCA samples were analysed. (C) Genome-wide DNA methylation and genome-wide CNA profiling was performed for precursor lesions of 51 patients and compared with 41 corresponding non-neoplastic bile duct (normal) and 27 corresponding invasive CCA samples. (D) Immunohistochemical and histomorphological subtyping was performed for the entire cohort; immunohistochemical characterisation of one representative IPNB (top row) and one representative ITPN (bottom row) case is depicted. (E) Kaplan-Meier survival curves of IPNB and ITPN cases. (F) Kaplan-Meier survival curves of IPNB/ITPN-CCA cases with UICC2 or UICC3 compared with non-IPNB/ITPN-CCA cases of an independent cohort with matched numbers of UICC2 or UICC3, respectively. CCA, cholangiocarcinoma; CNA, copy number alteration; IPNB, intraductal papillary neoplasm of the bile duct; ITPN, intraductal tubulopapillary neoplasm of the bile duct; NA, not available.

Figure 2

Massive parallel sequencing reveals frequent genetic alterations in intraductal neoplasms of the bile duct. (A) Circos plot representing co-occurrence of mutations in all 48 non-invasive IPNB and ITPN samples. A band connecting genes represents co-occurring mutations in a given patient. The width of the band represents the frequency of this mutation pair within the dataset. (B) Oncoplot of genomic alterations in IPNB (n=39) and ITPN (n=9) samples. Missense mutations, in-frame mutations, truncations, amplificationsand deletions of 48 patients with intraductal neoplasms are shown. AMP, amplification; DEL, deletion; INFRAME, in-frame mutation; IPNB, intraductal papillary neoplasm of the bile duct; ITPN, intraductal tubulopapillary neoplasm of the bile duct; TRUNC, truncation.

Figure 3

IPNB/ITPN and corresponding invasive CCA tissue samples share genetic molecular profiles. (A) Oncoplot of genomic alterations in IPNB/ITPN (n=26) and (B) in the corresponding invasive CCA samples (n=26). Missense mutations, in-frame mutations, truncations, amplifications and deletions are shown. Genes (rows) of both oncoplots (A, B) were sorted by the total number of mutations in all samples. In addition, the order of samples (columns) in both oncoplots is based on the number of alterations in the precursor samples; thus, invasive CCA are sorted identical to the corresponding precursor lesions. (C) Overlap of specific mutations, that is, mutation of a certain amino acid in the respective gene, present in IPNB/ITPN precursor lesions and corresponding invasive CCA. (D) Shown are the proportions of mutations observed exclusively in the precursor lesions, exclusively in the invasive CCA or shared by the precursor lesion and corresponding invasive CCA in the indicated genes. (E) Diagram depicting the mutational alterations during progression from non-neoplastic normal bile to precursor and invasive CCA. AMP, amplification; DEL, deletion; INFRAME, in-frame mutation; IPNB, intraductal papillary neoplasm of the bile duct; ITPN, intraductal tubulopapillary neoplasm of the bile duct; ITPN, intraductal tubulopapillary neoplasm of the bile duct; TRUNC, truncation.

Figure 4

Copy number alteration profiles of normal, IPNB or ITPN and invasive CCA samples. (A) Copy number alterations of normal bile duct controls, (B) IPNB, (C) ITPN, (D) iCCA, (E) pCCA and (F) dCCA samples are shown along the chromosomes on the x-axes. The relative frequency of observed gains (green) and losses (red) are depicted above and below the horizontal line, respectively. CCA, cholangiocarcinoma; dCCA, distal CCA; iCCA, intrahepatic CCA; IPNB, intraductal papillary neoplasm of the bile duct; ITPN, intraductal tubulopapillary neoplasm of the bile duct; pCCA, perihilar CCA.

Figure 5

DNA methylation profiles of IPNB and ITPN precursor lesions. (A) Unsupervised hierarchical clustering analysis of the triplet normal bile duct, precursor and corresponding invasive CCA of 24 patients. The total number of samples is 72. (B) t-SNE plot of the DNA methylation profiles of bile duct controls of the corresponding IPNB, ITPN and ITPN-P cases (n=50), IPNB (n=41), ITPN (n=10) and ITPN-P (n=9). (C) MeDeCom analysis of IPNB (n=41), ITPN (n=10) and ITPN-P (n=9) samples and (D) of 27 IPNB and ITPN samples with corresponding invasive CCA. CCA, cholangiocarcinoma; IPNB, intraductal papillary neoplasm of the bile duct; ITPN, intraductal tubulopapillary neoplasm of the bile duct; ITPN-P, ITPN of the pancreas.