Summary: cholangiocarcinogenesis and FGFR2 fusion signalling

Cholangiocarcinoma arises predominantly from cholangiocytes and can also develop from peribiliary glands and hepatocytes. Chronic inflammation, growth factors, and bile acids in the tumour microenvironment drive carcinogenesis, and the tumours are highly desmoplastic. Genomic alterations track with anatomic subtype: FGFR2 fusions, IDH1 and IDH2 mutations, and BAP1 mutations characterize intrahepatic disease, and in one sequenced cohort the most commonly altered genes in intrahepatic cholangiocarcinoma were IDH1 (30%), ARID1A (23%), BAP1 (20%), TP53 (20%), and FGFR2 fusions (14%).

FGFR2 fusions retain the extracellular and kinase domains of FGFR2 and lose the C-terminal negative regulatory region, and truncation of FGFR2 after exon 17 is oncogenic in itself. Fusion partners exhibit oligomerization capability, and fusion proteins interact in the absence of FGF ligand, so kinase activation is ligand-independent. Signalling proceeds through the MAPK (RAS, RAF, MEK, ERK) and PI3K, AKT, and mTOR pathways; expression of FGFR2 fusion kinases activated MAPK and conferred transforming ability in cell models, and patient-derived organoids were dependent on Ras-Erk signalling. Fusion partners are diverse: 56 different partners were identified among 107 participants in FIGHT-202, 42 (75%) unique to one participant, with BICC1 the most common (29%). FGFR2 fusions usually co-occur with inactivation of TP53, CDKN2A, or BAP1, and in a Japanese series of 102 cholangiocarcinomas they were mutually exclusive with KRAS and BRAF mutations. All tested FGFR2 rearrangement partners were oncogenic and sensitive to FGFR2 inhibition in vitro, which the authors interpret as indicating that the FGFR2 fusion, irrespective of partner gene, is the clinically important variable.

Pathophysiology: cell of origin and desmoplastic tumour immune microenvironment

“CCA displays features of cholangiocyte differentiation and probably arises predominantly from the epithelial cells lining the bile ducts, which are termed cholangiocytes; however, the cancers may also develop from peribiliary glands and hepatocytes, depending on the underlying liver disease and location” (opens the source at this quote in a new tab)
“These cancers are highly desmoplastic and are enmeshed in dense networks of inflammatory cells and matrix termed the tumour immune microenvironment” (opens the source at this quote in a new tab)

“CCA tumours, and particularly iCCAs and pCCAs, are characterized by a reactive desmoplastic stroma containing cancer-associated fibroblasts (CAFs) that crosstalk with CCA cells secreting paracrine factors such as heparin-binding EGF-like growth factor, stromal-cell derived factor 1 (SDF1), platelet-derived growth factor (PDGF)-B and extracellular matrix (ECM) proteins” (opens the source at this quote in a new tab)
“CAFs secrete a multitude of signalling molecules (such as IL-1β, PDGF-B, heparin-binding EGF-like growth factor and SDF1) that promote cancer progression by enhancing proliferation, survival, chemotaxis and angiogenesis” (opens the source at this quote in a new tab)
“Following stimulation by PDGF-D originated from the tumoural cholangiocytes in vitro, CAFs secrete VEGF-A and VEGF-C, which recruit and assemble lymphatic endothelial cells in vascular structures susceptible to tumour cell intravasation” (opens the source at this quote in a new tab)

Pathophysiology: cholangiocarcinogenesis driven by inflammatory and growth factor signalling

“Cholangiocarcinogenesis is orchestrated by a complex interplay of extracellular ligands (such as pro-inflammatory cytokines, growth factors and bile acids, among others), which are present in the tumour microenvironment (TME), and increased expression and/or aberrant activation of cell surface receptors and the deregulation of intracellular signalling pathways, finally leading to cell proliferation, survival and genetic and/or epigenetic alterations” (opens the source at this quote in a new tab)
“The most common genes that might be mutated or amplified resulting in the overactivation of some of these pathways are KRAS, BRAF, ARID1, PBRM1, BAP1, IDH1 and IDH2.” (opens the source at this quote in a new tab)
“The main oncogenic networks comprised WNT-CTNNB1, MYC, ERBB, TNF and VEGF signalling, emphasizing cell survival signalling pathways in patients with poor OS” (opens the source at this quote in a new tab)

Pathophysiology: genomic alterations differ by anatomic subtype

“Among CCAs, genetic aberrations differ depending on their anatomic locations; for example, FGFR2 fusions are almost exclusively found in iCCAs, whereas PRKCA–PRKCB fusions are observed in pCCAs and dCCAs” (opens the source at this quote in a new tab)

“Small bile duct iCCA can be characterized by isocitrate dehydrogenase (IDH1, IDH2) mutations or fibroblast growth factor receptor 2 (FGFR2) fusions” (opens the source at this quote in a new tab)

Pathophysiology: frequency of recurrent alterations in a real-world profiling series

“The most frequently altered genes were TP53 (51/91, 56.0%), KRAS (21/91, 23.1%), CDKN2A (18/91, 19.8%), SMAD4 (14/91, 15.4%), and ARID1A/PBRM1 (12/91, 13.2%).” (opens the source at this quote in a new tab)

FGFR2 fusion structure: retained kinase domain and loss of the C-terminal regulatory region

“Approximately 10%–15% ICCs show genetic alterations of FGFR2, most commonly involving in-frame fusions of FGFR2 exons 1–17 with a diversity of partners” (opens the source at this quote in a new tab)
“These fusions, as well as other truncating events that are sometimes observed, contain the entire FGFR2 extracellular and kinase domains but lack a C-terminal negative regulatory domain encoded by the final exon 18, leading to constitutive kinase activity” (opens the source at this quote in a new tab)

“FGFR2 fusions are the most common FGFR2 alteration in iCCA, occurring in 10% to 15% of cases” (opens the source at this quote in a new tab)
“It was recently demonstrated that the dimerization domain of a fusion partner is not a requirement for oncogenicity; rather the truncation of FGFR2 after exon 17” (opens the source at this quote in a new tab)
“is an oncogenic alteration itself, in which loss of the C-terminus is the key determinant of oncogenicity” (opens the source at this quote in a new tab)

Ligand-independent oligomerization of FGFR fusion proteins

“All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation.” (opens the source at this quote in a new tab)
“As examples, while FGFR3-BAIAP2L1, FGFR3-TACC3, FGFR2- BICC1, and FGFR2-CCDC6 interact in vitro, wild-type FGFR2 and FGFR3 do not in the absence of FGF ligands (Fig. 3A, Supplementary Fig. 2).” (opens the source at this quote in a new tab)

Transforming activity of FGFR2 fusion kinases and MAPK activation

“We performed massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations and identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1.” (opens the source at this quote in a new tab)
“Expression of the fusion kinases in NIH3T3 cells activated MAPK and conferred anchorage-independent growth and in vivo tumorigenesis of subcutaneous transplanted cells in immune-compromised mice. This transforming ability was attributable to its kinase activity.” (opens the source at this quote in a new tab)

“Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation.” (opens the source at this quote in a new tab)

FGFR2-AHCYL1 and FGFR2-BICC1 fusions: genomic origin, constitutive activation, and mutual exclusivity with KRAS and BRAF mutations

“The FGFR2-AHCYL1 and FGFR2-BICC1 fusion proteins are likely to form homodimers through the coiled-coil motif of AHCYL1” (opens the source at this quote in a new tab)
“FGFR2 and BICC1 are located on the long arm of chromosome 10 in opposite directions, suggesting that the FGFR2-BICC1 fusion is generated by intrachromosomal inversion (Supporting Fig. 1B).” (opens the source at this quote in a new tab)
“The wild-type fusion expressing cells showed constitutive tyrosine phosphorylation in the activation loop of the FGFR kinase domain.” (opens the source at this quote in a new tab)
“Immunoblot analysis revealed that activation of MAPK, but not AKT or STAT3, was induced in clones expressing FGFR2-AHCYL1 and FGFR2-BHCC1.” (opens the source at this quote in a new tab)
“KRAS mutations were detected in 19 cases (19/102, 17.8%) and BRAF mutations in one (1/102, 1%); these mutations were mutually exclusive with the FGFR2 fusions (Fig. 3A; Supporting Table 4).” (opens the source at this quote in a new tab)
“The identification of two recurrent FGFR2 fusions (FGFR2-AHCYL1 and FGFR2-BICC1) that are mutually exclusive with KRAS/BRAF mutations warrants a new molecular classification of cholangiocarcinoma and suggests a novel therapeutic approach in cholangiocarcinomas driven by these fusions.” (opens the source at this quote in a new tab)
“Association between FGFR2 fusion positivity and hepatitis virus infection may suggest an involvement of the virus in the chromosomal rearrangements in CC.” (opens the source at this quote in a new tab)

FGFR2 fusion-driven transformation of mouse liver organoids and ligand-independent signalling

“Chromosomal rearrangements generate FGFR2 fusions (FFs) in 12–15% of intrahepatic CCAs.” (opens the source at this quote in a new tab)
“In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation.” (opens the source at this quote in a new tab)
“It is thought that the fusion partner forces constitutive dimerization of adjacent FGFR2 sequences, triggering constitutive activation of the FGFR2 tyrosine kinase domain (TKD) and attendant oncogenic signaling.” (opens the source at this quote in a new tab)
“When expressed at levels comparable to those of F-TACC3, wild-type FGFR2IIIb required ligand stimulation in order to undergo catalytic activation (Fig. 1D). Thus, constitutive FF signaling in the above liver organoid models is caused by the ligand-independent mode of activation typical of FGFR2 fusions.” (opens the source at this quote in a new tab)
“In line with this observation, mutations affecting tumor suppressor genes (TSGs), most notably TP53, CDKN2A or BAP1, co-occur in the large majority of iCCA carrying FGFR2 fusions.” (opens the source at this quote in a new tab)
“Thus, FGFR2 fusions drive oncogenic conversion of liver bipotent precursors along the cholangiocellular lineage.” (opens the source at this quote in a new tab)
“All of the above phosphorylation events required FF catalytic activity, because they were suppressed by BGJ398 (Fig. 5A and S9A-C).” (opens the source at this quote in a new tab)
“The above data support a model whereby Erk1/2 activation plays a necessary role in shaping addiction of iCCA cells to FF oncogenic signaling.” (opens the source at this quote in a new tab)

Oncogenicity and pemigatinib sensitivity across FGFR2 fusion partners

“In this study, in vitro analysis demonstrated the oncogenicity of all tested FGFR2 rearrangement partners by foci formation and sensitivity to FGFR2 inhibition. Combined, these data suggest the clinically important variable is the FGFR2 fusion irrespective of partner gene.” (opens the source at this quote in a new tab)

Downstream MAPK and PI3K/AKT signalling from FGFR2 fusion proteins

“Constitutive activation of FGFR2 fusion proteins leads to persistent, ligand-independent stimulation of downstream oncogenic signaling, most prominently the MAPK (RAS/RAF/MEK/ERK) and PI3K/AKT/mTOR axes, thereby promoting sustained cellular proliferation, survival, and resistance to apoptosis (Figure 1)” (opens the source at this quote in a new tab)

Diversity of FGFR2 fusion partners and BICC1 as the most common partner

“In these 107 patients, 56 different partners were identified, 42 (75%) of which were unique to individual patients. The most common FGFR2 partner was BICC1 (31 [29%] patients; appendix p 11).” (opens the source at this quote in a new tab)

“Consistent with previous reports, BICC1, which resides near FGFR2 on chromosome 10, was the most common fusion partner gene (26% of FGFR2 fusions), but was only one of 34 partner genes found (Fig. 4B).” (opens the source at this quote in a new tab)