Intended for European healthcare professionals only

Fibroblast growth factor receptor 2 (FGFR2) fusions in intrahepatic cholangiocarcinoma (iCCA)

Genomic alterations in fibroblast growth factor receptors (FGFRs)

FGFRs are a family of receptor tyrosine kinases.^1,2
FGFR signalling pathways play a central role in multiple cellular processes, including cell proliferation, migration and survival^1,2

Alterations in FGFR genes have emerged as tumourigenic drivers in cancers including iCCA, urothelial carcinoma, myeloid/lymphoid neoplasms and other malignancies^1,3,4

FGFR amplifications, mutations and fusions have been observed in all FGFR subtypes (FGFR1–4).⁵ Chromosomal rearrangements involving FGFR2 – resulting in the creation of oncogenic fusion proteins – have frequently been identified in iCCA⁶
- Gene fusions are a type of genomic alteration where two independent genes or portions of genes are juxtaposed, resulting in a hybrid gene^7,8
- The development of fusion proteins with oncogenic potential can result from gene fusion events involving a range of different partner genes⁷

FGFR genomic alterations

Figure based on Jain A, et al. 2018,⁵ Lowery MA, et al. 2018,⁹ and Shibata T, et al. 2018¹⁰

FGFR2 fusions

FGFR2 fusions or rearrangements occur in 10–16% of iCCA cases^5,11-13
FGFR2 fusions result in ligand-independent activation of downstream signalling pathways, leading to tumourigenesis^1,14,15

Abnormal FGFR2 signalling pathway

Figure adapted from Babina IS, Turner NC. 2017,¹ Moeini A, et al. 2015,¹⁴ and Touat M, et al. 2015¹⁵

Tumour molecular profiling is necessary to identify FGFR2 fusions.^5,9 Assessment for FGFR2 fusion positivity should be performed with an appropriate diagnostic test⁷
FGFR2 fusions involve a wide range of fusion partners.⁹
To identify patients with FGFR2 fusion-positive cholangiocarcinoma (CCA), it is important to select an assay that:
- Specifically detects FGFR2 fusions (distinct from FGFR2 point mutations)^16,17
- Detects FGFR2 fusions with a wide range of fusion partners^16,17
The molecular diversity of CCA supports the use of DNA- or RNA-based next-generation sequencing (NGS) assays as standard to detect both known and novel FGFR2 fusions or rearrangements¹⁸

Testing methodologies for the detection of FGFR2 fusions

A number of methods with varying specificity can be used to detect FGFR2 fusions⁷

Click on a testing method to view the advantages and challenges

Least appropriate^7,19–27

Most appropriate^7,19–27

Least appropriate^7,19–27

Most appropriate^7,19–27

The European Society for Medical Oncology (ESMO) recommends routine use of NGS to detect FGFR2 fusions in advanced CCA²⁸

Proposed algorithm of how FGFR2 fusion testing can be incorporated into a diagnostic work-up

CCA, cholangiocarcinoma; FGFR2, fibroblast growth factor receptor 2

External quality assurance programmes are essential to ensure accurate and reliable clinical biomarker testing³¹

REFERENCES: 1. Babina IS, Turner NC. Nat Rev Cancer. 2017;17:318–32. 2. Turner N, Grose R. Nat Rev Cancer. 2010;10:116–29. 3. Pandith AA, et al. Urol Oncol. 2013;31:398–406. 4. Gallo LH, et al. Cytokine Growth Factor Rev. 2015;26:425–49. 5. Jain A, et al. JCO Precis Oncol. 2018;2:1–12. 6. Fangda L, et al. Cytokine Growth Factor Rev. 2020;52:56–67. 7. DeLuca A, et al. Int J Mol Sci. 2020;21:6856. 8. Latysheva S, Babu M. Nucleic Acids Research. 2016;10:4487–50. 9. Lowery MA, et al. Clin Cancer Res. 2018;24:4154–61. 10. Shibata T, et al. Cancer Sci. 2018;109:1282–91. 11. Ross JS, et al. Oncologist. 2014;19:235–42. 12. Farshidfar F, et al. Cell Rep. 2017;18:2780–94. 13. Graham RP, et al. Hum Pathol. 2014;45:1630–8. 14. Moeini A, et al. Clin Cancer Res. 2015;22:291–300. 15. Touat M, et al. Clin Cancer Res. 2015;21:2684–94. 16. Silverman IM, et al. Cancer Discov. 2021;11:326–39. 17. Barr FG. Expert Rev Mol Diagn. 2016;16:921–3. 18. Abou-Alfa GK, et al. Lancet Oncol. 2020;21:671–84. 19. Malka D, et al. EMJ Oncol. 2020;8:82–94. 20. Peter M, et al. Lab Invest. 2001;91:905–12. 21. Arai Y, et al. Hepatology. 2014;59:1427–34. 22. Abel H, et al. J Mol Diagn. 2014;16:405–17. 23. Beadling C. J Mol Diagn. 2016;18:165–75. 24. Hu L, et al. Biomark Res. 2014;2:3. 25. Maruki Y, et al. J Gastroenterol. 2021;56:250–60. 26. Serratì S, et al. Onco Targets Ther. 2016;9:7355–65. 27. Jennings LJ, et al. J Mol Diagn. 2017;19:341–65. 28. Vogel A, et al. Ann Oncol. 2023;34:127–40. 29. Patel T. Nat Rev Gastroenterol Hepatol. 2011;8:189–200. 30. Damodaran S, et al. Am Soc Clin Oncol Educ Book. 2015;e175–82. 31. Dufraing K, et al. Virchows Arch. 2021;478:553–65.

Immunohistochemistry (IHC)^7,17

Advantages	Challenges
Inexpensive process Can detect fusions when rearrangements lead to overexpression of the fused protein Can provide information about specific fusions depending on protein localisation	Very low sensitivity for identifying rare fusions Many IHC approaches use antibodies that cannot distinguish wild-type FGFR2 from fusion proteins No IHC method has been proven to have sufficient sensitivity and specificity to detect FGFR fusions

Reverse transcriptase polymerase chain reaction (RT-PCR)^17,20,21

Advantages	Challenges
Highly sensitive Assay can be multiplexed to cover a range of mutations within a single reaction Can easily be performed using clinical formalin-fixed paraffin-embedded samples	Methodology is limited to FGFR2 gene fusions with known fusion partners Requires prior knowledge of both fusion partners; novel fusion partners cannot be detected Assay probes have to be designed for each specific fusion combination Sensitive to cross-contamination linked to the carry-over of PCR products

Fluorescence in situ hybridisation (FISH)^7,22-25

Advantages	Challenges
Inexpensive process Well-established methodology and widely available within clinical laboratories Does not require living cells Can be easily performed on clinical formalin-fixed paraffin-embedded samples Break-apart FISH probes can detect unknown fusion partners Relatively fast turnaround time	Low-resolution method Mainly restricted to the detection of DNA Complex rearrangements are usually not easily detectable Intrachromosomal rearrangements, which account for about 50% of FGFR2 fusions in intrahepatic cholangiocarcinoma, can lead to false-negative results Break-apart FISH probes cannot identify the fusion partner Labour intensive and requires experienced pathologists

Next-generation sequencing (NGS)^{7,22,23,26,27}

Advantages	Challenges
Multiple targets simultaneously analysed in a single sample High sensitivity and specificity Detects both known and novel fusions, regardless of breakpoints or fusion partners (depending on library prep method) Commercial kits covering gene fusions are available RNA-based: can distinguish in-frame, transcribed gene fusions versus out-of-frame fusions and avoid difficulties of sequencing large intronic regions	Slow turnaround time Not cost effective for small sample numbers Requires bioinformatics and trained personnel DNA-based: detection of novel fusions might be limited, especially when large intronic regions are involved RNA-based: sensitivity depends on the expression levels of the novel fusion gene; RNA is less stable than DNA

Advantages

Challenges

Multiple targets simultaneously analysed in a single sample
High sensitivity and specificity
Detects both known and novel fusions, regardless of breakpoints or fusion partners (depending on library prep method)
Commercial kits covering gene fusions are available
RNA-based: can distinguish in-frame, transcribed gene fusions versus out-of-frame fusions and avoid difficulties of sequencing large intronic regions

Slow turnaround time
Not cost effective for small sample numbers
Requires bioinformatics and trained personnel
DNA-based: detection of novel fusions might be limited, especially when large intronic regions are involved
RNA-based: sensitivity depends on the expression levels of the novel fusion gene; RNA is less stable than DNA

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