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FGFR2 Fusions Testing in Intrahepatic Cholangiocarcinoma: ESMO Biomarker Factsheet

ESMO Factsheets on Biomarkers

Cholangiocarcinoma (CCA) is a relatively rare cancer associated with an extremely poor prognosis. Based on the anatomical location, CCA is usually classified as intrahepatic cholangiocarcinoma (iCCA) or extrahepatic cholangiocarcinoma (eCCA), which includes perihilar cholangiocarcinomas (pCCAs) and distal cholangiocarcinomas (dCCAs) [1]. Genomic profiling studies demonstrated that the molecular and genomic alterations of iCCA and eCCA are profoundly different, even with respect to actionable mutations that offer possibilities for therapeutic intervention [2]. In particular, fibroblast growth factor receptor 2 (FGFR2) fusions are usually detected only in iCCA. 

Actionable genomic alterations in iCCA

Several genomic alterations of iCCA have been classified as level I according to the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT), including IDH1 mutations, FGFR2 fusions, microsatellite instability (MSI-H) and NTRK fusions [3]. Mutations of IDH1 occur in up to 20% of iCCA, and clinical activity of ivosidenib in iCCA patients carrying IDH1 mutations within a phase III randomised clinical trial has been recently reported [4]. Pemigatinib, a selective FGFR inhibitor, showed a 35% response rate in patients with FGFR2 fusion-positive advanced cholangiocarcinoma in a prospective phase II trial [5]. Based on these findings, both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved pemigatinib for treatment of advanced or metastatic cholangiocarcinoma characterised by fusion or rearrangements of FGFR2. iCCA patients with either MSI-H (frequency 1-2%) or NTRK fusions (1-2%) benefited treatment with immune checkpoint inhibitors or TRK inhibitors in tumour-agnostic basket trials [6-8]. Other targetable genomic alterations of iCCA include BRAF mutations (class II, 3-5%), and the class III mutations/amplifications of ERBB2 (8-10%), PIK3CA mutations (7%), BRCA mutations (3%) and MET amplification [3, 9].

Characteristics of FGFR2 fusions in iCCA

FGFR2 fusions are determined by genomic rearrangements of the FGFR2 gene located on chromosome 10. FGFR2 fusions have been reported in up to 15% of iCCA, occur mostly in fluke-negative iCCA and are usually mutually exclusive with IDH1 mutations [10]. The FGFR2 can be involved in rearrangements with different genes. Although BICC1 is the partner in approximately 30% of FGFR2 fusions detected in iCCA, over 150 partners have been described up to now, with many partners found in few cases [9, 11]. Another important feature of FGFR2 fusions in iCCA is that approximately 50% of the rearrangements are intra-chromosomal, meaning the fusion partner is on the same chromosome as the FGFR2 gene [12].

Testing methods for FGFR2 fusions

A number of different techniques have been used to detect fusions in cancer.

Immunohistochemistry (IHC)

Antibodies used for immunohistochemical detection of fusions recognise both wild-type and fusion proteins. Therefore, fusions can be detected by IHC if the rearrangement induces an increased expression of the protein in the tumour cells as compared with normal surrounding cells, which happens in many but not all fusions. IHC methods are routinely used to detect fusions (ALK) or as screening technology to select cases with suspected fusions (ROS1, NTRK) that will next be confirmed with techniques that are more specific. However, no IHC technique has been validated up to now for the detection of FGFR2 fusions.

Fluorescent in situ hybridisation (FISH)

The most common approach to detect gene fusions in cancer is break-apart FISH, which employs two differently labeled (usually red and green) DNA probes complementary to 5’ and 3’ sequences of the gene. When the gene is unaltered, an orange signal deriving from overlapping red and green fluorescence is evident. Rearrangements increase the distance between the 5’ and 3’ probes, resulting in a “split” red and green fluorescent signal. FISH has shown a good sensitivity and specificity to detect FGFR2 fusions [13]. However, intrachromosomal rearrangements may lead to false negative FISH results, if the distance between the 5’ and 3’ probes after rearrangement remains too short.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

RT-PCR detects gene expression by first converting RNA into complementary DNA (cDNA) using reverse transcriptase, followed by amplification of newly synthesised cDNA by PCR. The amplification products are next separated by electrophoresis to identify the target sequence. Because unique primers are needed to identify each respective fusion partner, the fusion partner and the location of the fusion break point must be known prior to testing. Due to the high number of FGFR2 partners in iCCA, RT-PCR is not feasible for FGFR2 fusion testing in this disease.

Next generation sequencing (NGS)

Targeted approaches using NGS allow the sequencing of a subset of genes and, therefore, the identification of the different genomic alterations involved in cancer pathogenesis (point mutations, copy number alterations and gene fusions). A number of different multigene panels for targeted sequencing covering from a few genes to hundreds of genes are commercially available. Targeted sequencing multigene panels use different technologies for library preparation, such as hybrid capture, amplicon or Anchored Multiplex PCR (AMP). In addition, NGS panels are based on DNA and/or RNA sequencing for detection of fusions. The presence of intronic regions might limit the possibility to detect fusions by DNA sequencing. Therefore, technologies based on RNA sequencing have usually a better coverage for gene fusions as compared with DNA sequencing [14]. In addition, the AMP technique by using a single specific primer and primers that are complementary to adaptors, allows to identify gene fusions independently from the fusion partner [14]. Recently, novel NGS techniques with improved sensitivity became available for the analysis of the circulating cell-free DNA (cfDNA) that can be isolated from peripheral blood or other body fluids [15]. These NGS panels can detect different types of genomic alterations in cfDNA, including FGFR2 fusions [16].

Algorithm for FGFR2 fusion testing in iCCA

ESMO recommends the use of multigene NGS for the genomic profiling of iCCA patients in advanced stage of disease [3]. In fact, different types of actionable genomic alterations (point mutations, gene fusions, gene amplifications) can be identified in over 40% of iCCA. Given the availability of small biopsies in the majority of iCCA patients, NGS testing is the best option for comprehensive genomic profiling of iCCA. NGS-based RNA sequencing offers the most adequate coverage for RNA fusion detection. If NGS is not available, FISH might represent an alternative for FGFR2 fusion testing. In patients with no tissue available for genomic profiling, NGS testing of cfDNA is an option, although the limited sensitivity might lead to false negative results.

References

  1. Rizvi S, Khan SA, Hallemeier CL, et al. Cholangiocarcinoma - evolving concepts and therapeutic strategies. Nat Rev Clin Oncol 2018;15(2):95-111.
  2. Valle JW, Lamarca A, Goyal L, et al. New Horizons for Precision Medicine in Biliary Tract Cancers. Cancer Discov 2017;7(9):943-962. 
  3. Mosele F, Remon J, Mateo J, et al. Recommendations for the use of next-generation sequencing (NGS) for patients with metastatic cancers: a report from the ESMO Precision Medicine Working Group. Ann Oncol 2020;31(11):1491-1505. 
  4. Abou-Alfa GK, Macarulla T, Javle MM, et al. Ivosidenib in IDH1-mutant, chemotherapy-refractory cholangiocarcinoma (ClarIDHy): a multicentre, randomised, double-blind, placebo-controlled, phase 3 study. Lancet Oncol 2020;21(6):796-807. 
  5. Abou-Alfa GK, Sahai V, Hollebecque A, et al. Pemigatinib for previously treated, locally advanced or metastatic cholangiocarcinoma: a multicentre, open-label, phase 2 study. Lancet Oncol 2020;21(5):671-684.
  6. Marabelle A, Le DT, Ascierto PA, et al. Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study. J Clin Oncol 2020;38(1):1-10.
  7. Doebele RC, Drilon A, Paz-Ares L et al. Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials. Lancet Oncol 2020;21(2):271-282.
  8. Hong DS, DuBois SG, Kummar S et al. Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials. Lancet Oncol 2020;21(4):531-540.
  9. Silverman IM, Hollebecque A, Friboulet L, et al. Clinicogenomic Analysis of FGFR2-Rearranged Cholangiocarcinoma Identifies Correlates of Response and Mechanisms of Resistance to Pemigatinib. Cancer Discov 2021;11:326-339.
  10. Jusakul A, Cutcutache I, Yong CH, et al. Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma. Cancer Discov 2017;7(10):1116-1135.
  11. Javle MM, Murugesan K, Shroff RT, et al. Profiling of 3,634 cholangiocarcinomas (CCA) to identify genomic alterations (GA), tumor mutational burden (TMB), and genomic loss of heterozygosity (gLOH). Journal of Clinical Oncology 2019;37(Suppl 15):4087-4087.
  12. Hollebecque A, Silverman I, Owens S, et al. Comprehensive genomic profiling and clinical outcomes in patients (pts) with fibroblast growth factor receptor rearrangement-positive (FGFR2+) cholangiocarcinoma (CCA) treated with pemigatinib in the fight-202 trial. Annals of Oncology 2019;30(Suppl 5):v276.
  13. Maruki Y, Morizane C, Arai Y, et al. Molecular detection and clinicopathological characteristics of advanced/recurrent biliary tract carcinomas harboring the FGFR2 rearrangements: a prospective observational study (PRELUDE Study). Journal of Gastroenterology 2021;56(3):250-260.
  14. De Luca A, Esposito Abate R, Rachiglio AM, et al. FGFR Fusions in Cancer: From Diagnostic Approaches to Therapeutic Intervention. Int J Mol Sci 2020;21(18):6856.
  15. Normanno N, Cervantes A, Ciardiello F, et al. The liquid biopsy in the management of colorectal cancer patients: Current applications and future scenarios. Cancer Treat Rev 2018;70:1-8.
  16. Nakamura Y, Taniguchi H, Ikeda M, et al. Clinical utility of circulating tumor DNA sequencing in advanced gastrointestinal cancer: SCRUM-Japan GI-SCREEN and GOZILA studies. Nat Med 2020;26(12):1859-1864.

 

Disclosure of Interest:

Nicola Normanno reports receipt of honoraria as invited speaker from MSD, Illumina, BMS, Merck, Thermofisher, Sanofi, Eli Lilly; receipt of honoraria for participation in Advisory Board from Qiagen, Bayer, Biocartis, Incyte, Roche, AstraZeneca, Novartis; receipt of institutional research grants from Merck, Termofisher, Qiagen, Roche, AstraZeneca, Biocartis, Illumina, Incyte, Blueprint; non-financial interest for his leadearship role as a President of the International Quality Network for Pathology (IQN Path), and President of the Italian Cancer Society (SIC).

Last update: 12 Apr 2021

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