What is the role of Fluorescence In Situ Hybridization (FISH) testing in targeted therapy for cholangiocarcinoma?

FISH in Cholangiocarcinoma for Targeted Therapy

Role of FISH Testing

FISH testing in cholangiocarcinoma serves two distinct and critical purposes: (1) detecting FGFR2 fusions/rearrangements to guide targeted therapy selection with FGFR inhibitors like pemigatinib, and (2) diagnosing malignancy in indeterminate biliary strictures through polysomy detection, though RNA-based NGS is now the preferred method for identifying actionable FGFR2 alterations. 1, 2

FISH for FGFR2 Fusion Detection (Targeted Therapy Selection)

Primary Testing Approach

• RNA-based NGS is the preferred testing method for identifying FGFR2 fusions in intrahepatic cholangiocarcinoma, as it detects oncogenic fusions in 9.7-11.8% of cases and identifies novel fusion partners that FISH or DNA-based NGS may miss 3, 4

• FGFR2 fusions occur in approximately 10-20% of intrahepatic cholangiocarcinomas and represent the primary indication for pemigatinib therapy, which is FDA-approved for previously treated, unresectable locally advanced or metastatic cholangiocarcinoma with FGFR2 fusion or rearrangement 1, 5

FISH Performance for FGFR2 Detection

• FISH demonstrates 95.2% sensitivity and 98.5% specificity for detecting FGFR2 rearrangements when compared to oncogenic fusions confirmed by RNA-based NGS 4

• Break-apart FISH detects FGFR2 rearrangements in 10.2% of intrahepatic cholangiocarcinoma cases, with high concordance (95.1%) when compared to NGS methods 4

• Critical limitation: FISH cannot identify the specific fusion partner gene, which may be clinically relevant for understanding resistance mechanisms and treatment planning 6, 4

When to Use FISH vs. NGS

• Use FISH when:

  • Tumor tissue is insufficient for comprehensive NGS testing 3
  • Rapid turnaround time is critical and the patient needs immediate treatment decision for FGFR inhibitor therapy 3
  • NGS testing is unavailable at your institution 3

• Use RNA-based NGS when:

  • Adequate tissue is available for comprehensive molecular profiling 3, 4
  • Identifying specific fusion partners and other actionable alterations (IDH1, BRAF, HER2) is desired 4
  • The patient is treatment-naïve and comprehensive molecular characterization will guide first-line therapy selection 5

Comparative Testing Performance

• Only 57.7% of FGFR2 fusion-positive cases are detected by all three methods (RNA-NGS, DNA-NGS, and FISH), highlighting the importance of methodology selection 4

• DNA-based NGS shows lower sensitivity (71.4%) compared to FISH for detecting FGFR2 rearrangements, making it an inferior choice for this specific alteration 4

• RNA-based NGS identified five novel oncogenic FGFR2 fusions not previously reported, demonstrating its superiority for comprehensive fusion detection 4

FISH for Malignancy Diagnosis in Biliary Strictures

Diagnostic Performance

• For diagnosing cholangiocarcinoma in indeterminate biliary strictures, FISH detects polysomy (duplication of two or more chromosomes in ≥5 cells) with 41-51% sensitivity and 93-98% specificity 7, 2

• When combined with conventional brush cytology, FISH increases overall diagnostic sensitivity to 50-69% while maintaining excellent specificity of 91-100% 2

• The American Gastroenterological Association recommends FISH as an adjunctive test to standard brush cytology during ERCP to improve diagnostic sensitivity for cholangiocarcinoma 2

Clinical Application for Diagnosis

• A positive FISH result (polysomy) confidently diagnoses malignant biliary stricture in the appropriate clinical context, given its near-perfect specificity 2

• A negative FISH result does not exclude cholangiocarcinoma and requires additional sampling methods or serial monitoring, as sensitivity remains below 70% even when combined with cytology 2

• In primary sclerosing cholangitis patients with dominant strictures, FISH should be performed on biliary brushings obtained during ERCP, alongside CA 19-9 measurement (>129 U/mL threshold) and MR imaging 7

Superior Alternatives for Diagnosis

• Bile DNA methylation panels achieve 100% sensitivity and 90% specificity in PSC-related cholangiocarcinoma, representing a significant advancement over FISH for diagnostic purposes 2, 8

• Single-operator cholangioscopy-guided biopsies show 65% sensitivity and 97% specificity in PSC, potentially superior to FISH in this population 2

Critical Pitfalls and Caveats

Testing Methodology Issues

• Bioinformatic pipelines not optimized for FGFR2 fusion detection represent a major obstacle, with only 81% of centers successfully passing proficiency testing for NGS-based FGFR2 fusion detection 6

• Assays incapable of detecting unknown fusion partners will miss clinically relevant FGFR2 alterations, as 35% of fusion partners are located on chromosome 10 (most commonly BICC1), while the remaining 65% are distributed across 9 other chromosomes 6, 4

• FISH testing should never be requested for gallbladder masses, as this test is only validated for biliary strictures and cholangiocarcinoma, not gallbladder cancer 9

Clinical Context Requirements

• FGFR2 fusion-positive intrahepatic cholangiocarcinomas are typically microsatellite stable (MSS) with low tumor mutational burden (median 2.1 mut/Mb), making them poor candidates for immunotherapy 3

• FGFR2 fusion-positive cases are associated with small duct type intrahepatic cholangiocarcinoma, particularly in HBsAg-positive patients without cholangiolocarcinoma components 4

Resistance Considerations

• Acquired resistance to FGFR inhibitors remains a major barrier, requiring consideration of liquid biopsy for serial monitoring of treatment response when tissue biopsy is contraindicated or high-risk 8, 10