What next-generation sequencing (NGS) panel is recommended for advanced gallbladder cancer?

NGS Panel Recommendations for Advanced Gallbladder Cancer

For advanced gallbladder cancer, perform comprehensive next-generation sequencing (NGS) using a focused multigene panel that includes at minimum: IDH1, ERBB2 (HER2), BRAF, PIK3CA for hotspot mutations; FGFR2 and NTRK for gene fusions (preferably at RNA level); and MSI/dMMR status via immunohistochemistry or DNA-based analysis. 1, 2, 3

Essential Panel Components

The NGS panel must include specific genes based on ESMO 2024 guidelines for biliary tract cancers:

DNA-Based Hotspot Mutations

• IDH1 - actionable with ivosidenib (ESCAT level IA), present in ~19% of patients 1, 2, 3
• ERBB2 (HER2) - actionable with HER2-directed therapies, present in 13-18% of patients 1, 2, 3
• BRAF - actionable with BRAF inhibitors for V600E mutations 1, 2
• PIK3CA - frequently altered (17-33% of patients) and potentially actionable 1, 3

Gene Fusions (RNA-Based Testing Preferred)

• FGFR2 fusions - actionable with pemigatinib, futibatinib, or infigratinib (ESCAT level IA), present in ~10% of patients 1, 2, 3
• NTRK fusions - actionable with TRK inhibitors (tumor-agnostic indication) 1, 2
• RNA-based sequencing using hybrid capture or anchored multiplex PCR technology identifies both known and unknown fusion partners and provides functional information 1, 2
• DNA-based testing should specifically cover FGFR2 breakpoints in exons 17 and 18 1, 2

Microsatellite Instability Testing

• MSI-H/dMMR status via immunohistochemistry for MLH1, MSH2, MSH6, and PMS2, or DNA-based microsatellite analysis 1, 2
• MSI-H predicts response to PD-1 checkpoint inhibitors (ESCAT level IA) 1

Recommended Testing Technology

Use focused NGS panels with hybrid capture or anchored multiplex PCR technology on formalin-fixed paraffin-embedded (FFPE) tumor tissue. 1, 2, 3

• FFPE tissue from core biopsies (not fine needle aspiration alone) provides adequate material for both diagnostic pathology and molecular profiling 1, 2
• Liquid biopsies using cell-free circulating DNA should only be considered when insufficient tumor tissue is available 1, 2
• RNA-based sequencing is superior to DNA-only approaches for detecting gene fusions, as it identifies fusion transcripts, expression levels, and functionality 1, 2

Clinical Implementation Strategy

Timing of Testing

• Obtain tissue for NGS at initial diagnosis during ERCP/PTC-guided biopsies or EUS-guided fine needle biopsy 1, 2
• Never delay tissue acquisition - obtain biopsies during biliary drainage procedures for simultaneous diagnosis and molecular profiling 1, 2
• Avoid relying solely on biliary brush cytology, which has limited sensitivity for both diagnosis and NGS 1, 2

Actionability of Results

• Approximately 35-50% of gallbladder cancer patients harbor clinically actionable alterations that can guide targeted therapy selection 2, 3
• The most common actionable targets include: TP53 (57-73%), CDKN2A/B (25-26%), ERBB2 (13-18%), PIK3CA (14-20%), and FGFR2 fusions (10%) 3, 4, 5
• Use the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT) to prioritize treatment decisions, focusing on level I-II alterations 2, 3

Treatment Algorithm Based on NGS Results

First-Line Therapy

• Initiate gemcitabine/cisplatin with durvalumab or pembrolizumab for ECOG 0-1 patients while awaiting NGS results (typical turnaround time 10-14 days) 2, 3
• Do not delay systemic therapy in rapidly progressing disease 2

Subsequent Therapy Based on Molecular Findings

• IDH1 mutations: Ivosidenib in previously treated patients (ESCAT IA) 3
• FGFR2 fusions: Pemigatinib, futibatinib, or infigratinib (ESCAT IA) 3
• ERBB2 amplification/overexpression: HER2-directed therapies including anti-HER2 monoclonal antibodies, TKIs, or antibody-drug conjugates 2, 3
• NTRK fusions: TRK inhibitors (larotrectinib or entrectinib) 2
• BRAF V600E mutations: BRAF inhibitors with EGFR inhibitors 1
• MSI-H/dMMR: PD-1 checkpoint inhibitors 1

Critical Pitfalls to Avoid

• Never perform single-gene testing when NGS panels are available - parallel sequencing is preferred and more cost-effective 1, 2
• Do not use inadequate tissue - core biopsies are mandatory, not fine needle aspiration alone 2, 3
• Ensure the panel includes RNA-based fusion detection - DNA-only panels may miss FGFR2 and NTRK fusions due to variable breakpoint locations 1, 2
• Verify NRG1 fusions are included in the panel, as these are emerging actionable targets 1
• Discuss technology selection with a molecular pathologist based on available tissue and specific targets of interest 1, 2

Additional Considerations

• Tumor mutational burden (TMB) is relatively high in gallbladder cancer (17-18% with TMB ≥10 mutations/Mb), suggesting potential benefit from immune checkpoint inhibitors 4
• Over 76-83% of patients have at least one potentially actionable alteration when comprehensive profiling is performed 4, 5
• Cost-effectiveness should be assessed at the local level, particularly regarding access to tumor-agnostic targeted therapies 1