Molecularly guided therapy in gastrointestinal malignancies: From established targets to resistance and emerging precision strategies

Table 7 Biomarker testing considerations for molecularly targeted therapy in gastrointestinal malignancies

Biomarker	Principal GI malignancies	Preferred/commonly used assay	Key interpretation considerations	Tissue–liquid biopsy considerations	Role in treatment selection
HER2 amplification/overexpression	Gastric/GEJ, colorectal, biliary	IHC ± ISH/FISH; NGS may assess amplification	IHC scoring and predefined positivity thresholds should follow the assay/clinical trial context; heterogeneity may affect interpretation	Plasma NGS may identify amplification but does not necessarily replace tissue assessment	Selects patients for HER2-directed therapy
CLDN18.2 expression	Gastric/GEJ	IHC using validated antibody/scoring approach	Positivity depends on staining intensity, proportion of positive tumor cells, and the threshold used in the supporting clinical study; spatial heterogeneity may occur	Plasma testing is not an established substitute for tissue protein-expression assessment	Selects patients for CLDN18.2-directed therapy
FGFR2 fusion/rearrangement	Intrahepatic cholangiocarcinoma	NGS, RNA-based fusion testing, FISH or other validated molecular assays	Assay choice influences detection of fusion/rearrangement; DNA-only assays may have limitations for some structural alterations	Plasma ctDNA can identify some alterations but may have lower sensitivity for fusions/rearrangements, particularly in low-shedding tumors	Identifies candidates for FGFR-directed therapy
IDH1 mutation	Intrahepatic cholangiocarcinoma	PCR-based assay or NGS	Specific pathogenic IDH1 alterations should be distinguished from non-actionable variants	Plasma testing may be complementary but tissue remains important when plasma is negative or inconclusive	Selects patients for IDH1-directed therapy
BRAF V600E	Colorectal, biliary and other selected GI cancers	PCR, IHC where validated, or NGS	Specific V600E alteration should be confirmed using an appropriate validated assay	Plasma ctDNA can provide complementary mutation detection; negative results may require tissue confirmation	Selects patients for BRAF-directed therapy
KRAS/NRAS mutations	Colorectal	PCR-based hotspot testing or NGS	RAS status should include relevant KRAS and NRAS alterations when determining eligibility for EGFR-directed therapy	ctDNA may provide a non-invasive alternative or complementary assessment, but false negatives can occur with low tumor shedding	Determines eligibility for or resistance to selected targeted therapies
KRAS G12C	Colorectal; rare pancreatic and other GI tumors	PCR or NGS	Detection of the specific G12C alteration is required; broader KRAS positivity is not equivalent	Plasma NGS may detect G12C but negative plasma testing does not necessarily exclude a tissue alteration	Selects patients for KRAS G12C-directed strategies
NTRK fusion	Rare across GI malignancies	RNA-based NGS, DNA/RNA hybrid NGS, FISH or validated fusion assay	Fusion detection requires an assay capable of identifying relevant structural events; IHC may be useful for screening but generally requires molecular confirmation	Plasma detection is possible but less established than tissue-based fusion testing	Supports tissue-agnostic TRK-directed therapy
RET fusion	Rare GI malignancies	NGS or other validated fusion assays	Fusion detection requires appropriate structural-variant methodology	Plasma testing may be complementary but sensitivity depends on tumor shedding and assay characteristics	Supports RET-directed therapy in molecularly selected patients
KIT/PDGFRA mutations	GIST	NGS or targeted molecular testing	Mutation subtype and exon are clinically relevant because sensitivity to individual TKIs varies	Plasma ctDNA may help identify secondary resistance alterations but is not a universal substitute for tumor genotyping	Guides initial TKI selection and reassessment after resistance
BRCA1/2 and other HRR alterations	Pancreatic cancer and selected GI malignancies	Germline testing plus tumor NGS where appropriate	Germline and somatic alterations should be distinguished; pathogenicity and clinical relevance require appropriate interpretation	Plasma testing may complement tissue analysis but should not automatically replace germline assessment	Identifies selected patients for platinum/PARP or other DNA-repair-directed strategies
MSI-H/dMMR	Colorectal, gastric/GEJ and other GI malignancies	MMR IHC and/or MSI PCR/NGS	Assay concordance and appropriate interpretation of equivocal results are important	Plasma MSI assessment remains complementary and less established than validated tissue testing in many settings	Biomarker for precision immunotherapy rather than conventional targeted therapy
TMB-H	Multiple GI malignancies	Validated NGS-based genomic profiling	Thresholds are assay-specific and should not be assumed to be interchangeable across platforms	Plasma-derived TMB may differ from tissue TMB because of assay and biological factors	May identify selected patients for precision immunotherapy where clinically validated
ctDNA/molecular residual disease	Multiple GI malignancies	Plasma NGS or other validated ctDNA platforms	Primarily a monitoring/longitudinal biomarker rather than a therapeutic target; interpretation depends on assay sensitivity and timing	Particularly vulnerable to false negatives in low-shedding or localized disease; serial testing may improve longitudinal assessment	May support molecular monitoring and detection of emerging resistance; treatment-changing utility remains context dependent

Biomarker thresholds and assay requirements should be interpreted according to the validated assay, pivotal clinical study, and applicable regulatory/clinical diagnostic framework. Tissue and plasma assays are complementary rather than universally interchangeable. MSI-H/dMMR and TMB-H are included because they guide precision immunotherapy rather than conventional molecularly targeted therapy. CISH: Chromogenic in situ hybridization; ctDNA: Circulating tumor DNA; FISH: Fluorescence in situ hybridization; GEJ: Gastroesophageal junction; GI: Gastrointestinal; GIST: Gastrointestinal stromal tumor; HRR: Homologous recombination repair; IHC: Immunohistochemistry; ISH: In situ hybridization; MSI-H: Microsatellite instability-high; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; TKI: Tyrosine kinase inhibitor; TMB-H: Tumor mutational burden-high; CLDN18.2: Claudin 18.2.