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

Table 1 Literature search strategy used for the narrative review

Component	Description
Review type	Narrative review
Databases	PubMed/MEDLINE, Scopus, Web of Science, and Google Scholar
Search period	January 2000 to July 30, 2026
Language	English
Search approach	Structured keyword-based searches using Boolean operators (AND/OR), with MeSH terms used where applicable
Core search terms	“Gastrointestinal cancer”, “gastrointestinal malignancy”, “targeted therapy”, “molecular targeted therapy”, and “precision oncology”
Cancer-specific terms	Esophageal cancer, gastric cancer, gastroesophageal junction cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, cholangiocarcinoma, biliary tract cancer, and GIST
Molecular target terms	EGFR, HER2, VEGF, FGFR, KIT, PDGFRA, BRAF, KRAS, IDH1, CLDN18.2, FGFR2b, PARP, NTRK, RET, MET, ALK, ROS1, PI3K, AKT, and mTOR
Additional therapeutic terms	Antibody-drug conjugates, bispecific antibodies, DNA damage repair, genomic profiling, next-generation sequencing, and liquid biopsy
Additional searches	Cancer-specific and drug-specific searches addressing pivotal trials, regulatory evidence, resistance mechanisms, and emerging therapies
Evidence prioritized	Randomized clinical trials, phase II/III studies, prospective studies, meta-analyses, international guidelines, landmark trials, and high-quality reviews
Study selection	Studies were selected based on relevance, methodological quality, clinical significance, molecular specificity, and contribution to established or emerging targeted treatment
Screening process	Independent screening of titles and abstracts, followed by full-text assessment by two reviewers
Disagreement resolution	Discussion and consensus, with consultation of a third reviewer when required
Evidence synthesis	Narrative synthesis organized by gastrointestinal malignancy and molecular target, addressing mechanism of action, clinical role, evidence maturity, biomarkers, resistance mechanisms, and emerging strategies
Evidence grading	Grade A: Randomized phase III evidence and/or established standard-of-care treatment. Grade B: Prospective phase II evidence or substantial clinical evidence. Grade C: Early-phase, retrospective, or otherwise limited clinical evidence. Grade D: Preclinical or experimental evidence

MeSH: Medical Subject Headings; GIST: Gastrointestinal stromal tumor; EGFR: Epidermal growth factor receptor; HER2: Human epidermal growth factor receptor 2; VEGF: Vascular endothelial growth factor; FGFR: Fibroblast growth factor receptor; PDGFRA: Platelet-derived growth factor receptor alpha; PARP: Poly (ADP-ribose) polymerase; NTRK: Neurotrophic tyrosine receptor kinase; RET: Rearranged during transfection; MET: Mesenchymal-epithelial transition factor; ALK: Anaplastic lymphoma kinase; ROS1: ROS proto-oncogene 1; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B; mTOR: Mechanistic target of rapamycin; KIT: KIT proto-oncogene receptor tyrosine kinase; CLDN18.2: Claudin 18.2.

Table 2 Molecularly targeted therapies in colorectal cancer

Molecular target/pathway	Biomarker/alteration	Therapeutic agent(s)	Current clinical role	Evidence grade	Key resistance mechanisms/emerging strategies	Representative sources
EGFR	RAS wild-type mCRC; BRAF status should also be considered for treatment selection and sequencing	Cetuximab; panitumumab	Established therapy in molecularly selected mCRC, used with chemotherapy or as monotherapy in appropriate later-line settings	A	Primary or acquired resistance may involve emergent KRAS/NRAS or BRAF alterations, HER2 or MET amplification, PI3K/PTEN pathway alterations, EGFR extracellular-domain mutations, gene fusions, and intratumoral heterogeneity. ctDNA-guided rechallenge is an emerging strategy in selected patients	Kasi et al[8]; Khelwatty et al[9]; Leite et al[10]; Li et al[14]
VEGF-A	No validated predictive biomarker	Bevacizumab	Established anti-angiogenic therapy, generally combined with chemotherapy in first-line and subsequent-line treatment strategies	A	Adaptive angiogenesis, vascular heterogeneity, hypoxia-driven adaptation, and activation of alternative pro-angiogenic pathways. Biomarker discovery and combinations with immunotherapy remain under investigation	Colombo and Porretto[13]; Li et al[14]; Jacobsen et al[16]
VEGF-A/VEGF-B/PlGF	No validated predictive biomarker	Aflibercept	Established second-line treatment in combination with chemotherapy after progression on an oxaliplatin-containing regimen in appropriate patients	A	Alternative angiogenic pathway activation, vascular remodeling, and endothelial adaptation	Li et al[14]; Fakih[17]
VEGFR-2	No validated predictive biomarker	Ramucirumab	Established second-line treatment in combination with chemotherapy in selected patients previously treated with bevacizumab-, oxaliplatin-, and fluoropyrimidine-based therapy	A	Compensatory angiogenic signaling, vascular remodeling, and microenvironmental adaptation	Li et al[14]
VEGFR1–3 and multiple angiogenic/tumor-signaling kinases	No validated predictive biomarker	Regorafenib	Established later-line treatment for previously treated or refractory mCRC	A	Multikinase pathway escape, adaptive angiogenesis, and tumor–microenvironment interactions. Toxicity and performance status are important treatment considerations	Li et al[14]; Jacobsen et al[16]
VEGFR1–3	No validated predictive biomarker	Fruquintinib	Established later-line treatment for previously treated or refractory mCRC	A	Adaptive angiogenesis and vascular pathway redundancy; combination strategies are under investigation	Li et al[14]; Jiao et al[18]
BRAF/MAPK	BRAF V600E mutation	Encorafenib + cetuximab, with or without binimetinib depending on the treatment context	Established targeted therapy for previously treated BRAF V600E-mutant mCRC	A	MAPK pathway reactivation, feedback signaling, and bypass activation. Combined BRAF/EGFR inhibition, with or without MEK inhibition, is intended to suppress pathway escape	Kopetz et al[24]; Ambrosini et al[22]; Li et al[14]
HER2	HER2 amplification and/or overexpression; assessment requires an appropriate validated assay	Trastuzumab-based dual HER2 blockade; tucatinib-based combinations; trastuzumab deruxtecan and other HER2-directed ADCs	Clinically actionable target in selected HER2-positive mCRC, particularly in RAS wild-type disease and appropriate later-line settings	B	HER2 heterogeneity, HER2 loss, bypass signaling, downstream pathway reactivation, and acquired resistance. Next-generation ADCs and combination HER2-directed strategies are under investigation	Sartore-Bianchi et al[34]; Pizzamiglio et al[33]; Benli et al[38]
KRAS G12C	KRAS G12C mutation	KRAS G12C inhibitor combined with EGFR inhibition	Molecularly selected targeted therapy for KRAS G12C-mutant mCRC, generally in previously treated disease	A	EGFR-mediated feedback, MAPK pathway reactivation, secondary KRAS alterations, and downstream pathway escape. Combination strategies aim to improve pathway suppression and delay resistance	Li et al[14]; Manzi et al[39]
NTRK	NTRK gene fusion	Larotrectinib; entrectinib	Tissue-agnostic targeted therapy for fusion-positive tumors, including rare CRC cases	B	Acquired kinase-domain mutations and bypass signaling may lead to resistance; next-generation TRK inhibitors are being developed for resistant disease	Li et al[40]
RET	RET fusion	Selective RET inhibitors	Rare molecularly selected indication; CRC-specific evidence remains limited	B	Acquired resistance, limited tumor-specific clinical evidence, and uncertainty regarding optimal sequencing; prospective evaluation continues	Li et al[40]
BRAF/MAPK	Non-V600E BRAF alterations	Molecularly matched BRAF-, MEK-, and/or EGFR-directed combinations	Limited clinical evidence in selected patients; not equivalent to the established BRAF V600E treatment pathway	C	Biological heterogeneity and variable sensitivity according to the specific BRAF alteration. Molecularly matched combinations require further clinical validation	Sasaki et al[26]; Ambrosini et al[22]
MET/PI3K/PTEN and related bypass pathways	MET amplification, PIK3CA alterations, PTEN loss, or other putative resistance-associated alterations	Molecularly matched combinations	Investigational strategies aimed at overcoming resistance to EGFR inhibition	C	These alterations may contribute to intrinsic or acquired resistance, although their predictive and therapeutic value is context-dependent. Multi-target inhibition and biomarker-guided approaches remain under investigation	Khelwatty et al[9]; Leite et al[10]

Evidence classification: Evidence levels were assigned according to the maturity and clinical validation of the available data. Established: Supported by prospective randomized trials and/or incorporated into clinical guidelines or routine standard-of-care practice; Clinically supported: Supported by prospective clinical studies, non-randomized comparative studies, or consistent clinical evidence but with more limited validation; Emerging/investigational: Supported by early-phase clinical trials, small prospective or retrospective studies, or preliminary clinical data. Preclinical: Supported primarily by in vitro, animal, organoid, or other laboratory-based studies without sufficient clinical validation. Evidence grades describe the maturity of the evidence and do not necessarily indicate the magnitude of treatment benefit. ADC: Antibody–drug conjugate; BRAF: B-Raf proto-oncogene; EGFR: Epidermal growth factor receptor; FGFR: Fibroblast growth factor receptor; MAPK: Mitogen-activated protein kinase; mCRC: Metastatic colorectal cancer; PDGFR: Platelet-derived growth factor receptor; PI3K: Phosphatidylinositol-3-kinase; PlGF: Placental growth factor; PTEN: Phosphatase and tensin homolog; RET: Rearranged during transfection; T-DXd: Trastuzumab deruxtecan; TRK: Tropomyosin receptor kinase; VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptor; ctDNA: Circulating tumor DNA.

Table 3 Molecularly targeted therapies in cholangiocarcinoma: Clinical role, evidence, resistance, and emerging strategies

Molecular target/alteration	Approximate frequency	Representative therapy	Current clinical role	Evidence grade	Major resistance mechanisms/emerging strategies	Representative sources
FGFR2 fusion/rearrangement	10%-16%, predominantly intrahepatic CCA	Pemigatinib; futibatinib	Established targeted therapy for previously treated, unresectable, or metastatic FGFR2 fusion/rearranged CCA	A	Acquired FGFR2 kinase-domain mutations, bypass signaling, polyclonal resistance, and intratumoral heterogeneity. Next-generation FGFR inhibitors, ctDNA-guided monitoring, combination approaches, and earlier-line treatment are under investigation	Li et al[65]; Mie et al[57]; Goyal et al[42]; Ellis et al[41]; Xin and Miao[47]
IDH1 mutation	10%–20%, predominantly intrahepatic CCA	Ivosidenib	Established targeted therapy for previously treated IDH1-mutated CCA	A	Clonal evolution, metabolic adaptation, secondary pathway alterations, epigenetic remodeling, and bypass signaling. Combinations with immunotherapy, epigenetic agents, or PARP-directed therapy, as well as earlier-line treatment and ctDNA-guided monitoring, remain investigational	Abou-Alfa et al[66]; Bray and Sahai[51]; Salati et al[50]; Li et al[65]
HER2 amplification/overexpression	5%-15%; more frequent in extrahepatic CCA and gallbladder cancer	Trastuzumab-based combinations; pertuzumab; tucatinib-based combinations; trastuzumab deruxtecan	Molecularly selected treatment option for HER2-positive biliary tract cancers; clinical evidence is emerging and varies according to the agent and study population	B	HER2 heterogeneity, HER2 loss, bypass signaling, and downstream pathway reactivation. Next-generation ADCs, bispecific antibodies, and combination HER2-directed therapies are under investigation	Inoue et al[62]; Theocharopoulos et al[58]
BRAF V600E mutation	3%-5%	Dabrafenib + trametinib	Established molecularly selected targeted therapy for BRAF V600E-mutated biliary tract cancer	A	MAPK pathway reactivation, adaptive signaling, and bypass pathway activation. Additional combination strategies are under investigation	Mie et al[57]; Ellis et al[41]

Evidence grading: Grade A, randomized phase III evidence and/or established standard-of-care treatment; Grade B, prospective phase II evidence or substantial clinical evidence; Grade C, early-phase, retrospective, or otherwise limited clinical evidence; Grade D, preclinical or experimental evidence. Evidence grades reflect the maturity and clinical validation of the available evidence and do not directly indicate the magnitude of treatment benefit. ADC: Antibody–drug conjugate; CCA: Cholangiocarcinoma; ctDNA: Circulating tumor DNA; DDR: DNA damage response; dMMR: Mismatch repair deficient; FGFR2: Fibroblast growth factor receptor 2; IDH1: Isocitrate dehydrogenase 1; MSI-H: Microsatellite instability-high; NTRK: Neurotrophic tyrosine receptor kinase; PARP: Poly (ADP-ribose) polymerase; RET: Rearranged during transfection; TMB-H: Tumor mutational burden-high; TRK: Tropomyosin receptor kinase.

Table 4 Molecularly targeted therapies in gastric and gastroesophageal junction adenocarcinoma

Molecular target/pathway	Biomarker/alteration	Representative therapy	Current clinical role	Evidence grade	Major resistance mechanisms/emerging strategies	Representative sources
HER2	HER2 overexpression and/or amplification, assessed using validated IHC and ISH-based methods	Trastuzumab + platinum/fluoropyrimidine chemotherapy ± pembrolizumab; T-DXd	Established first-line HER2-directed therapy based on ToGA and KEYNOTE-811. T-DXd is an established treatment option after trastuzumab-based therapy based on DESTINY-Gastric01	A	HER2 heterogeneity or loss, MET/EGFR/FGFR bypass signaling, PI3K pathway activation, and clonal evolution. HER2-low disease, next-generation ADCs, bispecific antibodies, CAR-T approaches, ctDNA-guided monitoring, and combination immunotherapy remain under investigation	Bang et al[72]; Shitara et al[74]; Nakamura et al[95]; Janjigian et al[73]
VEGFR-2	Not biomarker selected	Ramucirumab ± paclitaxel	Established second-line therapy based on REGARD and RAINBOW	A	Adaptive angiogenic resistance, alternative pro-angiogenic pathway activation, and vascular remodeling. Combinations with chemotherapy and immune checkpoint inhibitors are under investigation	Fuchs et al[77]; Wilke et al[78]; Nakamura et al[95]
VEGFR-2	Not biomarker selected	Apatinib	Later-line anti-angiogenic therapy, with regulatory approval in China	B	Alternative angiogenic pathway activation and adaptive vascular signaling	Wang et al[96]; Yuan et al[97]
VEGFR1–3	Not biomarker selected	Fruquintinib	Emerging anti-angiogenic therapy under clinical evaluation in gastric and GEJ cancer	C	Adaptive angiogenesis and pathway redundancy; combination approaches remain investigational	Wang et al[96]
CLDN18.2	CLDN18.2-positive, generally HER2-negative disease	Zolbetuximab + chemotherapy	Established first-line targeted therapy for appropriately selected CLDN18.2-positive, HER2-negative advanced gastric or GEJ adenocarcinoma based on SPOTLIGHT and GLOW	A	Heterogeneous antigen expression, variable assay thresholds, antigen loss, and treatment-related gastrointestinal toxicity. CLDN18.2 ADCs, bispecific antibodies, CAR-T therapy, and improved biomarker classification are under investigation	Shitara et al[74]; Nakamura et al[95]; Derks and van Laarhoven[88]
FGFR2b	FGFR2b overexpression	Bemarituzumab + chemotherapy	Clinical evidence from the phase II FIGHT study; further validation and treatment positioning remain necessary	B	FGFR2b expression heterogeneity, pathway bypass, and uncertainty regarding optimal biomarker thresholds. Earlier-line treatment and combination strategies are under investigation	Wainberg et al[100]; Yuan et al[97]
MET	MET amplification or other activating MET alterations	MET inhibitors	Early clinical or investigational development; no established routine role	C	Biomarker heterogeneity, pathway redundancy, and limited predictive consistency. Biomarker refinement and combination targeted therapy are under investigation	Wang et al[96]; Hsu et al[98]
HER2	HER2-positive refractory disease	Disitamab vedotin	Clinical activity reported in selected HER2-positive disease, with ongoing development	B	HER2 heterogeneity, antigen loss, payload resistance, and altered intracellular trafficking. ADC-based strategies are being developed to improve activity in heterogeneous or treatment-resistant disease	Shao et al[75]; Zhang et al[99]
HER2	HER2-positive disease	Zanidatamab; tucatinib-based therapy; pyrotinib	Emerging or region-specific clinical evidence; not uniformly established across gastric/GEJ cancer treatment settings	C	HER2 heterogeneity, downstream pathway reactivation, and acquired resistance. Dual or multi-agent HER2 blockade and treatment-sequencing strategies remain under investigation	Hu et al[68]; Shao et al[75]; Zhang et al[99]
KRAS/PI3K and related signaling pathways	KRAS or PI3K pathway alterations	Selective pathway inhibitors and molecularly matched combinations	Early clinical or investigational development	C	Pathway redundancy, feedback activation, and compensatory signaling. Combination pathway inhibition requires biomarker refinement and prospective validation	Yuan et al[97]; Kawakami[93]
VEGF/VEGFR and immune pathways	Not biomarker selected	VEGF/VEGFR inhibitor + immune checkpoint inhibitor	Investigational strategies with preliminary activity	C	Uncertain biomarker selection, adaptive angiogenesis, immune resistance, and overlapping toxicity. Optimal sequencing and combination partners remain under investigation	Yuan et al[97]; Kawakami[93]
VEGF + PD-1/PD-L1	Not biomarker selected	VEGF/PD-1 or PD-L1 bispecific antibodies	Early clinical investigation	C	Requires prospective validation of efficacy, safety, biomarker selection, and treatment sequencing	Yuan et al[97]; Kawakami[93]
MET/EGFR/FGFR/PI3K	Molecularly selected or pathway-driven disease	Combination targeted strategies	Investigational	C	Bypass signaling, clonal evolution, pathway redundancy, and intratumoral heterogeneity	Nakamura et al[95]

Evidence grading: Grade A, randomized phase III evidence and/or established standard-of-care treatment; Grade B, prospective phase II evidence or substantial clinical evidence; Grade C, early-phase, retrospective, or otherwise limited clinical evidence; Grade D, preclinical or experimental evidence. Evidence grades reflect the maturity and clinical validation of the available evidence and do not directly indicate the magnitude of treatment benefit. ADC: Antibody–drug conjugate; CAR-T: Chimeric antigen receptor T-cell therapy; CLDN18.2: Claudin 18.2; ctDNA: Circulating tumor DNA; EGFR: Epidermal growth factor receptor; FGFR: Fibroblast growth factor receptor; FGFR2b: Fibroblast growth factor receptor 2b; GC/GEJ: Gastric/gastroesophageal junction; HER2: Human epidermal growth factor receptor 2; MET: Mesenchymal–epithelial transition factor; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; PI3K: Phosphatidylinositol-3-kinase; T-DXd: Trastuzumab deruxtecan; VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptor.

Table 5 KIT proto-oncogene receptor tyrosine kinase- and platelet-derived growth factor receptor alpha-targeted therapies in gastrointestinal stromal tumors

Molecular alteration	Approximate frequency	Preferred targeted therapy	Clinical role	Evidence grade	Resistance/emerging strategy	Representative sources
KIT exon 11 mutation	60%–70%	Imatinib 400 mg/day	Established first-line therapy; generally associated with the greatest sensitivity to imatinib	A	Secondary KIT mutations, clonal evolution, and acquired resistance; molecular reassessment may guide subsequent therapy	Li and Raut[104]; Wang et al[105]
KIT exon 9 mutation	10%–15%	High-dose imatinib 800 mg/day	Dose escalation may improve disease control, particularly in advanced disease	A	Secondary KIT mutations and clonal evolution; NGS-guided treatment selection and sequencing	Li and Raut[104]; Jakhetiya et al[103]
PDGFRA D842V mutation	5%–6%	Avapritinib	Preferred targeted therapy; this genotype is characteristically resistant to imatinib	A	Secondary resistance and clonal evolution; molecular monitoring and resistance-directed strategies remain under investigation	Mazzocca et al[106]; Wang et al[105]
Other PDGFRA mutations	Rare	Imatinib or alternative therapy according to mutation subtype	Variable sensitivity based on the specific PDGFRA alteration	B	Mutation-specific sensitivity and resistance require detailed molecular characterization	Li and Raut[104]
Wild-type GIST, including SDH-deficient, NF1-associated, and BRAF-associated tumors	10%–15%	Individualized treatment and clinical-trial enrollment	Limited response to conventional KIT-directed TKIs; management depends on the underlying molecular subtype	C	Alternative molecular drivers and genotype-specific treatment strategies are under investigation	Schaefer et al[107]; Wang et al[105]
Secondary KIT/PDGFRA resistance mutations	Variable	Sequential TKI therapy guided by molecular profiling	Molecular characterization can support selection and sequencing of subsequent therapies	B	Intratumoral heterogeneity, polyclonal resistance, and clonal evolution	Li and Raut[104]; Wang et al[105]

Evidence classification: Grade A, established therapy supported by prospective randomized evidence and/or incorporated into clinical guidelines or routine standard-of-care practice; Grade B, clinically supported therapy based on prospective clinical studies: Non-randomized comparative studies: Or consistent clinical evidence with more limited validation; Grade C, emerging or investigational strategy supported by early-phase trials: Small prospective or retrospective studies: Or preliminary clinical data; Grade D, preclinical or experimental strategy supported primarily by laboratory or animal studies without sufficient clinical validation. Evidence grades describe the maturity and clinical validation of the evidence and do not necessarily indicate the magnitude of treatment benefit. BRAF: B-Raf proto-oncogene; GIST: Gastrointestinal stromal tumor; KIT: KIT proto-oncogene receptor tyrosine kinase; NF1: Neurofibromatosis type 1; NGS: Next-generation sequencing; PDGFRA: Platelet-derived growth factor receptor alpha; SDH: Succinate dehydrogenase; TKI: Tyrosine kinase inhibitor.

Table 6 Molecularly targeted therapies in pancreatic ductal adenocarcinoma: Established targets, therapeutic agents, evidence, and emerging strategies

Molecular target/pathway	Approximate frequency	Biomarker/molecular subgroup	Representative therapy	Evidence grade	Current clinical role	Major challenges/emerging strategies	Representative sources
KRAS	90%–95%	KRAS-mutant PDAC	Direct KRAS inhibitors and pathway-directed strategies	B	Principal molecular target under active clinical development	Adaptive pathway reactivation and stromal resistance; combinations with SHP2, SOS1, MEK or EGFR inhibition	Li et al[108]; Leroux and Konstantinidou[109]; Xing et al[110]
KRAS G12C	1%–2%	KRAS G12C	Sotorasib, adagrasib	B	Early clinical activity in molecularly selected PDAC	Adaptive resistance and rational combination strategies	Li et al[108]; Leroux and Konstantinidou[109]
KRAS G12D	Approximately 40%	KRAS G12D	Mutation-specific KRAS inhibitors	C	Early clinical development	Allele-specific inhibition, pathway reactivation, and acquired resistance	Li et al[108]; Xing et al[110]
KRAS G12V/G12R	Common	KRAS G12V/G12R	Allele-specific inhibitors	C	Preclinical and early clinical development	Development of effective mutation-specific inhibitors	Leroux and Konstantinidou[109]; Li et al[108]
BRCA1/2 and HRR	5%–10% germline BRCA1/2; other HRR alterations less common	Germline BRCA1/2 or selected HRR-deficient PDAC	Olaparib maintenance after platinum-based chemotherapy; other PARP/DDR-directed strategies	A	Olaparib is an established maintenance option for appropriately selected germline BRCA1/2-mutated disease; broader HRR-directed approaches remain investigational	Acquired resistance and identification of PARP-sensitive tumors beyond BRCA1/2	Golan et al[114]; Li et al[108]; Leroux and Konstantinidou[109]
PALB2	Rare	PALB2 mutation	PARP inhibitors and platinum-based strategies	C	Selected/investigational patients	Prospective validation of predictive value and definition of PARP-sensitive subgroups	Leroux and Konstantinidou[109]; Li et al[108]
EGFR	Common	EGFR-expressing PDAC	Erlotinib + gemcitabine	A	Randomized evidence demonstrates a modest benefit, with limited contemporary clinical role	Constitutive KRAS signaling limits efficacy; combinations with KRAS/SHP2/MEK/PI3K inhibition	Li et al[108]; Leroux and Konstantinidou[109]
NTRK	< 1%	NTRK fusion	TRK inhibitors	B	Tissue-agnostic targeted therapy for fusion-positive tumors	Comprehensive genomic profiling to identify rare fusion-positive patients	Li et al[108]
RET	< 1%	RET fusion	Selective RET inhibitors	B	Rare molecularly selected indication	Limited PDAC-specific evidence; prospective validation	Li et al[108]
HER2	Rare	HER2 amplification, particularly KRAS-wild-type PDAC	HER2-targeted therapy, trastuzumab-based regimens, ADCs	C	Early clinical/case-based evidence	Biomarker refinement and combination HER2-targeted strategies	Li et al[108]; Xing et al[110]
BRAF	Rare	BRAF-mutant PDAC	BRAF ± MEK inhibitors	C	Limited molecularly selected evidence	Combination MAPK inhibition and prospective validation	Leroux and Konstantinidou[109]; Li et al[108]

Evidence classification: Grade A, established therapy supported by prospective randomized evidence and/or incorporated into clinical guidelines or routine standard-of-care practice; Grade B, clinically supported therapy based on prospective clinical studies, non-randomized comparative studies, or consistent clinical evidence with more limited validation; Grade C, emerging or investigational strategy supported by early-phase trials, small prospective or retrospective studies, or preliminary clinical data; Grade D, preclinical or experimental strategy supported primarily by laboratory or animal studies without sufficient clinical validation. Evidence grades describe the maturity and clinical validation of the evidence and do not necessarily indicate the magnitude of treatment benefit. ADC: Antibody–drug conjugate; ALK: Anaplastic lymphoma kinase; ApDC: Aptamer–drug conjugate; ATR: Ataxia telangiectasia and Rad3-related protein; BRAF: B-Raf proto-oncogene; DDR: DNA damage response/repair; EGFR: Epidermal growth factor receptor; HRD: Homologous recombination deficiency; HRR: Homologous recombination repair; MEK: Mitogen-activated protein kinase kinase; NGS: Next-generation sequencing; PARP: Poly (ADP-ribose) polymerase; PDAC: Pancreatic ductal adenocarcinoma; PDC: Peptide–drug conjugate; PI3K: Phosphatidylinositol 3-kinase; PROTAC: Proteolysis-targeting chimera; ROS1: ROS proto-oncogene 1; SHP2: Src homology region 2 domain-containing phosphatase 2; SOS1: Son of sevenless 1; TKI: Tyrosine kinase inhibitor; TRK: Tropomyosin receptor kinase.

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.