Non-exonuclease domain POLE mutations and their biological significance in colorectal cancer

Table 1 Classification of DNA polymerase epsilon mutations in colorectal cancer

Feature	Exonuclease domain mutations	Non-exonuclease domain mutations
Location	Exonuclease (proofreading) domain	Outside exonuclease domain
Mechanism	Loss of proofreading activity	Indirect effects (replication stress, altered polymerase dynamics)
Tumor mutational burden	Ultra-high (> 100 mut/Mb)	Intermediate to high (context-dependent)
Mutational signature	COSMIC signature 10	Heterogeneous/less defined
MSI status	Often MSI-H or independent	Predominantly MSS/MSI-L
Immunogenicity	High (strong neoantigen load)	Variable, potentially increased in subsets
Clinical relevance	Established biomarker for immunotherapy	Emerging, not yet standardized
Role in tumorigenesis	Early driver event	Likely modifier, context-dependent

MSI-H: High levels of microsatellite instability; MSI-L: Low levels of microsatellite instability; MSS: Microsatellite stable.

Table 2 Co-mutational landscape of non-exonuclease domain mutations DNA polymerase epsilon-mutant colorectal cancer

Gene	Pathway/function	Role in CRC	Interaction with POLE non-EDM
MLH3	Mismatch repair	DNA repair	Partial repair deficiency → synergistic mutagenesis
MSH3	Mismatch repair	Indel repair	MSI-L phenotype, replication error accumulation
KRAS	MAPK signaling	Oncogenic driver	Sustains proliferation in unstable genome
BRAF	MAPK signaling	Prognostic/driver	Less frequent, may define subgroups
PIK3CA	PI3K/AKT pathway	Growth and survival	Supports tumor progression alongside instability

CRC: Colorectal cancer; EDM: Exonuclease domain mutation; MAPK: Mitogen-activated protein kinase; MLH3: MutL homolog 3; MSH3: MutS homolog 3; MSI-L: Low levels of microsatellite instability; PIK3CA: Phosphoinositide 3-kinase catalytic subunit alpha; POLE: DNA polymerase epsilon.

Table 3 Clinical implications and proposed integration of non-exonuclease domain DNA polymerase epsilon mutations in colorectal cancer

Clinical domain	Current evidence	Key supporting findings	Proposed interpretation/recommendation
Immunotherapy eligibility	MSS CRC generally resistant to ICIs	MSS/pMMR tumors show poor response rates to ICIs (systematic reviews)	Do not exclude all MSS tumors; consider molecular subgroups
	POLE mutations linked to immunogenicity	POLE EDM tumors show high TMB and strong immune infiltration[25,30]	POLE status should be considered alongside MSI
	Non-EDM POLE may confer benefit	Non-exonuclease POLE mutations associated with ICI response in selected cases[26,31]	Selected MSS + non-EDM + high TMB → potential ICI candidates (clinical trials)
TMB	High TMB predicts ICI response	POLE-mutant tumors show elevated TMB even in MSS context[28,42]	TMB should be interpreted with POLE status, not MSI alone
	MSS tumors usually low TMB	Subset of MSS + POLE non-EDM show intermediate/high TMB	Identify “hidden hypermutated” MSS subgroup
Biomarker strategy	MSI/MMR used as binary biomarker	MSI classification fails to capture full genomic instability spectrum	Move toward composite biomarker model
	POLE not routinely integrated	Non-EDM variants often underreported or labeled VUS	Include POLE (full-length or extended panels) in selected cases
Molecular testing	Hotspot POLE testing (EDM-focused)	Non-EDMs more frequent than EDMs[42]	Expand testing beyond exonuclease domain in high-risk scenarios
	TMB/TNB testing limited	Requires WES or large panels[55]	Use tiered strategy: Prioritize selected patients
Co-mutation-informed therapy	Targeted therapy used independently	KRAS, PIK3CA, BRAF commonly co-occur with POLE[42,46]	Consider combination approaches (ICI + targeted therapy)
	Limited integration of pathways	Co-mutations reflect pathway activation (MAPK, PI3K)	Adopt pathway-informed therapeutic strategies
Prognostic implications	MSI-H → favorable prognosis	POLE EDM tumors associated with improved outcomes	Prognostic role of non-EDM unclear
	MSS → poorer prognosis	Some POLE non-EDM tumors show aggressive features[28]	Avoid overinterpreting mutation alone without context
Reporting practices	POLE often reported alone or omitted	Co-mutations (MLH3, MSH3) influence biological behavior	Always report POLE with co-mutational profile
	Variants labeled as VUS	Functional impact unclear but context-dependent	Do not ignore non-EDMs-interpret within genomic context
Clinical trial design	MSI-based stratification	POLE-mutant tumors span MSI categories	Future trials should stratify by POLE + co-mutations
Clinical caution	Biomarker-driven decisions increasing	Many non-EDM variants lack functional validation	Do not use POLE alone for treatment decisions
	Evidence still emerging	Small cohorts, retrospective data	Integrate molecular + clinical parameters (stage, TME, response)

CRC: Colorectal cancer; EDM: Exonuclease domain mutation; ICI: Immune checkpoint inhibitor; MAPK: Mitogen-activated protein kinase; MMR: Mismatch repair; MSI: Microsatellite instability; MSI-H: High levels of microsatellite instability; MSS: Microsatellite stable; PIK3CA: Phosphoinositide 3-kinase catalytic subunit alpha; pMMR: Proficient mismatch repair; POLE: DNA polymerase epsilon; TMB: Tumor mutational burden; TME: Tumor microenvironment; TNB: Tumor neoantigen burden; VUS: Variant of uncertain significance; WES: Whole exome sequencing.