The patient first noticed abnormal bleeding on March 7, 2026, and hysteroscopy at a local hospital quickly confirmed endometrioid adenocarcinoma. Imaging at Qilu Hospital showed an irregular endometrial mass of roughly 4.6 by 3.7 by 2.5 centimeters with internal branching vessels, and pelvic magnetic resonance imaging revealed a slightly larger lesion with the restricted diffusion typical of malignancy. Her only laboratory abnormality was a mildly elevated CA125 of 49.7 U/mL. She had no family history of gynecologic or hereditary cancers, and after undergoing a total hysterectomy with bilateral salpingo-oophorectomy and sentinel lymph node biopsy, pathology showed a FIGO grade 2 endometrioid carcinoma invading about 20 percent of the myometrium, with focal lymphovascular invasion but no lymph node involvement and a final stage of pT1aN0, corresponding to FIGO 2009 stage IA.

What transformed this from an ordinary early-stage cancer case into a scientific puzzle was next-generation sequencing. Using a 572-gene hybrid-capture panel on tumor tissue containing roughly 45 percent tumor cells, the team identified a rare variant in the exonuclease domain of POLE, the gene encoding DNA polymerase epsilon: c.833C>A, resulting in a threonine-to-lysine substitution at position 278, or p.T278K. This variant sat at a variant allele frequency of 40.42 percent, and manual review of the raw sequencing data confirmed 440 reads at the locus with 187 supporting the alternate T allele. Alongside it, sequencing uncovered two separate frameshift variants in MSH6, a key mismatch repair gene, both hitting the same nucleotide position c.3261 within a low-complexity sequence but predicting differently truncated proteins.

The genomic consequences were dramatic. The tumor’s mutational burden reached 370.0 mutations per megabase, firmly in the ultramutated range reserved for POLE-driven cancers, and microsatellite analysis showed profound instability with an MSI score of 1279.88, far above the positivity threshold of 200. The substitution profile reinforced the POLE story: 26.35 percent of coding variants were C>A transversions and 8.87 percent were T>G substitutions, the classic nucleotide fingerprint left by a failed DNA proofreading enzyme. Additional driver mutations in PTEN, PIK3CA, ARID1A, PPP2R1A, and FGFR2, plus alterations in DNA damage response genes including BRCA2, ATM, ATR, and RAD54L, rounded out a genomic landscape typical of endometrial carcinoma, and a TP53 missense variant was detected by sequencing even though p53 immunostaining looked wild-type.

Immunohistochemistry added a second layer of complexity. The tumor showed subclonal loss of the mismatch repair proteins MLH1, PMS2, and MSH6, with retained MSH2 expression, and methylation-specific PCR detected MLH1 promoter methylation. Taken together, these findings meant the tumor carried features of not one but two of the four molecular classifiers established by The Cancer Genome Atlas: the POLE-ultramutated group, known for excellent prognosis, and the microsatellite instability-hypermutated group driven by mismatch repair deficiency. Such multiple-classifier endometrial carcinomas are uncommon, and interpreting a rare POLE variant within one of them is where the diagnostic challenge truly begins.

Most pathogenic POLE mutations cluster at well-known hotspot residues such as p.P286R, p.V411L, p.S297F, p.A456P, and p.S459F. The p.T278K variant is different. It lies within the Exo I motif of the exonuclease domain, immediately adjacent to the catalytic residue E277 and inside the DNA-binding cleft, a location that raises suspicion but does not by itself prove pathogenicity. To address exactly this problem, León-Castillo and colleagues developed an evidence-based scoring framework that integrates tumor mutational burden, the proportion of C>A and T>G substitutions, insertion-deletion frequency, and recurrence data to separate genuine proofreading-deficient variants from variants of uncertain significance.

Applying that framework to this tumor, the researchers assigned one point each for the ultrahigh tumor mutational burden, the C>A proportion, the T>G proportion, and the C>G proportion, but no points for the indel proportion of 9.17 percent or for recurrence, since p.T278K was absent from the TCGA endometrial carcinoma cohort and represented by only a single record in COSMIC. The resulting exploratory POLE score of 4, combined with the characteristic substitution spectrum, aligns the tumor squarely with the POLE-ultramutated profile, though the authors caution that the framework was built on whole-exome data and its use with targeted panels should be regarded as supportive rather than definitive. Prior evidence strengthens the case: before this report, p.T278K had been documented in only two patients with endometrial carcinoma, one of whom showed the same ultramutated phenotype and a POLE score of 4, and a yeast-based reversion assay has demonstrated a mutator phenotype for this variant, providing functional evidence of impaired proofreading.

The dual MSH6 frameshift variants tell their own fascinating story. Because they arise as reciprocal insertion and deletion events at the same nucleotide within a repetitive, low-complexity sequence, and because their variant allele frequencies differ unequally at 17.64 and 34.94 percent, they are most consistent with secondary somatic events arising through replication slippage in an already hypermutable genome, a phenomenon previously described in tumors that lose both polymerase proofreading and mismatch repair. Yet both variants are classified in ClinVar as pathogenic in the germline context, and because sequencing was performed on tumor tissue without matched normal DNA, a germline origin could not be excluded. The team therefore recommended peripheral-blood germline testing, a step with major implications for the patient’s relatives. Similarly, MLH1 promoter methylation plausibly explains the subclonal MLH1 and PMS2 loss, making this apparently only the third reported endometrial carcinoma with co-occurring POLE alteration and MLH1 promoter methylation, although the bulk-tumor assay used cannot establish spatial correspondence between the methylation and the patchy protein loss.

Why does all this molecular detective work matter clinically? Because molecular classification now directly determines treatment intensity. Under the 2023 FIGO molecular staging system, a pathogenic POLE mutation in a tumor like this one places it in stage IAmPOLEmut, a category with such an excellent prognosis that adjuvant therapy can often be safely omitted. Even setting the POLE question aside, the tumor’s grade 2 endometrioid histology, limited myometrial invasion, focal lymphovascular invasion, negative sentinel nodes, and mismatch repair deficiency would satisfy FIGO 2023 IA2 criteria and place the patient in the low-risk category. Accordingly, the patient received no adjuvant therapy, a decision consistent with the 2025 ESGO-ESTRO-ESP guidelines, and at four months of follow-up her CA125 had normalized, pelvic ultrasound showed no disease, and there was no evidence of recurrence. Her mismatch repair-deficient status nonetheless remains clinically valuable: should the cancer ever return or progress, MSI-H tumors are prime candidates for immune checkpoint inhibitors.

The case does carry limitations. Tumor-only sequencing cannot definitively distinguish somatic from germline variants, targeted-panel analysis rather than whole-exome sequencing constrains mutational signature analysis, the MLH1 methylation test was qualitative on bulk tissue, and follow-up remains short. The discordance between the TP53 sequencing result and the wild-type p53 staining pattern also remains unexplained, possibly reflecting intratumoral heterogeneity or differences in protein regulation, though it did not alter classification or management. Still, by converging from three independent directions, genomic phenotype, prior clinical observations, and functional assay data, the report substantially strengthens the argument that p.T278K is a genuine proofreading-deficient variant rather than a passenger, and it offers a practical template for pathologists confronting rare POLE variants in tumors that carry multiple molecular fingerprints. For the growing number of patients whose cancers are now classified at the DNA level, that template may help ensure the right patients receive less treatment, and the right ones receive more.

Subject of Research: Molecular classification of endometrial carcinoma harboring a rare POLE p.T278K variant and MSH6 frameshift mutations

Article Title: Case Report: Endometrial carcinoma harboring a rare POLE p.T278K variant and dual MSH6 frameshift variants

Article References: Yang, Y., Luo, H., Yan, H., Yu, H., Ma, X., Teng, Q., Zhang, L., Ye, J., Yuan, Z., & Zhang, Q. (2026). Case Report: Endometrial carcinoma harboring a rare POLE p.T278K variant and dual MSH6 frameshift variants. Frontiers in Oncology, 16, Article 1964637. https://doi.org/10.3389/fonc.2026.1964637

Image Credits: AI Generated

DOI: 10.3389/fonc.2026.1964637

Keywords: endometrial carcinoma, POLE, p.T278K, MSH6, mismatch repair deficiency, microsatellite instability, tumor mutational burden, MLH1 promoter methylation, molecular classification, León-Castillo scoring, next-generation sequencing, gynecologic oncology