POLE mutations in endometrial carcinoma from a tertiary cancer care centre in eastern India

Methods

This was a retrospective observational study undertaken by the department of Molecular Pathology, Tata Medical Centre, Kolkata, India. The archived clinical and pathological data from digital hospital records of Tata Memorial Centre (TMC) was used for undertaking the secondary analysis. An ethics waiver was obtained for carrying out this analysis from the Institutional Review Board of the TMC, Kolkata. This study analysed data from cases of endometrial carcinoma referred for POLE gene mutation testing at between August 2022 and December 2024. During the study period, molecular analysis was requested for 339 cases of endometrial carcinoma, of which 327 were tested for mutations in the POLE exonuclease domain. The sample rejection criteria for POLE exonuclease domain mutation analysis at our centre were as follows: FFPE tissue was excluded if tumour cellularity after macrodissection was below 40%, if the FFPE block was more than two years old, or if extensive mucin or necrosis was present, as these act as PCR inhibitors. Samples were also rejected when the extracted DNA was of poor quality or quantity, when amplification failed, or when Sanger sequencing quality-control metrics were not met. In our cohort, 12 cases with inadequate tumour tissue or failed amplification of exons 9 to 14 were excluded from the study.

Results

During the study period, 339 patients were treated for endometrial carcinoma at our institute. Of these, POLE mutation testing was successfully performed in 327 cases, which formed the study cohort. The mean patient age was 59.9 years (range: 37–81 yr), with a median of 60 years.

Endometrioid carcinoma was the most common histological subtype, accounting for 234 cases (71.6%), followed by serous carcinoma (45 cases, 13.8%), carcinosarcoma (17 cases, 5.2%), dedifferentiated carcinoma (7 cases, 2.1%), and clear cell carcinoma (7 cases, 2.1%). Mixed and ambiguous histologies comprised the remaining cases. All ambiguous cases were independently reviewed by two pathologists to confirm classification. Tumour grade was available in 320 of 327 cases. Low-grade tumours were observed in 197 cases (60.2%), while 123 cases (37.6%) were high-grade.

POLE mutations were detected in 15 of 327 tumours (4.6%). The majority occurred in endometrioid carcinomas (12 cases), with one case each of serous carcinoma, ambiguous histology, and mixed histology (endometrioid+serous).

Spectrum of POLE mutations

Thirteen of the 15 cases harboured pathogenic POLE mutations, while two cases harboured variants of uncertain significance. One of these harboured dual variants (D301N and L280R) identified by Sanger sequencing. The cases with variants of uncertain significance were not included in the POLE-mutated category for molecular classification ( Table).

Table. Distribution of the 15 POLE hotspot mutations in endometrial carcinoma.

Mutation/variant	Number of cases	Variant classification
P286R	7	Pathogenic
S459F	2	Pathogenic
V411L	2	Pathogenic
Y458H	1	VUS
D301N and L280R (Both mutations found in the same patient)	1	VUS (both)
S297F	1	Pathogenic
A456P	1	Pathogenic

Hotspot Mutations: The numbers in the mutation nomenclature refer to the position at which the amino acid change has occurred. P, proline; R, arginine; S, serine; F, phenylalanine; V, valine; L, leucine; Y, tyrosine; H, histidine; D, aspartic acid; N, asparagine; A, alanine; VUS, variant of uncertain significance

Figure shows the representative electropherograms of the two most observed POLE exonuclease domain mutations in our series (P286R and V411L), demonstrating the characteristic dual peaks seen in a heterozygous base substitution.

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Figure. Electropherogram of (A) forward and (B) reverse sequence of exon 9 of the POLE gene showing missense mutation at position c.857C>G (P286R) (Blue arrows). Electropherogram of (C) forward and (D) reverse sequence of exon 13 of the POLE gene showing missense mutation at position c.1231T>G (V411L) (Grey arrows).

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No significant association was found between POLE mutation status and tumour grade [Odds ratio (OR)=1.07, 95% confidence interval (CI): 0.37–3.09; P=1.00]. Similarly, no statistically significant association was found between POLE mutation and tumour type (OR= 2.39, 95% CI: 0.53–10.84; P=0.37).

Discussion

We identified POLE exonuclease domain mutations in a small but clinically important subset of tumours, aligning with the well-established prevalence of ∼5–10% reported globally. The mutations were most often seen in endometrioid carcinomas, with P286R and V411L representing the most frequent alterations. Endometrial carcinoma shows a rising incidence worldwide, with the mean age at diagnosis varying across populations. In India, prior studies have reported mean ages in the mid-50s to early 60s, and our cohort of 60 years corroborates this pattern, suggesting no significant regional deviation.^17,18

The prevalence observed in our series closely mirrors that from neighbouring Asian populations while appearing somewhat lower than pooled global estimates. A Thai cohort reported a prevalence of 5.1%, while two South Indian studies documented 2.7% and 10.4%, placing our findings intermediate between these extremes.^19-21 Large-scale meta-analyses from predominantly Western cohorts report higher pooled rates of ∼8%, highlighting regional variability. A Japanese cohort described POLE mutations in 8.7% of cases, confined to P286R and V411L. However, their analysis was limited to exons 9 and 13, the two known hotspots, and thus did not capture additional rare variants.²² In contrast, our comprehensive interrogation of the entire exonuclease domain identified both common and rare alterations, underscoring how methodological scope can influence reported prevalence. This distinction also has clinical implications: hotspot-restricted testing may underestimate mutation frequency, whereas broader sequencing provides a more complete profile for molecular stratification. A large Chinese study reported a prevalence of 8.9%, where P286R and V411L were the most frequent mutations, while Q453R also emerged as a recurrent variant, further underscoring geographic heterogeneity in mutation spectra.²³

Consistent with the broader literature, most POLE-mutated tumours in our cohort displayed endometrioid morphology. Although the association was not statistically significant, likely owing to small numbers, meta-analyses with larger datasets have likewise observed a predominance of endometrioid histology without reaching significance.²⁴ Histological subtype remains clinically relevant as recent pooled evidence from previously published literature indicates that within the same molecular subgroup, non-endometrioid tumours portend worse outcomes compared with endometrioid carcinomas.²⁵ Another key observation was that 40% of our POLE-mutated cases were histologically high grade. This paradox of tumours with aggressive morphology but indolent clinical behaviour has been consistently described in the literature and is attributed to the ultramutated phenotype, high neoantigen load, and robust immune response characteristic of POLE-mutated cancers.²⁶

The mutational spectrum in our study reaffirmed the predominance of P286R and V411L, while also documenting less common variants such as S297F and A456P.²⁷ Although we did not detect a founder mutation, subtle shifts in distribution compared with other populations may reflect population-specific variation that merits further investigation. Two cases harboured variants of uncertain significance of which one case showed dual variants (D301N and L280R) on Sanger sequencing. Although not classified as POLE-mutated, the location of these variants in the exonuclease domain suggests possible functional relevance. In line with TCGA recommendations, tumour mutational burden assessment could have further clarified their significance, but this was not feasible in our setting due to cost and resource constraints.³ Also, since PCR and Sanger sequencing were performed exclusively on tumour-derived DNA, and paired peripheral blood or normal tissue were not tested, the distinction between somatic and germline variants could not be ascertained in our study.

The landmark TCGA study, which classified endometrial cancers into the four prognostically distinct groups, requires extensive genomic testing, which is often impractical. To address this, simplified approaches such as the ProMisE classifier and the BAGP recommendations combine widely available techniques like MMR and p53 immunohistochemistry with targeted POLE sequencing. This stepwise strategy is also endorsed by the 2021 ESMO–ESGO–ESTRO consensus guidelines.^28,29 Our testing method follows this simplified yet effective model by systematically sequencing the entire POLE exonuclease domain, enabling detection of both common and rare variants. This is particularly relevant in resource-constrained settings, where cost-effective sequencing provides reliable results. While focused assays such as real-time PCR and digital PCR deliver sensitive and rapid detection of known POLE hotspots at lower cost, they cannot capture rare or novel variants. Overall, these findings reinforce the clinical utility of incorporating POLE mutation analysis into the molecular classification of endometrial carcinoma in India.