Context.—

Molecular stratification of endometrial carcinoma provides more accurate prognostic information than traditional clinicopathologic features. However, because next-generation sequencing is typically recommended for polymerase epsilon (POLE) mutation detection, the practical application of a test based on molecular stratification is limited in the clinical setting.

Objective.—

To evaluate a polymerase chain reaction (PCR)–based assay for POLE mutation detection in endometrial carcinoma.

Design.—

We developed a PCR-based technology called Dalton Mutation Identifier Technology (Dalton-MIT) that targets 9 mutation sites within POLE exons. Endometrial carcinoma specimens from 613 patients were tested for POLE mutations. Correlations between POLE mutations and patient clinicopathologic characteristics and prognosis were analyzed.

Results.—

PCR detection data showed that the incidence rate of POLE mutation was 11.4% (70 of 613). Patients with POLE mutations had better clinicopathologic characteristics and prognosis than those with non–POLE mutations. Comparison between Dalton-MIT and next-generation sequencing in 59.5% (365 of 613) of specimens showed that the sensitivity of Dalton-MIT for detecting POLE pathogenic mutations was 100%, the specificity was 99.3%, the Youden index was .993, and the κ value was .981 (P < .001).

Conclusions.—

Our data demonstrate that POLE mutation detection by Dalton-MIT correlates with next-generation sequencing. This suggests that Dalton-MIT represents a promising alternative assay for detecting POLE mutations and will facilitate the wider application of molecular stratification tools for endometrial carcinoma in the clinic.

Endometrial carcinoma is one of the most common gynecologic malignancies worldwide, with increasing mortality rates each year.1  Prognosis in cases of endometrial carcinoma depends on clinicopathologic features, such as histopathologic type and stage, as defined by the International Federation of Gynecology and Obstetrics (FIGO).2  However, an increasing amount of clinical data demonstrate that the prognostic value of simple clinicopathologic factors is limited.3  Four clinically significant subgroups of endometrial carcinoma are identified through studies using next-generation sequencing and multiomics at the genomic level, including polymerase epsilon (POLE) mutation, microsatellite instability high secondary to deficient mismatch repair (dMMR), copy number high (p53 mutation), and copy number low (p53 wild-type).4,5  Retrospective evidence has shown that molecular stratification provides more accurate prognostic information than clinicopathologic features, which improves the precision of therapies used to treat endometrial carcinoma.6,7  However, next-generation sequencing techniques are typically used for molecular stratification,3,6,8  which restricts the practical application of this technique in clinical settings. In 2020, guidelines published by the National Comprehensive Cancer Network recommended immunohistochemistry analysis for mismatch repair (MMR) and p53 proteins in place of next-generation sequencing of MMR and p53 gene detection.7,9–12  Simpler methods for performing molecular stratification in cases of endometrial carcinoma are needed. However, a convenient and accurate alternative to next-generation sequencing techniques for detecting POLE mutations has not been developed.13 

Polymerase chain reaction (PCR) is a widely used laboratory technique that can accurately identify mutations in genes with known mutations.14–16  POLE mutation is defined as a frequency of 100 or more mutations per megabase in the exonuclease domain of the replicative DNA POLE.4  The 5 most common POLE mutations (P286R, V411L, S297F, A456P, and S459F) have been defined.4,13,17  However, other POLE variants with less frequent mutations are still undefined.18,19  Furthermore, the detection of POLE mutation by PCR is challenging.

In this study, a PCR-based technology targeting 11 types of nucleotide substitutions toward 9 mutation sites in POLE exons was developed and called Dalton Mutation Identifier Technology (Dalton-MIT). Using Dalton-MIT, POLE mutations were detected in formalin-fixed, paraffin-embedded (FFPE) tissues from 613 patients with endometrial carcinoma. The correlation of POLE mutations with clinicopathologic characteristics and prognosis was analyzed. Next-generation sequencing was also performed to detect POLE pathogenic mutations in 365 of 613 specimens (59.5%). The accuracy of the 2 detection methods was compared. This study aimed to evaluate the accuracy of Dalton-MIT in detecting POLE mutations in order to provide a cost-effective, feasible, practical, and accurate alternative for molecular stratification in endometrial carcinoma.

Patients and Sample Collection

This study enrolled a total of 649 patients who received a diagnosis pathologically of endometrial carcinoma and underwent standard staging surgery at the Women’s Hospital Zhejiang University School of Medicine (Hangzhou, China) between January 2015 and December 2020. Clinicopathologic data were collected from all patients, except 9 (1.4%) who were excluded because of incomplete clinicopathologic data. Among the remaining 640 patients (98.6%), FFPE tissue specimens were resected, with 6 serial sections obtained from each specimen. One slice was used for hematoxylin-eosin staining and pathologic review to identify regions with greater than 30% tumor and less than 20% necrosis. The remaining 5 slices were used for DNA extraction (27 patients [4.2%] were further excluded because of insufficient DNA quality). Thus, 613 specimens (95.8%) were included in the final analysis (Figure 1).

Figure 1.

Flow chart of patient enrollment and sample collection. Abbreviations: FIGO, International Federation of Gynecology and Obstetrics; MIT, Mutation Identifier Technology; POLE-mut, polymerase epsilon mutation.

Figure 1.

Flow chart of patient enrollment and sample collection. Abbreviations: FIGO, International Federation of Gynecology and Obstetrics; MIT, Mutation Identifier Technology; POLE-mut, polymerase epsilon mutation.

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All pathologic diagnoses were reviewed independently by 2 pathologists, who followed the World Health Organization classification of tumors for endometrial carcinoma.20  The study was approved by the ethics committee of the hospital (reference No. IRB-20200363-R).

DNA Extraction

DNA was extracted from 5 serial slices 10 μm thick for each specimen using a NuClean FFPE DNA kit (CWbio, catalog No. CW 2646) according to the manufacturer’s instructions. The purity and concentration of the DNA extracts were determined using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific).

Dalton-MIT Assay

A total of 9 nucleotide mutation sites in exons 9 to 14 of the POLE gene were selected for detection based on a literature review and reference to The Cancer Genome Atlas (https://www.cancer.gov/ccg/research/genome-sequencing/tcga), the Clinvar database (http://www.ncbi.nlm.nih.gov/clinvar), and the Cosmic database (https://cancer.sanger.ac.uk/cosmic). The 9 mutation sites were c.857C>G, c.890C>T, c.1100T>C, c.1231G>T/C, c.1270C>G/A, c.1307C>G, c.1331T>A, c.1366G>C, and c.1376C>T, referring to 11 types of nucleotide mutations and corresponding to 10 amino acid variations, namely, P286R, S297F, F367S, V411L, L424V/I, P436R, M444K, A456P, and S459F, respectively (Supplemental Table 1, see Supplemental Digital Content file containing 1 figure and 5 tables, at https://meridian.allenpress.com/aplm in the August 2024 table of contents). Among these, c.1231G>T and c.1231G>C shared the same amino acid variation (V411L). Primers specific to 11 types of nucleotide substitutions were designed, synthesized, and assembled into the Dalton-MIT kit (Supplemental Table 2). The kit contained 6 PCR tubes with the corresponding primer probe combinations. DNA (1.5 µL) was added to each tube for PCR. The PCR procedure was as follows: 37°C for 5 minutes, 95°C for 3 minutes (1 cycle), 95°C for 10 seconds, 53°C for 10 seconds, and 72°C for 35 seconds (40 cycles). Signals of the channels FAM, VIC, and Cy5 for each tube, signals of FAM and VIC for tube C, and signals of VIC for the external control tube F (globin as internal reference gene) were collected in the second stage at 72°C. The ΔCT for each mutation site was calculated as the CT value of a specific site minus the CT value of the external control. Next-generation sequencing of the pathogenic mutations for each corresponding site was used as the standard for comparison. The threshold (ΔCT) value of the Dalton-MIT assay to identify a POLE mutation was determined for each mutation site, and an equal to or less than certain ΔCT value was defined as POLE mutation positive (POLE-mut). Greater than certain ΔCT values was defined as POLE mutation negative (non–POLE-mut) (Supplemental Table 3). All 613 samples were tested using the Dalton-MIT assay.

Next-Generation Sequencing

Among 613 specimens, 384 (62.6%) were selected for next-generation sequencing. Of these, 70 specimens (11.4%) that were defined as POLE-mut by the Dalton-MIT assay were included, and 314 specimens (51.2%) that were classified as non–POLE-mut were randomly assigned by computer at a ratio of approximately 2:1. A total of 19 specimens (4.9%) failed sequencing. Thus, 365 specimens (95.1%) yielded high-quality sequencing data and were included in the final analysis (Figure 1).

Multigene target sequencing was performed using VariantPro Capture Technology by LC Sciences (Hangzhou, China), as described previously.21–23  The presence of the desired fragments and the purity of the indexed libraries were analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies) using a High-Sensitivity DNA Analysis Kit (Agilent Technologies). The concentrations of the libraries were measured using a Qubit Fluorometer (ThermoFisher Scientific). Sequencing was performed using the Illumina MiSeq Reagent v2 kit (500 cycles) on the Illumina MiSeq instrument following the manufacturer’s instructions (Illumina). Mutation analysis of exons 9 to 14 was performed using Mutation Surveyor software (SoftGenetics, State College, Pennsylvania) following manual inspection.

Statistical Analysis

Fisher exact test and independent t tests were used to analyze the correlation between POLE mutation status and clinicopathologic data. Nonparametric testing was used for variables not meeting the assumptions of the t test equivalent. Survival analysis was performed by using Kaplan-Meier curves and log-rank tests. All analyses were performed using SPSS version 25.0 (IBM). All P value tests were 2 sided, and a P value <.05 was considered statistically significant.

The Positive Rate, Frequency, and Percentage of POLE-mut by Dalton-MIT

Of 613 specimens, Dalton-MIT detected 70 POLE-mut specimens (11.4%), all of which were single-nucleotide mutations (Supplemental Table 4). Among the 9 detected mutation sites, the 3 most common were c.857C>G (5.38%), c.1231G>T/C (3.26%), and c.1366G>C (1.14%). The top 3 sites for mutation frequencies in all POLE-mut specimens were c.857C>G (47.14%), c.1231G>T/C (28.57%), and c.1366G>C (10.00%), and their corresponding amino acid changes were P286R, V411L, and A456P, respectively.

Comparison of Clinicopathologic Characteristics Between POLE-mut and Non–POLE-mut Patients by Dalton-MIT

The clinicopathologic characteristics of 613 patients are shown in Table 1. Compared with POLE-mut patients, non–POLE-mut patients were at a significantly higher FIGO stage and had more lymph node metastasis (both P < .05). These results suggest that POLE-mut patients present less aggressive clinical and pathologic characteristics than non–POLE-mut patients.

Table 1.

Comparison of Clinicopathologic Characteristics Between Polymerase Epsilon Mutation–Positive (POLE-mut) and Non–POLE-mut Patients by Dalton Mutation Identifier Technology

Comparison of Clinicopathologic Characteristics Between Polymerase Epsilon Mutation–Positive (POLE-mut) and Non–POLE-mut Patients by Dalton Mutation Identifier Technology
Comparison of Clinicopathologic Characteristics Between Polymerase Epsilon Mutation–Positive (POLE-mut) and Non–POLE-mut Patients by Dalton Mutation Identifier Technology

Survival Analysis for POLE-mut and Non–POLE-mut Patients by Dalton-MIT

All 613 patients were followed up for a median of 22 months (range, 5–75 months). In total, 26 patients (4.2%) were lost to follow-up. The overall survival (OS) and progression-free survival (PFS) of POLE-mut and non–POLE-mut patients were analyzed. Of the 587 patients (95.8%) who completed follow-up, 22 (3.7%) experienced recurrence, and 14 (2.4%) died during the follow-up period. None of these patients were in the POLE-mut group. POLE-mut patients had higher OS (mean ± SD, 25.88 ± 1.786 versus 23.89 ± 0.565 months) and PFS (mean ± SD, 25.88 ± 1.786 versus 23.37 ± 0.557 months) than non-POLE-mut patients, but the difference was not significant (Figure 2, A and B). In patients with high-grade (G3) endometrioid carcinoma or FIGO stages II to IV, differences were more obvious but remained nonsignificant (Figure 2, C through F). Our results indicate that patients with POLE-mut may have a better prognosis than those with non-POLE-mut.

Figure 2.

Survival analysis. A, Comparison of overall survival between endometrial carcinoma patients with polymerase epsilon mutation (POLE-mut) and non–POLE-mut patients, in all patients. B, Comparison of progression-free survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in all patients. C, Comparison of overall survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with grade 3 cancer. D, Comparison of progression-free survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with grade 3 cancer. E, Comparison of overall survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with stages II to IV. F, Comparison of progression-free survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with stages II to IV.

Figure 2.

Survival analysis. A, Comparison of overall survival between endometrial carcinoma patients with polymerase epsilon mutation (POLE-mut) and non–POLE-mut patients, in all patients. B, Comparison of progression-free survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in all patients. C, Comparison of overall survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with grade 3 cancer. D, Comparison of progression-free survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with grade 3 cancer. E, Comparison of overall survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with stages II to IV. F, Comparison of progression-free survival between endometrial carcinoma POLE-mut and non–POLE-mut patients, in patients with stages II to IV.

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POLE Pathogenic Mutations as Detected by Next-Generation Sequencing

POLE pathogenic mutations were detected in 64 of 365 specimens (17.5%; Supplemental Table 5). The detected mutation sites were c.857C>G, c.1231G>T/C, c.1366G>C, c.1376C>T, c.1100T>C, c.890C>T, c.1270C> G, c.1331T>A, and c.1307C>G. The 3 most common sites were c.857C>G (45.3%), c.1231G>T/C (29.7%), and c.1366G>C (9.4%), and the top 3 mutation frequencies were c.890C>T (33.4%), c.1331T>A (24.5%), and c.1307C>G (23.5%).

Comparison of Mutation Detections Between Dalton-MIT and Next-Generation Sequencing

Furthermore, we performed a direct comparison between Dalton-MIT and next-generation sequencing in 365 specimens (59.5%). All 9 mutation sites were detected by both assays, but only 10 of 11 types of nucleotide mutations were detected by next-generation sequencing (Table 2). In 2 samples, POLE-mut was detected by Dalton-MIT but not next-generation sequencing. The POLE-mut was further verified by Sanger sequencing in these 2 samples (Supplemental Figure 1).

Table 2.

Consistency of Polymerase Epsilon Mutation (POLE-mut) Detected by Dalton Mutation Identifier Technology (Dalton-MIT) and POLE Pathogenic Mutation Detected by Next-Generation Sequencing (NGS)

Consistency of Polymerase Epsilon Mutation (POLE-mut) Detected by Dalton Mutation Identifier Technology (Dalton-MIT) and POLE Pathogenic Mutation Detected by Next-Generation Sequencing (NGS)
Consistency of Polymerase Epsilon Mutation (POLE-mut) Detected by Dalton Mutation Identifier Technology (Dalton-MIT) and POLE Pathogenic Mutation Detected by Next-Generation Sequencing (NGS)

POLE mutation detection by Dalton-MIT was compared to next-generation sequencing as the standard. The sensitivity of the Dalton-MIT assay for detecting POLE pathogenic mutations was 100%, the specificity was 99.3%, the Youden index was .993, and the κ value was .981 (P < .001), as shown in Figure 3, A through C. Our data demonstrate good consistency between Dalton-MIT and next-generation sequencing for detecting POLE mutations.

Figure 3.

Performance of Dalton Mutation Identifier Technology (Dalton-MIT) for identifying polymerase epsilon mutation (POLE) pathogenic mutation (POLE-mut) compared with next-generation sequencing (NGS). A, Number of POLE-mut and non–POLE-mut specimens detected by Dalton-MIT and NGS, respectively. B, Receiver operating characteristic (ROC) curve of Dalton-MIT. C, Evaluation indicators for accuracy of Dalton-MIT. Abbreviations: NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio; PPV, positive predictive value; YI, Youden index.

Figure 3.

Performance of Dalton Mutation Identifier Technology (Dalton-MIT) for identifying polymerase epsilon mutation (POLE) pathogenic mutation (POLE-mut) compared with next-generation sequencing (NGS). A, Number of POLE-mut and non–POLE-mut specimens detected by Dalton-MIT and NGS, respectively. B, Receiver operating characteristic (ROC) curve of Dalton-MIT. C, Evaluation indicators for accuracy of Dalton-MIT. Abbreviations: NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio; PPV, positive predictive value; YI, Youden index.

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Primers for the most common mutations, including P286R, V411L, S297F, A456P, and S459F, were assembled in the Dalton-MIT kit. POLE mutations were detected in 70 of 613 patients (11.4%), which is consistent with previous reports that POLE mutations are found in 7% to 12% of endometrial cancer cases, as detected by next-generation sequencing.19  The most common mutation was P286R (47.14%), followed by V411L (28.57%) and A456P (10.00%). Next-generation sequencing was performed on 365 of 613 specimens (59.5%) in order to compare the 2 technologies; only 2 specimens showed discrepant results. The sensitivity of Dalton-MIT for detecting POLE pathogenic mutations was 100%, the specificity was 99.3%, the Youden index was .993, and the κ value was .981 (P < .001).

POLE is a catalytic subunit of DNA POLE and is involved in nuclear DNA repair and replication. Pathogenic mutations in the POLE gene identified a subtype of endometrial cancer with a nonprogression prognosis. Multiple POLE mutation sites are detected by next-generation sequencing, but only those that correlate with favorable prognosis are defined as pathogenic mutations.13  The 5 most common mutations (P286R, V411L, S297F, A456P, and S459F) have been confirmed. The pivotal study by The Cancer Genome Atlas found substitutions primarily in P286R and V411L, although S297F, A456P, M444K, and L424I substitutions were also detected.4,13  León-Castillo et al13  developed a scoring system to estimate the pathogenicity of novel POLE mutations based on the presence or absence of genomic alterations associated with known POLE pathogenic mutations. As reported, approximately 7% to 12% of endometrial carcinomas demonstrate pathogenic mutations in the POLE gene. In our study, POLE mutations were detected by the Dalton-MIT kit in 70 of 613 specimens (11.4%), which is consistent with previous reports.

The PCR technique is a fast, simple, and convenient technology with wide applications in disease diagnosis. However, it is limited by its ability to only detect genes with known mutated sites because it can only amplify preselected DNA segments.14  Kim and colleagues developed a droplet digital PCR assay for detecting 5 types of POLE mutations (P286R, V411L, S297F, A456P, and S459F), which only account for 84% of the 11 pathogenic mutations.24  However, the Dalton-MIT kit, covering a wider range of POLE mutations, has a better accuracy. The Dalton-MIT kit is also more applicable in a clinical setting because of its low cost and convenient handling.

The results of the current study demonstrate that Dalton-MIT detection in patients with endometrial cancer with POLE-mut presented with a better prognosis than those with non–POLE-mut. All of the 22 patients who experienced recurrence (3.7%) and the 14 who died during the follow-up period (2.4%) were non–POLE-mut patients. Survival analysis showed that both the OS and PFS of POLE-mut patients were longer than those of non–POLE-mut patients, even though the difference was not significant. A possible reason for this could be that 90.4% of patients enrolled in this study were classified at an early stage (stage I or II), and therefore had a good prognosis. In the current study, the recurrence rates of these patients were 9.9% (8 of 81) and 7.6% (10 of 131), respectively. This study is limited by its small single-center retrospective design. Future studies should include more cases and expand to multicenter prospective designs in order to validate the POLE mutation detection by the Dalton-MIT kit.

POLE mutations are generally identified by next-generation sequencing or Sanger sequencing. Both methods have certain limitations. Next-generation sequencing is expensive and time-consuming, whereas Sanger sequencing has low sensitivity. PCR is a technology that is easy to operate and is inexpensive. The Dalton-MIT kit was developed to address these limitations and is a convenient and straightforward tool for POLE mutation detection. Using the Dalton-MIT kit to detect POLE mutations can be widely applied for molecular stratification in the clinic to accurately predict prognosis.

The Dalton-MIT assay targeted 9 POLE mutation sites, and POLE mutations were detected in 11.4% of samples from 613 patients with endometrial cancer. Patients with POLE-mut had better clinicopathologic characteristics and prognosis than those with non–POLE-mut. There was a good consistency between PCR and next-generation sequencing for detecting mutations. Our results suggest that Dalton-MIT is a promising alternative assay to detect POLE mutations. It will facilitate the wider application of molecular classification of endometrial cancer in the clinical setting.

We gratefully acknowledge the kind cooperation of all the members in our department.

1.
Crosbie
 
EJ,
Kitson
 
SJ,
McAlpine
 
JN,
Mukhopadhyay
 
A,
Powell
 
ME,
Singh
 
N.
Endometrial cancer
.
Lancet
.
2022
;
399
(
10333
):
1412
1428
.
2.
Brooks
 
RA,
Fleming
 
GF,
Lastra
 
RR,
et al.
Current recommendations and recent progress in endometrial cancer
.
CA Cancer J Clin
.
2019
;
69
(
4
):
258
279
.
3.
León-Castillo
 
A,
de Boer
 
SM,
Powell
 
ME,
et al.
Molecular classification of the PORTEC-3 trial for high-risk endometrial cancer: impact on prognosis and benefit from adjuvant therapy
.
J Clin Oncol
.
2020
;
38
(
29
):
3388
3397
.
4.
Kandoth
 
C,
Schultz
 
N,
Cherniack
 
AD,
et al.
Integrated genomic characterization of endometrial carcinoma
.
Nature
.
2013
;
497
(
7447
):
67
73
.
5.
Church
 
DN,
Stelloo
 
E,
Nout
 
RA,
et al.
Prognostic significance of POLE proofreading mutations in endometrial cancer
.
J Natl Cancer Inst
.
2015
;
107
(
1
):
402
.
6.
Stasenko
 
M,
Tunnage
 
I,
Ashley
 
CW,
et al.
Clinical outcomes of patients with POLE mutated endometrioid endometrial cancer
.
Gynecol Oncol
.
2020
;
156
(
1
):
194
202
.
7.
Casey
 
L,
Singh
 
N.
POLE, MMR, and MSI testing in endometrial cancer: proceedings of the ISGyP Companion Society Session at the USCAP 2020 annual meeting
.
Int J Gynecol Pathol
.
2021
;
40
(
1
):
5
16
.
8.
Talhouk
 
A,
McConechy
 
MK,
Leung
 
S,
et al.
Confirmation of ProMisE: a simple, genomics-based clinical classifier for endometrial cancer
.
Cancer
.
2017
;
123
(
5
):
802
813
.
9.
Singh
 
N,
Piskorz
 
AM,
Bosse
 
T,
et al.
p53 immunohistochemistry is an accurate surrogate for TP53 mutational analysis in endometrial carcinoma biopsies
.
J Pathol
.
2020
;
250
(
3
):
336
345
.
10.
Stelloo
 
E,
Jansen
 
AML,
Osse
 
EM,
et al.
Practical guidance for mismatch repair-deficiency testing in endometrial cancer
.
Ann Oncol
.
2017
;
28
(
1
):
96
102
.
11.
McConechy
 
MK,
Talhouk
 
A,
Li-Chang
 
HH,
et al.
Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas
.
Gynecol Oncol
.
2015
;
137
(
2
):
306
310
.
12.
Uterine neoplasms, version 1
.
2020
.
National Comprehensive Cancer Network Web site
. https://www.nccn.org/professionals/physician_gls/. Accessed March 6, 2020.
13.
León-Castillo
 
A,
Britton
 
H,
McConechy
 
MK,
et al.
Interpretation of somatic POLE mutations in endometrial carcinoma
.
J Pathol
.
2020
;
250
(
3
):
323
335
.
14.
Matsuda
 
K.
PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement
.
Adv Clin Chem
.
2017
;
80
:
45
72
.
15.
de Oliveira Cavagna
 
R,
Leal
 
LF,
de Paula
 
FE,
Bernardinelli
 
GN,
Reis
 
RM.
A PCR-based approach for driver mutation analysis of EGFR, KRAS, and BRAF genes in lung cancer tissue sections
.
Methods Mol Biol
.
2021
;
2279
:
109
126
.
16.
Corné
 
J,
Le Du
 
F,
Quillien
 
V,
et al.
Development of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients
.
Sci Rep
.
2021
;
11
(
1
):
17316
.
17.
Church
 
DN,
Briggs
 
SE,
Palles
 
C,
et al.
DNA polymerase ε and δ exonuclease domain mutations in endometrial cancer
.
Hum Mol Genet
.
2013
;
22
(
14
):
2820
2828
.
18.
Billingsley
 
CC,
Cohn
 
DE,
Mutch
 
DG,
Stephens
 
JA,
Suarez
 
AA,
Goodfellow
 
PJ.
Polymerase ɛ (POLE) mutations in endometrial cancer: clinical outcomes and implications for Lynch syndrome testing
.
Cancer
.
2015
;
121
(
3
):
386
394
.
19.
Rayner
 
E,
van Gool
 
IC,
Palles
 
C,
et al.
A panoply of errors: polymerase proofreading domain mutations in cancer
.
Nat Rev Cancer
.
2016
;
16
(
2
):
71
81
.
20.
Female Genital Tumours. 5th ed. Lyon,
France
:
IARC Press
;
2020
.
World Health Organization Classification of Tumours
; vol.
4
.
21.
Dai
 
Y,
Wang
 
C,
Nie
 
Z,
et al.
Mutation analysis of Leber's hereditary optic neuropathy using a multi-gene panel
.
Biomed Rep
.
2018
;
8
(
1
):
51
58
.
22.
Németh
 
K,
Darvasi
 
O,
Likó
 
I,
et al.
Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas
.
J Endocrinol Invest
.
2019
;
42
(
8
):
931
940
.
23.
Li
 
X,
Liu
 
L,
Xi
 
Q,
et al.
Short-term serum deprivation causes no significant mitochondrial DNA mutation in vascular smooth muscle cells revealed by a new next generation sequencing technology
.
Acta Biochim Biophys Sin (Shanghai)
.
2016
;
48
(
9
):
862
864
.
24.
Kim
 
G,
Lee
 
SK,
Suh
 
DH,
et al.
Clinical evaluation of a droplet digital PCR assay for detecting POLE mutations and molecular classification of endometrial cancer
.
J Gynecol Oncol
.
2022
;
33
(
2
):
e15
.

Author notes

Supplemental digital content is available for this article at https://meridian.allenpress.com/aplm in the August 2024 table of contents.

Chen and Y. Li contributed equally to this work

This study was financially supported by the National Natural Science Foundation of China (No. 82072858 to Y. Li).

The authors have no relevant financial interest in the products or companies described in this article.

Supplementary data