Clinical impact of endometrial cancer stratified by genetic mutational profiles, POLE mutation, and microsatellite instability
Clinical impact of endometrial cancer stratified by genetic mutational profiles, POLE mutation, and microsatellite instability
Tomoko Haruma 0 1
Takeshi Nagasaka 1
Keiichiro Nakamura 0 1
Junko Haraga 0 1
Akihiro Nyuya 1
Takeshi Nishida 0 1
Ajay Goel 1
Hisashi Masuyama 0 1
Yuji Hiramatsu 0 1
0 Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama , Japan , 2 Department of Clinical Oncology, Kawasaki Medical School , Kurashiki , Japan , 3 Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute , Dallas, Texas , United States of America, 4 Charles A Sammons Cancer Center, Baylor University Medical Center , Dallas, Texas , United States of America
1 Editor: Amanda Ewart Toland, Ohio State University Wexner Medical Center , UNITED STATES
The molecular characterization of endometrial cancer (EC) can facilitate identification of various tumor subtypes. Although EC patients with POLE mutations reproducibly demonstrate better prognosis, the outcome of patients with microsatellite instability (MSI) remains controversial. This study attempted to interrogate whether genetic stratification of EC can identify distinct subsets with prognostic significance.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by MEXT/JSPS
KAKENHI (15K20144 to TH and 16K11141 to KN).
Competing interests: The authors have declared
that no competing interests exist.
Materials and methods
A cohort of 138 EC patients who underwent surgical resection with curative intent was
enrolled. Sanger sequencing was used to evaluate mutations in the POLE and KRAS
genes. MSI analysis was performed using four mononucleotide repeat markers and
methylation status of the MLH1 promoter was measured by a fluorescent bisulfite polymerase
chain reaction (PCR). Protein expression for mismatch repair (MMR) proteins was
evaluated by immunohistochemistry (IHC).
Extensive hypermethylation of the MLH1 promoter was observed in 69.6% ECs with MLH1
deficiency and 3.5% with MMR proficiency, but in none of the ECs with loss of other MMR
genes (P < .0001). MSI-positive and POLE mutations were found in 29.0% and 8.7% EC
patients, respectively. Our MSI analysis showed a sensitivity of 92.7% for EC patients with
MMR deficiency, and a specificity of 97.9% for EC patients with MMR proficiency. In
univariate and multivariate analyses, POLE mutations and MSI status was significantly associated
with progression-free survival (P = 0.0129 and 0.0064, respectively) but not with endometrial
This study provides significant evidence that analyses of proofreading POLE mutations and
MSI status based on mononucleotide repeat markers are potentially useful biomarkers to
identify EC patients with better prognosis.
Endometrial cancer (EC) is one of the most common gynecologic malignancy in the western
world and Japan, and its prevalence has increased in recent years [
]. Lately, several important
advances have been made in defining the molecular alterations that contribute towards
endometrial tumorigenesis [2±10]. The Cancer Genome Atlas Research Network (TCGA) has
provided new insights that ECs can be divided into four categories according to various genetic
and epigenetic features: an ultramutated phenotype caused by POLE mutations, a
hypermutator phenotype caused by the DNA mismatch repair deficiency (dMMR) leading to
microsatellite instability (MSI), a copy number low phenotype, and a copy number high phenotype [
Among these alterations, the POLE gene is a catalytic subunit of DNA polymerase epsilon
that is involved in nuclear DNA replication and repair. Hotspot mutations are located in the
exonuclease domain of POLE (exons 9±14) which cause an ultramutated phenotype in
colorectal and endometrioid tumors. Especially of EC cases, hotspot mutations in exon 9 (P286R and
S297F) and exon 13 (V411L, L424V and L424I) were reported and EC patients with such
POLE mutations demonstrate a better progression-free survival [
2, 3, 10, 11
MSI is caused by dMMR, which results in greatly increased rates of strand-slippage
mutations, the so-called hypermutator phenotype compared with ECs harboring POLE mutations.
Although the majority of ECs with dMMR are sporadic, 3% to 5% of cases develop disease
because of inherited mutations in MMR genes (Lynch syndrome) [
]. Universal screening by
evaluating tumor MSI status and MMR immunohistochemistry (IHC) has been widely
adapted to screen Lynch syndrome, especially in patients with colorectal cancer [
addition to identifying potential germline mutation carriers, MMR analysis of colorectal and
non-colorectal tumors is used as both a prognostic and a predictive approach for PD-1
targeted therapies [
]. Although ECs with ultramutator phenotype consistently demonstrate
better outcomes, patent survival in EC patients with hypermutated dMMR/MSI remain
controversial [2, 3, 5, 9, 16±23].
In this retrospective study, we initially analyzed genetic mutations in the POLE gene,
evaluated tumor MSI status, MLH1 promoter methylation profile, and MMR expression status in all
138 ECs. Finally, we classified ECs according to the genetic profiles based on POLE mutations
and MSI status to determine their precise relationship with various clinic-pathological
Materials and methods
A cohort of 138 patients with EC resected at Okayama University Hospital (Okayama, Japan)
from 2006 to 2009 was enrolled in this study. All patients underwent surgery followed by
adjuvant chemotherapy and/or radiation if indicated. Institutional review board approval was
granted by the ethics committee of the Okayama University, and written informed consent
was obtained from all patients to use their tissues for research. The medical records of the
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patients were retrospectively explored and matched with clinical and pathological data.
Standard post-treatment surveillance included serial physical examination with pap smears and
computed tomography (CT) including positron emission tomography-computed tomography
(PET-CT) every 3 to 6 months. Clinical data was abstracted from hospital records and
included age at diagnosis, surgical International Federation of Gynecology and Obstetrics
(FIGO) stage, adjuvant treatment and outcomes. Experienced gynecologic pathologists
evaluated all cases for pathological information such as tumor grade, histologic subtype, depth of
myometrial invasion, cervical stromal invasion, and lymphovascular space invasion (LVSI)
and confirmed diagnoses.
DNA extraction and bisulfite modification
We collected formalin-fixed, paraffin-embedded (FFPE) tissue specimens of primary EC from
the cohort of 138 patients who had undergone surgery. DNA was extracted by the TaKaRa
DEXPAT kit (Takara Bio Inc., Otsu, Japan) from EC tissue macro-dissected manually from
FFPE tissue sections. Prior to sodium bisulfite modification, all genomic DNA was purified
and concentrated by ethanol precipitation. Thereafter, genomic DNA was subjected to sodium
bisulfite modification using the EZ DNA Methylation Kit (ZYMO Research, Irvine, CA).
POLE, KRAS, and BRAF mutation analysis
Exon 9 and 13 in the POLE gene, KRAS exon 2 and BRAF exon 15 mutation status were
analyzed in all 138 EC patients. Primer sequences for the POLE mutation analyses are shown in S1
Table. KRAS exon 2 and BRAF exon 15 mutation status were analyzed by using primer sets
described previously [
]. The amplified PCR products were electrophoresed on an ABI 3100
Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
The MSI status was analyzed in all 138 ECs by using four mononucleotide repeat markers
(BAT26, NR21, NR27, and CAT25), as described previously [
]. When at least one or
more mononucleotide repeat markers displayed MSI, tumors were defined to have an MSI
phenotype and the tumors without MSI in the four mononucleotide repeat markers were
defined to have a non-MSI phenotype according to our previous studies.
Methylation analysis for the MLH1 promoter
The MLH1 gene promoter was divided into two regions (5'-region and 3'-region) as described
24, 27, 28
]. The combined bisulfite restriction analysis was modified to measure
methylation density quantitatively by a capillary sequencer. PCR products digested with HhaI
or RsaI (New England BioLabs, Ipswitch, MA, USA) were loaded simultaneously onto an ABI
310R or 31000 Genetic Analyzer (Applied Biosystems, California, USA). Signals from
individual PCR products were distinguished by the unique fluorescent PCR signal from each target
and their fragment length, and the data were analyzed using GeneMapper software version 4.0
(Applied Biosystems, Foster City, CA, USA). In this study, the percentages of methylated HhaI
or RsaI sites were calculated by determining the ratios between the HhaI/RsaI-cleaved PCR
products and the total amount of PCR product in each locus and methylation positive was
defined when the percentages of methylated HhaI or RsaI sites over 5.0%.
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We examined protein expression for MLH1, MSH2, PMS2, and MSH6 in 138 tumor tissues by
immunohistochemical (IHC) staining using DAKO EnVision System-HRP polymer system
kit (DakoCytomation California, Inc., Carpinteria, CA, USA). Staining was performed
manually with FFPE specimens. Thin (5 μm) sections of representative blocks were deparaffinized
and dehydrated using gradient solvents. Following antigen retrieval in the citrate buffer (pH
6.0), endogenous peroxidase was blocked with 3% H2O2. Thereafter, slides were incubated
overnight in the presence of purified mouse monoclonal antibodies against MLH1 (clone
G168-15, BD Pharmingen, San Diego, CA, USA; dilution 1:50), MSH2 (clone G219-1129, BD
Pharmingen; dilution 1:200), PMS2 (clone A16-4, BD Pharmingen; dilution 1:200), and MSH6
protein (clone 44/MSH6, BD Pharmingen; dilution 1:100), respectively. A further incubation
was performed with a secondary antibody and the avidin±biotin±peroxidase complex (Vector
Laboratories, Burlingame, CA, USA) and then incubated with biotinyltyramide, followed by
streptavidin±peroxidase. Diaminobenzidine was used as a chromogen and hematoxylin as a
nuclear counterstain. Tumor cells were scored negative for MMR protein expression only if
the epithelial cells within the tumor tissue lacked nuclear staining, while the surrounding
stromal cells still showed positive staining. Samples showing proficiency in expression of all MMR
proteins were defined as pMMR, and samples showing deficiency in at least one of the four
MMR proteins were defined as dMMR.
Statistical analyses were performed using JMP software (version 10.0; SAS Institute, Inc., Cary,
NC, USA). First, methylation levels were analyzed as continuous variables. Next, the
methylation status was analyzed as a categorical variable (positive, methylation level 5.0%; negative,
methylation level < 5.0%). Categorical variables were compared by Fisher's exact test.
Endometrial cancer-specific survival (ECS) was calculated from the length of time from treatments
including neo-adjuvant therapies or surgical resection to the date of death due to EC or last
follow-up for censored patients. Progression-free survival (PFS) was defied as the time from
surgical resection to recurrence or progression by CT and/or PET-CT routinely performed every
3 to 6 months. ECS and PFS were univariately estimated with the Kaplan±Meier method.
Univariate and multivariate analyses for ECS and PFS were performed by Cox's proportional
hazard regression. Clinically accepted prognostic factors significant on univariate analysis were
included in the model, including age, stage, tumor grade, histology, depth of invasion, LVSI,
cervical stromal invasion, adjuvant treatment, KRAS status, and POLE mutation/MSI status.
All reported P values were two-sided and a P value of less than 0.05 was considered statistically
Expression status of mismatch repair proteins
Clinicopathological findings and outcomes of 138 EC patients enrolled in this study are
summarized in Table 1 and S1 Fig. In total, 123 endometrioid tumors (89.1%) and 15 others
including clear cell and serous (10.9%) endometrial cancers were included. By FIGO staging
criteria, stage I, II, III, and IV were 93 (67.4%), 11 (7.8%), 24 (17.4%), and 10 (7.2%),
respectively. Expression status of the four MMR proteins (MLH1, MSH2, PMS2, and MSH6) was
confirmed in all 138 EC tissues by IHC. Representative examples of IHC staining results are
shown in Fig 1. By the IHC analysis, 97 tumors (70.3%) were classified as MMR-proficient
(pMMR) and 41 (29.7%) as MMR-deficient (dMMR). Of 41 dMMR tumors, 23 (56.1% in
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Depth of myometrial invasion
LSVI and BMI denote lymphovascular space invasion and body mass index, respectively. MMR denotes mismatch repair.
P values were calculated by chi-squire test.
Fig 1. Representative examples of immunohistochemistry staining for the four MMR proteins. Tumors with MLH1-deficiency (dMLH1) show negative expression
in both MLH1 and PMS2 IHC, those with MSH2-deficiency (dMSH2) show negative expression in both MSH2 and MSH6 IHC, with PMS2-deficiency (dPMS2) they
show negative expression only in PMS2, and tumors with MSH6-deficiency show negative expression only in MSH6.
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dMMR) showed both MLH1- and PMS2-deficiency (dMLH1), 8 tumors (19.5%) both
MSH2and MSH6-deficiency (dMSH2), 8 tumors (19.5%) MSH6-deficiency alone (dMSH6), and 2
tumors (4.9%) PMS2-deficiency alone (dPMS2).
Association between methylation profiles of two discrete promoter regions
in MLH1 and MLH1 protein expression
In view of the published evidence that to inactivate MLH1 expression, extensive methylation
towards whole promoter CpG region in the MLH1 gene is required [
24, 27, 29
investigated the methylation status of both the 5'- and the 3'-regions of the MLH1 promoter in a
cohort of 138 ECs. Results of a panel of representative fluorescent bisulfite PCRs following
restriction enzyme analysis are depicted in Panel A in Fig 2, and these results were analyzed
with methylation as a continuous and a categorical variable. Partial methylation in MLH1 (i.e.
affecting the 5'-region only) was observed in 24 of 138 ECs (17.4%) and extensive methylation
Fig 2. Methylation analysis of the promoter region in the MLH1 gene. (A) Schematic depiction of two regions (5'-region and 3'-region) of the MLH1 promoter for
methylation and results of a panel of representative fluorescent bisulfite PCR following restriction enzyme analysis. Methylated samples had the new fragment cleaved by
the restriction enzyme. (B) The frequencies of MLH1 promoter methylation according to MLH1 expression status. The top panel shows the results of the MLH1-5'
region, the middle panel shows the MLH1-3' region and the bottom panel shows partial (i.e. only MLH1-5' methylation) and extensive methylation (i.e. both MLH1-5'
and -3' methylation).
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(i.e. affecting both the 5'-region and the 3'-region) was confirmed in 18 of 138 ECs (13.0%).
Partial methylation in MLH1 was observed in 13 of 97 (13.4%) pMMR-ECs, 4 of 23 (17.4%)
ECs with dMLH1, 4 of 8 (50.0%) ECs with dMSH2, 2 of 8 (25.0%) ECs with dMSH6, and 1 of
2 (50.0%) ECs with dPMS2, whereas extensive methylation was detected in 2 of 97 (2.1%) ECs
with pMMR, 16 of 23 (69.6%) ECs with dMLH1, none of the other dMMR (P < .0001, Panel B
in Fig 2).
KRAS/BRAF mutation status
Because sporadic MSI/dMMR phenotype in colorectal cancer is strongly associated with BRAF
V600E mutation [
]. We analyzed mutations in the KRAS and BRAF genes (Panel A in S2
Fig). In this cohort, no BRAF mutation were observed in exon 15 while KRAS mutations in
exon 2 were present in 20 EC patients (14.5%), and the spectrum of relative frequency of
individual mutations was 7 (35.0% in all KRAS mutant), 7 (35.0%), 2 (10.0%), 2 (10.0%), 1 (5.0%),
and 1 (5.0%) for the G12D, G12V, G12A, G12C, G13D, and G13S mutations, respectively.
POLE mutation status
As determination of ECs with POLE mutations was the first step in the stratification based
upon their genomic features, we examined proofreading POLE mutations in exon 9 and 13 [
By conventional Sanger sequencing, a total of 12 ECs (8.7%) with POLE mutations was
observed; the spectrum of relative frequencies of individual mutations was 7 (58.3% of POLE
mutations) for P286R and 5 (41.7% of POLE mutations) for V411L, all of the POLE mutations
we confirmed was exonuclease domain hotspot mutations (Panel B in S2 Fig).
Association between MSI, POLE mutation, MLH1 methylation, and MMR
protein expression status
According to our previous studies[
], tumors with MSI-positive (MSI) status were defined
when at least one or more mononucleotide repeat markers displayed allelic variations and the
tumors without MSI in the four mononucleotide repeat markers were defined as a non-MSI.
By this criterion, we detected MSI tumors in 40 (29.0%) of the 138 EC patients. Panel A in Fig
3 shows representative examples of both pMMR and dMMR cases for each marker. By the
four mononucleotide repeat markers, 92.7% (38 of 41) of MSI tumors showed dMMR, and
97.9% (95 of 97) of non-MSI tumors showed pMMR. Similar with previous studies, 12 ECs
with POLE mutations defined as non-MSI by the four mononucleotide makers and positive
staining in the four MMR proteins (Panel B in Fig 3). Regarding dMMR, only three cases did
not show MSI signatures; one case was dMLH1 epigenetically silenced by MLH1 promoter
methylation and the other two cases were dMSH6.
Clinical outcomes of EC patients with respect to stratification by
Since POLE mutations and dMMR were mutually exclusive in our cohort, ECs were classified
into the following three subsets; ECs with POLE mutations (POLE-mt), MSI, or non-MSI
(Panel A in Fig 4). Table 1 shows the associations of clinicopathological features among the
The median follow-up for PFS and ECS were 62 and 64 months, respectively (follow-up
periods for both PFS and ECS: 1±105 months). For ECs with POLE-mutations, MSI and
nonMSI, five-year PFSs were 100%, 89.5%, and 74.5% (P = 0.0420), five-year ECSs were 100%,
88.7%, and 84.5% (P = 0.3162), respectively (Panel B in Fig 4). In our cohort, there was no
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Fig 3. Detection of MSI and distribution of number of MSIs in 138 EC patients. (A) Example of MSI and non-MSI cases analyzed by four mononucleotide repeat
markers (BAT26, NR21, NR27, and CAT25). (B) Association between MSI, POLE mutation, MLH-1 promoter methylation and MMR protein expression. The number
of mononucleotide repeat markers showing MSI are shown by color.
association between clinicopathologiclal findings and POLE-mutant/MSI status. In contrast,
MLH1 methylation status and MMR expression status were obviously associated with tumors
with MSI. Finally, univariate and multivariate analysis are shown in Table 2. In the univariate
analyses, FIGO stage, grade, histology, depth of myometrial invasion, LVSI, and POLE/MSI
status were significantly associated with PFS. On the other hand, among those valuables, KRAS
mutation status and POLE/MSI status was not associated with ECS. We next considered all
variables to construct a multivariate model. The multivariate analysis for PFS demonstrated
that POLE/MSI status, histology and adjuvant therapy are significantly associated with PFS, so
these are considered as the prognostic factors. Again, the multivariate analysis for ECS
demonstrated that only histology is associated with ECS.
EC had historically been categorized into two pathogenic subtypes; type I and type II [
This classification lacks sufficient discriminative ability to categorize tumors or to guide the
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Fig 4. Molecular and clinic-pathological features of 138 ECs. (A) Molecular and clinic±pathological landscape of 138 ECs. Genetic analysis, focusing on frequent
hotspot mutations in the POLE gene, and MSI status result in the identification of three molecular subgroups: (1) POLE-mutant, (2) MSI and (3) non-MSI. (B)
Progression-free survival and endometrial cancer-specific survival of 138 EC patients stratified by genetic profiles. P values were calculated by the log-rank test.
treatment decision of EC patents [
]. Currently, all major risk-stratification systems for
EC patients, the treatment recommendations are based on a combination of histological type,
stage, and grade [33±35]. However, it has been demonstrated that these systems lack the
discriminative ability to determine outcomes .
Several studies have performed molecular characterization of ECs and have identified
mutational profiles to help distinguish EC subtypes [2±8]. The most comprehensive molecular
study of EC to date has been from the TCGA, which included a combination of whole-genome
sequencing, exome sequencing, MSI analyses, copy number analyses, and proteomics [
Molecular information was used to classify 232 patients with EC into four groupsÐPOLE-mt,
MSI, copy number low, and copy number highÐwhich were correlated with PFS [
However, it would be cost-prohibitive and impractical to apply the range and extent of genomic
and molecular tests used in the TCGA study to patients in a clinical setting.
ECs with proofreading POLE mutations showed better prognosis [
2, 3, 10, 11
examined POLE mutations in exons 9 and 13. By the cBioPortal FOR CANCER GENOMICS
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>60 (vs <60)
LSVI and MSI denote lymphovascular space invasion and microsatellite instability, respectively.
(http://www.cbioportal.org/index.do) website, a total of 92 POLE mutations including 46
duplicate mutations in patients with multiple samples were found in EC (TCGA, Nature 2013
and Provisional cohorts). Among them, 32 mutations were considered to be pathogenic
mutations; P286R and S297F located in exon 9 were found in 16 and 2 ECs, respectively; V411L,
L424V and L424I located in exon 13 were found in 10, 2 and 2 ECs respectively (Panel C in S2
Fig). Of our 138 ECs, only P286R and V411L were detected in 7 and 5 ECs.
Our study attempted to determine whether genetic stratification of ECs can effectively
identify distinct subsets with prognostic significance. Interestingly, we found highly significant and
clinically relevant differences in relapse and survival rates between genetically stratified
subgroups. Thus, our classification by the set of two mutational profiles, POLE mutation and MSI
status, is rather easy to practical for routine clinical practice. As ECs consist of a heterogeneous
group of tumors with diverse molecular alterations, our analyses further support previous
studies that showed an association of POLE proofreading mutations with favorable prognosis
2, 3, 5, 10, 37, 38
Defective DNA mismatch repair represents one of the most frequent molecular defects in
EC, and tumors with such defects are readily identifiable through MSI analysis [
]. In this
retrospective cohort, the population with MSI tumors was 29.0%, consistent with other studies
[2, 3, 5, 16, 17, 39±41]. In this study we used the four mononucleotide repeat markers for the
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detection of MSI phenotype. By this MSI assay, tumors could be divided into the two
phenotype; MSI and non-MSI, as we reported previously [
]. Historically, MSI status was
evaluated by the conventional MSI assay recommended by National Cancer Institute (NCI)
workshop by the use of a reference panel of five markers: two mononucleotide-repeat markers
(BAT26 and BAT25) and three dinucleotide repeat markers (D2S123, D5S346, and D17S250)
. By using this NCI recommended maker panel, tumors were divide into the three subtype;
MSI-high, MSI-low and microsatellite stable (MSS). MSI-high tumors were always
demonstrated dMMR and displayed MSI in almost of the five repeat makers irrespective of type of
microsattelite markers, such as mononucleotide or dinucleotide repeat markers [
26, 43, 44
contrast, MSI-low tumors showed pMMR and one or two sifted microsartellite markers
mainly in di-nucleotide markers, not common in mononucleotide markers [
]. Based on
those background, a pentaplex PCR system, as well as our MSI assay based on mononucleotide
markers was developed to detect MMR deficient tumors [
25, 26, 44
The MLH1 gene has a large CpG island within its promoter that clearly divides it into at
least 2 discrete regions of methylation (Panel A in Fig 2). The methylation pattern is not
homogeneous among various CpG sites within a CpG island. Deng et al. examined the methylation
status of 3 regions (A, B and C) in the MLH1 promoter, and compared the methylation status
to the gene expression in 24 cell lines and concluded that only the C region was associated
with the loss of gene expression [
]. In particular, the methylation in regions A and B (the
5'region in this study) occurs in normal mucosa, and may spread toward region C (the 3'-region
in this study) during tumor progression in colorectal cancer [
24, 27, 46
]. In ECs, extensive
methylation (i.e. affecting both the 5'-region and the 3'-region) in the MLH1 promoter region
was detected in 69.6% of ECs with dMLH1 (dMMR by epigenetic alteration) and in none of
ECs with other dMMR (dMMR probably by MMR mutations).
Recently, McMeekin et al. demonstrated dMMR caused by an epigenetic alteration (MLH1
methylation) showed relatively worse prognosis compared with dMMR caused by probable
MMR mutations or pMMR in large clinical cohorts [
]. In contrast, in line with few previous
2, 3, 5, 16, 23
], our cohort demonstrated that ECs with MSI features (implicating
dMMR) were associated with a reduced risk of recurrence and distant metastases (Table 2 and
Fig 4). When we divided ECs with dMMR into two subclasses, dMMR by epigenetic alteration
and probable MMR mutations, ECs with dMMR by epigenetic alteration showed relatively
better outcome compared with ECs with dMMR by probable MMR mutations (data not
Our cohort demonstrated that ECs with POLE mutations would have the better outcome
among our three subsets. Indeed, the study by McMeekin et al lacks to stratify ECs with POLE
mutations. Therefore, ECs with POLE mutations are lost in ECs with pMMR subclass, having
a possibility to make clinical outcome of ECs with pMMR better.
Similar to our results, Stelloo et al demonstrated that ECs with POLE mutations and MSI
showed better prognosis compared with other ECs by analyzing their large cohort obtained
from three clinical trials (PORTEC-1, -2 and -3) and subclassifying EC patients with neither
POLE mutation nor MSI into two subtypes according to p53 mutational status; ECs with p53
mutations (p53-mutant) and no specific molecular profile (NSMP) [
]. Interestingly, the
cohort of earlier grades (86.8% was Grade 1/2; PORTEC-1 and -2) showed better prognosis in
the NSMP group compared with p53-mutant, whereas in the cohort of advanced grades
(84.5% was Grade 3; PORTEC-3), NSMP had a worse prognosis similar to p53-mutant [
Thus, although the prognostic character of NSMP varied, ECs with POLE mutations and MSI
constantly showed a better prognosis than the other subtypes.
This study provides robust genetic analyses that can easily be implemented in prospective
studies and clinical practice. Especially, we adhere to stratifying only by reproducible genetic
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analyses that are easily accessible for daily clinical practice. MSI analyses have some limitations
for detecting dMMR tumors. In our previous study, although our data suggested that a marker
panel consisting of BAT26, NR21 and NR27 markers was more accurate in detecting CRCs
with dMMR, we showed that the use of mono-markers missed identifying 3 of 8 (47%) CRCs
with dMSH6 [
]. Thus, we added a mononucleotide repeat marker, CAT25, to the three
mononucleotide marker panel (BAT26, NR21 and NR27) to try to increase the sensitivity for
detecting tumors with dMMR [
In conclusion, we acknowledge that this study has some limitations. For instance, the number
of analyzed samples was relatively small and from a retrospective cohort in a single hospital.
However, this study provides robust genetic analyses that can easily be implemented in
prospective studies and clinical practice. Although intratumor heterogeneity may interfere with
prediction of the patient's tumor genetic profile, our data suggest that analyses of proof reading
POLE mutations and MSI by mononucleotide markers will be useful as biomarkers for
identifying patients who have a good prognosis and may not require intensive postoperative
radiotherapy or even chemotherapy.
S1 Table. Primer sequence for POLE mutations.
S1 Fig. Progression free survival and endometrial cancer specific survival of 138 EC
patients. Progression free survival and endometrial cancer specific survival of 138 EC patients
stratified by tumor grade (A), FIGO stage (B) tumor type (C). and P values were calculated by
S2 Fig. KRAS and POLE mutation analyses. (A) Examples of KRAS mutations in EC
specimens. (B) Examples of POLE mutations in EC specimens.
The authors thank Mr. Toru Nakai and Mrs. Tae Yamanishi for technical assistance.
Data curation: Tomoko Haruma, Takeshi Nagasaka, Junko Haraga, Akihiro Nyuya.
Formal analysis: Takeshi Nagasaka, Takeshi Nishida.
Funding acquisition: Tomoko Haruma, Keiichiro Nakamura.
Investigation: Tomoko Haruma, Keiichiro Nakamura, Junko Haraga, Akihiro Nyuya, Takeshi
Nishida, Ajay Goel.
Methodology: Takeshi Nagasaka, Akihiro Nyuya, Takeshi Nishida.
Project administration: Takeshi Nagasaka, Ajay Goel, Hisashi Masuyama, Yuji Hiramatsu.
Supervision: Hisashi Masuyama, Yuji Hiramatsu.
Validation: Akihiro Nyuya, Ajay Goel, Yuji Hiramatsu.
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Writing ± original draft: Tomoko Haruma, Takeshi Nagasaka.
Writing ± review & editing: Keiichiro Nakamura, Junko Haraga, Akihiro Nyuya, Takeshi
Nishida, Ajay Goel, Hisashi Masuyama, Yuji Hiramatsu.
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