Gynecologic Oncology 162 (2021) 694–701

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Gynecologic Oncology

journal homepage: www.elsevier.com/locate/ygyno

Comprehensive immunomolecular profiling of endometrial carcinoma:

A tertiary retrospective study
Jasper Victoor a, Sara Vander Borght a,b, Lien Spans b, Stefan Lehnert b, Hilde Brems b, Annouschka Laenen c,
Ignace Vergote d, Toon Van Gorp d, Els Van Nieuwenhuysen d, Sileny Han d, Stefan Timmerman d,
Anne-Sophie Van Rompuy a,e,⁎,1, Isabelle Vanden Bempt b,1
a
Department of Pathology, University Hospitals Leuven, Leuven, Belgium
b
Department of Human Genetics, University Hospitals Leuven, Leuven, Belgium
c
KU Leuven, Biostatistics and Statistical Bioinformatics Centre, Leuven, Belgium
d
Department of Gynecology and Obstetrics, Division of Gynecological Oncology, University Hospitals Leuven, Leuven, Belgium
e
Laboratory of Translational Cell & Tissue Research, Department of Imaging and Pathology, KU Leuven – University of Leuven, Leuven, Belgium

H I G H L I G H T S

• Comprehensive NGS may help to determine pathogenicity of non-hotspot POLE variants.

• Comprehensive NGS can identify targetable alterations in endometrial cancer cases.
• POLEmut and MMRd endometrial cancers have higher numbers of TILs and PD-L1-expressing immune cells.
• Determination of CD3, CD8 and PD-L1 expression may reveal endometrial cancers benefitting from immune checkpoint inhibition.
• p53 abn endometrial cancers have higher L1CAM expression and lack ARID1A or PTEN variants.

a r t i c l e i n f o a b s t r a c t

Article history: Objective. Combined immunohistochemical and molecular classification using the Proactive Molecular Risk
Received 28 May 2021 Classifier for Endometrial Cancer (ProMisE) independently predicts prognosis in endometrial carcinoma (EC).
Accepted 28 June 2021 As next-generation sequencing (NGS) is entering clinical practice, we evaluated whether more comprehensive
Available online 10 July 2021
immunomolecular profiling (CIMP), including NGS and extended immunohistochemical analysis, could further
refine the current ProMisE classification.
Keywords:
Endometrial carcinoma
Methods. A series of 120 consecutive ECs, classified according to ProMisE, was stained immunohisto-
Comprehensive immunomolecular chemically for CD3, CD8, PD-L1, beta-catenin and L1CAM. An in-house 96 gene NGS panel was performed
profiling (CIMP) on a subset of 44 ECs, representing the 4 ProMisE subgroups (DNA polymerase epsilon catalytic subunit exonu-
POLE clease domain mutated (POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53 abn) and no specific
MMRd molecular profile (NSMP) ECs). Cases harboring non-hotspot POLE variants were analyzed with Illumina TruSight
p53 Oncology 500 NGS panel (TSO500) as a surrogate for whole-exome sequencing.
NSMP Results. Eight cases harbored POLE variants, half of which were hotspots. Using TSO500, non-hotspot POLE
variants were classified as pathogenic (3) or variant of unknown significance (1). POLEmut and MMRd ECs typ-
ically showed higher numbers of CD3+/CD8+ tumor-infiltrating lymphocytes and higher PD-L1 expression in
tumor-infiltrating immune cells. p53 abn ECs showed significantly higher L1CAM immunoreactivity and fre-
quently harbored gene amplifications including HER2 (25%), but typically lacked ARID1A or PTEN variants.
Beta-catenin-positivity and FGFR2 variants were predominantly found in NSMP ECs.
Conclusions. Our data show that CIMP adds significant value to EC characterization and may help to determine
pathogenicity of non-hotspot POLE variants, encountered more frequently than expected in our series. In addi-
tion, CIMP may reveal ECs benefitting from immune checkpoint inhibition and allows upfront identification of
targetable alterations, such as HER2 amplification in p53 abn ECs.
© 2021 Elsevier Inc. All rights reserved.

⁎ Corresponding author at: Department of Pathology, Herestraat 49, 3000 Leuven, Belgium.
E-mail address: annexsophie.vanrompuy@uzleuven.be (A.-S. Van Rompuy).
1
Shared last authorship.

https://doi.org/10.1016/j.ygyno.2021.06.030
0090-8258/© 2021 Elsevier Inc. All rights reserved.
J. Victoor, S.V. Borght, L. Spans et al.
1. Introduction
Endometrial carcinoma (EC) is the second most common gynecologi-
cal cancer worldwide [1]. It comprises a heterogeneous group of histolog-
ical subtypes with endometrioid, serous and clear cell carcinomas being
most frequent [2–4]. Traditionally, ECs are classified based on histological
subtype together with histological grade and clinicopathological stage,
which is required to guide clinical decision making [2–4]. However, deter-
mination of histological subtype and grade is poorly reproducible, making
this traditional classification system less reliable [2,5].
In the last decade, the Cancer Genome Atlas (TCGA) provided more in-
sight into the molecular landscape of EC by identifying 4 major molecular
subgroups: (1) ECs with variants in the exonuclease domain of the DNA
polymerase epsilon catalytic subunit (POLE) gene representing ‘ultra-mu-
tated’ tumors with a favorable outcome (POLEmut); (2) ‘hyper-mutated’
ECs characterized by DNA mismatch repair deficiency (MMRd) and hav-
ing an intermediate progression-free survival; (3) ECs with a very high
degree of somatic copy number alterations, a serous-like morphology
and frequent TP53 variants (90%) having significantly worse prognosis
(p53 abnormal, abn) and (4) a heterogeneous group of ECs lacking any
of the characteristic abnormalities observed in the other 3 subgroups
(no specific molecular profile, NSMP) but showing frequent activating
CTNNB1 variants (52%) and an intermediate progression-free survival
[6,7]. These subgroups can be identified using the Proactive Molecular
Risk Classifier for Endometrial Cancer (ProMisE) proposed by Talhouk
et al.: a pragmatic, relatively low-cost and TCGA-based classification sys-
tem for EC, applicable to formalin-fixed paraffin-embedded (FFPE) tumor
tissue, using sequencing for POLE exonuclease domain variants and im-
munohistochemistry (IHC) for p53 and the mismatch repair proteins
(MLH1, PMS2, MSH2 and MSH6) [8]. Because of the clear prognostic
value and given its high reproducibility, ProMisE is a much more reliable
classification method compared to the one based on histology alone, and
ultimately represents an important tool for improving patient manage-
ment [6,9]. Kommoss et al. were able to further stratify risk among ECs
of the NSMP subgroup by determining the status of L1 cell adhesion mol-
ecule (L1CAM/CD171) [10]. They found L1CAM immunoreactivity to be
predictive of worse outcome in this otherwise heterogeneous and poorly
defined subgroup, making it a specific prognostic marker and potential
therapeutic target. L1CAM is a target gene of beta-catenin. Both L1CAM
overexpression and nuclear beta-catenin immunoreactivity have been as-
sociated with epithelial to mesenchymal transition and thus aggressive
tumor behavior and poor prognosis [10–12].
Recently, it has been shown that MMRd ECs and POLEmut ECs are as-
sociated with an elevated number of CD3+/CD8+ tumor-infiltrating
lymphocytes (TILs), which seems to be compensated by overexpression
of immune checkpoint molecules such as PD-L1 on tumor-infiltrating
immune cells, making these ECs ideal candidates for immune check-
point inhibition [13–21]. Still, identifying true POLEmut ECs can be chal-
lenging as pathogenicity of POLE variants may be unclear [22] and
important overlap exists between different molecular EC subtypes as
previously shown by our group [23].
Since NGS panels used for routine diagnostic analysis of solid tumors
are becoming more comprehensive and costs are decreasing, we
wanted to investigate whether the use of a more extensive NGS panel
currently used in cancer diagnostics adds significant value to the current
molecular classification of ECs. In addition, we extended immunohisto-
chemical analyses by adding CD3, CD8, PD-L1, L1CAM and beta-catenin
stainings to further characterize the different ProMisE subgroups of EC
and their immune landscape.
2. Materials and methods
2.1. Patient selection
Hematoxylin and eosin (H&E) stained slides and corresponding FFPE
tissue blocks of 120 consecutive EC cases were collected from the
695
Gynecologic Oncology 162 (2021) 694–701
Department of Pathology at the University Hospitals Leuven, Belgium.
Tumor tissue was obtained by hysterectomy (n = 116) or endometrial
biopsy (n = 4) between June 2017 and June 2019. Three patients with
advanced disease received chemotherapy prior to hysterectomy. A suf-
ficient amount of tumor tissue was available for all cases. This study was
approved by the Ethics Committee Research University Hospitals Leu-
ven / Catholic University of Leuven, Leuven, Belgium (S61501).
2.2. Histopathological analysis
Endometrial tumors were histopathologically classified and graded
according to the World Health Organization (WHO) criteria, and staged
according to the guidelines of the Fédération Internationale des
Gynaecologistes et Obstetristes (FIGO) [3].
2.3. ProMisE classification
Detailed methodology including germline testing in the context of
cancer predisposition has been described elsewhere [23]. Briefly, IHC
was performed for p53 and the mismatch repair proteins (MLH1,
PMS2, MSH6 and PMS2), and Sanger sequencing was used to identify
POLE variants. Cases with abnormal MMR IHC were tested by polymer-
ase chain reaction for microsatellite instability (MSI) (MSI analysis sys-
tem, Promega). In order not to miss MMRd ECs with POLE variants, we
decided to swap the first two steps in the decision tree, following the
approach of Murali et al. [24] and Vermij et al. [25].
2.4. Extended and comprehensive molecular genetic analysis
On a subset of 44 ECs, representing the 4 molecular subtypes and in-
cluding all cases harboring a POLE extracellular domain variant [23], we
performed NGS using an in-house developed 96 cancer gene panel (Uni-
versity Hospitals Leuven 96 gene assay or UHL96) [26], investigating
single nucleotide variants, small insertions/deletions and amplifica-
tions/deletions. Variants were classified according to Froyen et al. [27].
For cases harboring a POLE variant, parallel blood analysis was per-
formed. To determine pathogenicity of non-hotspot POLE variants, we
used the TruSight Oncology 500 (TSO500) DNA NGS panel (Illumina,
San Diego, CA, USA) according to the manufacturer's instructions. Path-
ogenicity was evaluated based on the POLE-scoring system recently
proposed for whole-exome sequencing (WES) by León-Castillo et al.,
taking into account tumor mutational burden over 100 variants per
megabase and percentage of C > A, T > G, C > G as well as indel propor-
tions [22].
2.5. Fabricating and processing TMAs
Six tissue microarrays (TMAs) were created from 106 out of 120
FFPE tissue blocks using an automated tissue microarrayer (TMA
Grand Master, 3DHistech/Sysmex Belgium, Hoeilaart, Belgium). For
the remaining 14 cases, insufficient tumor tissue was available for inclu-
sion in the TMA construction and entire tissue sections were used for
the immunohistochemical analyses. For the TMAs, three core biopsies
of 2 mm diameter were collected from the representative tumor areas
on each FFPE tissue block and inserted in predetermined array locations
in six blank paraffin blocks. These cores were taken randomly from dif-
ferent tumor areas to overcome tumor heterogeneity. Five μm-thick sec-
tions were cut from the TMAs and stained with H&E.
2.6. Immunohistochemical analyses
CD3 and CD8 stainings were performed on one 5 μm-thick section of
each of the 120 FFPE blocks before the TMAs were built. For PD-L1, beta-
catenin and L1CAM, stainings were performed on 5 μm-thick serial sec-
tions from the 6 TMAs and the 14 residual FFPE blocks. We used poly-
clonal rabbit anti-human CD3, ready-to-use, Dako Omnis; monoclonal
J. Victoor, S.V. Borght, L. Spans et al. Gynecologic Oncology 162 (2021) 694–701

mouse anti-human CD8, clone C8/144B, ready-to-use, Dako Omnis;
monoclonal mouse anti-human PD-L1, clone 22C3, ready-to-use, Dako
Omnis; monoclonal mouse anti-human beta-catenin, clone β-catenin-
1, ready-to-use, Dako Omnis; and monoclonal mouse anti-human
L1CAM/CD171, clone 74-5H7, dilution 1/50, BioLegend. All stainings
were performed on the Dako Omnis autostainer (Agilent Technologies
Belgium, Diegem, Belgium) according to standard protocols. Immunore-
activity was visualized with the EnVision FLEX, High pH system (Dako
Omnis). Hematoxylin (Dako Omnis) was used for counterstaining.
POLE variant cases were considered “multiple classifiers” until further
comprehensive analysis. Interestingly, cases 69 and 79 showed a patho-
genic germline variant in one of the mismatch repair genes confirming
Lynch syndrome in those cases [23]. Case 33 had only focal loss of
MSH6 on IHC, but did not show MSI nor the presence of a pathogenic
germline variant [23]. Thirty out of 120 cases were assigned to the
MMRd subgroup (25%), 26 cases were assigned to the p53 abn subgroup
(21.7%) and the remaining 56 cases were assigned to the NSMP subgroup
(46.7%).

2.7. Quantification of CD3+ and CD8+ T-lymphocytes

3.2. Extended molecular analysis
The CD3- and CD8-stained slides were first digitalized using a slide
Detailed results are presented in Supplementary Table S1.
scanner (Philips IntelliSite Pathology Solution Ultra Fast Scanner 1.6,
Results of the TSO500 NGS gene panel for the 4 cases carrying a non-
Best, the Netherlands). For both CD3 and CD8, the total number of im-
hotspot POLE variant is shown in Table 2. Based on the POLE-scoring sys-
munoreactive T-lymphocytes in the entire tumor area was counted, re-
tem [22], 3 out of 4 non-hotspot POLE variants were classified as patho-
gardless of stromal or intraepithelial localization. Counting was
genic (score of 4 in all 3 cases), while 1 variant was considered a variant
performed in a standardized way using computerized bioimage analysis
of unknown significance (VUS, score 3). In the latter case (case 12), the
software (QuPath) [28]. We determined the mean (± standard error
POLE extracellular domain variant (p.D287E) was found in the germline
(SE)) number of immunoreactive lymphocytes per mm2 tumor area
of the patient (Supplementary Table S1). This variant has a frequency in
for each case.
the European non-Finnish population of >0.1% and would therefore be
classified as likely benign according to Froyen et al. [27]. In addition,
2.8. Quantification of PD-L1
case 12 showed diffuse aberrant p53 staining and 2 underlying patho-
genic TP53 variants, a low mutational load, amplification of the CCNE1
Based on the method of Pasanen et al. [19], we defined PD-L1 ex-
gene and a serous morphology. Therefore, this case was finally classified
pression as at least partial membranous staining in tumor cells and at
as p53 abn. None of the other POLE variants were present in the germline.
least partial membranous and/or cytoplasmic staining in tumor-
Concerning the MMRd subgroup, 3 cases showed loss of MSH6 ex-
infiltrating immune cells. We determined the mean (±SE) percentage
pression: 1 with subclonal loss and 2 with complete loss of MSH6 expres-
of PD-L1 expressing cells separately for the (epithelial) tumor cells
sion by IHC (Supplementary Table S1; cases 46, 18 and 91 respectively). In
and the (stromal) tumor-infiltrating immune cells. Necrotic areas
both cases showing complete loss of MSH6 expression (cases 18 and 91),
were excluded from counting. We used a 1% threshold for positive stain-
a pathogenic MSH6 variant was found in the germline, confirming a diag-
ing and assigned a semiquantitative score as follows: 0: < 1% of the
nosis of Lynch syndrome. Interestingly, in one of those cases (case 91) no
cells; 1: 1–4%; 2: 5–9%; 3: 10–49%; 4: ≥ 50%.
MSI was found by polymerase chain reaction [23].
The p53 abn subgroup, now extended to 12 cases based on pathoge-
2.9. Quantification of L1CAM and beta-catenin
nicity analysis of the POLE variants, was characterized by aberrant p53
expression on IHC and lack of other aberrations used as surrogate
Counting was performed using QuPath [28]. We determined the
markers for molecular classification. In all 12 cases, a pathogenic TP53
mean (±SE) percentage of L1CAM expressing tumor cells and tumor
variant was present in the tumor. In addition, gene amplification was
cells with nuclear beta-catenin expression respectively. Necrotic areas
found for the HER2 (ERBB2) gene (cases 51, 56 and 88), the CCNE1
were excluded from counting. For L1CAM, we used a 10% threshold
gene (cases 12, 56 and 77) and the cMYC gene (case 41).
for positive staining, based on the findings of Zeimet et al., who deter-
In the NSMP subgroup, 1 case (case 16) showed a pathogenic TP53
mined this cut-off to be best correlating with prognosis [29]. For beta-
variant but no aberrant p53 expression could be shown by IHC. In 4
catenin, we used a 1% threshold for positive staining.
out of 12 cases (33%), a (likely) pathogenic FGFR2 variant was found.
Such FGFR2 variants were also found in one case (case 3) belonging to
2.10. Statistical analysis
the MMRd subgroup. Two more cases (cases 7 and 50) showed a path-
ogenic BRCA2 variant which was also present in the germline.
Comparisons between the 4 different ProMisE subgroups were per-
Extended NGS by UHL96 further revealed that ARID1A and PTEN loss-
formed using the Kruskal Wallis test for continuous or ordinal variables,
of-function variants were frequently found in 3 out of the 4 molecular
while the Mann-Whitney U test was used for pairwise comparisons.
subgroups with respectively 5 and 6 out of 7 POLEmut cases, 10 and 11
Categorical variables were analyzed using the Fisher exact test. All
out of 13 MMRd cases and 6 and 11 out of 12 NSMP cases carrying such
tests were two-sided and a 5% significance level was adopted for all
variants. In the p53 abn subgroups, no ARID1A or PTEN variants were
tests. Analyses have been performed using SAS software (version 9.4
found. Hotspot variants in PIK3CA and KRAS were found in each molecular
of the SAS System for Windows).
subgroup. Variants in the CTNNB1 gene were found in 5 cases: 2 POLEmut
cases, 1 MMRd case and 2 NSMP cases. The presence of a CTNNB1 variant
3. Results
only correlated with nuclear beta-catenin immunoreactivity in 1 NSMP
case. Of note, given the ultramutated profile of POLEmut cases, no other
3.1. ProMisE classification
individual actionable targets were evaluated as many variants were
found which may be the result of the ultramutator profile. Gene amplifi-
Eight cases presented with a POLE exonuclease domain variant (6.7%):
cations were only found in the p53 abn subgroup.
4 known hotspots and 4 non-hotspot variants [22] (Table 1). The 4 cases
with a non-hotspot POLE variant (cases 12, 33, 69 and 79) and 1 case with
a hotspot POLE variant (case 66) presented with multiple positive 3.3. Histopathological analysis
markers. Two cases had diffuse aberrant expression of p53 by IHC
(cases 12 and 66). Three other cases showed abnormal staining patterns An overview of all clinical-pathological data is given in Supplemen-
for MMR proteins as well as focal aberrant immunohistochemical expres- tary Table S2, according to definitive ProMisE classification after ex-
sion of p53 (cases 33, 69 and 79). According to ProMisE, the 4 non-hotspot tended molecular analysis.

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J. Victoor, S.V. Borght, L. Spans et al. Gynecologic Oncology 162 (2021) 694–701

Table 1
Overview of the 8 cases with a POLE variant.

Case 9 12 15 33 66 69 79 103

POLE (c.) 857C > G 861 T > A 1231G > T 1271 T > C 1231G > C 1381 T > A 1270C > G 1231G > T
POLE (p.) P286R D287E V411L L424P V411L S461T L424V V411L
POLE_ SOM GL SOM SOM SOM SOM SOM SOM
(SOM/GL)
POLE_Type Hotspot Non-hotspot Hotspot Non-hotspot Hotspot Non-hotspot Non-hotspot Hotspot
MMR_IHC MMRp MMRp MMRp Subclonal MSH6 loss MMRp Loss PMS2 Loss MSH6/ MMRp
MSH2
MSI_PCR – – – MSS – MSI-H MSI –
LYNCH (GL) – – – – – PMS2: c.1882C > T MSH6: c.1509del –
p.R628* p.K504Sfs*6
IHC_p53 WT Ab, diff WT Ab, focal Ab, diff Ab, focal Ab, focal WT
ProMisE POLEmut Multiple classifier? POLEmut Multiple classifier? POLEmut Multiple classifier? Multiple classifier? POLEmut

POLE_(SOM/GL); POLE variant Somatic/Germline; POLE_Type; type of exonuclease domain variant in POLE; MMR_IHC, immunohistochemistry for mismatch repair proteins; MMRp, mis-
match repair proficient; MSI_PCR: microsatellite instability analysis as described elsewhere [23]; MSS, microsatellite stable; MSI-H, microsatellite instability high; GL, presence of a
germline variant in one of the mismatch repair genes; WT, wild-type; Ab, aberrant; diff, diffuse; ProMisE, Proactive Molecular Risk Classifier for Endometrial Cancer; POLEmut, POLE-
mutated.

Table 2 cases in the NSMP subgroup, not all data were available due to tissue de-
Results for the pathogenicity of non-hotspot POLE variants using the POLE-score [28] pletion. The results of the pairwise comparisons of the immunohisto-
based on TSO500 NGS analysis.
chemical data between the different ProMisE subgroups are listed in
Case 12 33 69 79 Supplementary Table S3.
POLE (c.) 861 T > A 1271 T > C 1381 T > A 1270C > G
POLE (p.) D287E L424P S461T L424V 3.5. CD3+/CD8+
Score_Recurrence 0 0 0 1
C > A > 20% % 2.92 14.31 4.16 7.15 The mean number of CD3+ TILs per mm2 tumor was significantly
Score 0 0 0 0
T > G > 4% % 4.87 5.16 5.86 3.01
higher in the POLEmut subgroup compared to the MMRd subgroup,
Score 1 1 1 0 the p53 abn subgroup and the NSMP subgroup (respectively 2214.2 ±
Indels < 5% % 0.39 1.07 2 1.13 1253.85; 1192.5 ± 1016.12, p = 0.038; 592.2 ± 605.36, p = 0.005;
Score 1 1 1 1 and 640.5 ± 441.98, p = 0.002). A similar result was found for CD8+
C > G > 0.6% % 10.92 5.26 4.24 6.65
TILs (respectively 1811.3 ± 1034.6; 912.6 ± 765.38, p = 0.035; 489.3
Score 1 1 1 1
TMB > 100mut/Mb Mut/Mb 5 270 426 161 ± 505.06, p = 0.008; and 361.4 ± 289.59, p = 0.002). Additionally,
Score 0 1 1 1 the number of both CD3+ and CD8+ TILs was significantly higher in
POLE_Score TSO 3 4 4 4 the MMRd subgroup compared to the p53 abn and NSMP subgroup
POLE_Score Ref Unknown Unknown Unknown 3 (p = 0.004 and < 0.001 for CD3 and p ≤ 0.001 and 0.015 for CD8). No
Definitive_Classification p53 abn POLEmut POLEmut POLEmut
CD3/mm2 471.4 2030.67 1500.31 2474.89
significant difference was detected between the p53 abn and NSMP
CD8/mm2 279.68 1940.52 1546.3 2309.82 subgroup (Fig. 1, Supplementary Fig. S1).
POLE_Score_TSO, POLE-scoring according to Illumina TSO500, based on Léon-Castillo et al.
[22]; POLE_Score_Ref, reference POLE-scoring according to Léon-Castillo et al. [22]; 3.6. PD-L1
Definitive_Classification, final classification of the 4 cases showing a non-hotspot POLE
variant after comprehensive molecular analysis and POLE-scoring, following the recently The distribution of the semiquantitative PD-L1 staining scores for
proposed decision tree [25].
(epithelial) tumor cells and (stromal) tumor-infiltrating immune cells
separately are listed in Supplementary Table S4, sorted according to
the definitive ProMisE subgroups.
3.4. Immunohistochemical analysis A higher PD-L1 expression was observed in the tumor-infiltrating
immune cells in the POLEmut and the MMRd subgroups compared to
The immunohistochemical data for CD3, CD8, PD-L1, L1CAM and the p53 abn and NSMP subgroups. The mean semiquantitative PD-L1
beta-catenin are listed in Table 3, sorted according to the final ProMisE score was respectively 3.1 for the POLEmut subgroup and 2.6 for the
subgroups, defined after more extensive molecular analysis. For 2 MMRd subgroup, compared to 1.6 for the p53 abn subgroup (p =

Table 3
Results of immunohistochemical stainings sorted by ProMisE subgroups.

Variable POLEmut (N = 7) MMRd (N = 30) p53 abn (N = 27) NSMP (N = 54) p-value

CD3+ (mean ± SE) (/mm2) 2214.2 ± 473.91 1192.5 ± 185.52 592.2 ± 116.5 640.5 ± 59.6 <0.001
CD8+ (mean ± SE) (/mm2) 1811.3 ± 391.04 912.6 ± 139.74 489.3 ± 97.2 361.4 ± 39.05 <0.001
PD-L1 strom (mean ± SE) 3.1 ± 0.55 2.6 ± 0.2 1.6 ± 0.25 1.1 ± 0.14 <0.001
PD-L1 epith (mean ± SE) 1.6 ± 0.3 0.9 ± 0.15 1.0 ± 0.16 0.4 ± 0.08 <0.001
L1CAM (mean ± SE) (%) 4.1 ± 1.93 1.9 ± 0.81 42.7 ± 7.35 2.6 ± 1.56 <0.001
<10% (n/N) (%) 6/7 (85.7%) 28/30 (93.3%) 10/27 (37%) 51/54 (94.4%) <0.001
≥10% (n/N) (%) 1/7 (14.3%) 2/30 (6.7%) 17/27 (63%) 3/54 (5.6%)

Beta-catenin
<1% (n/N) (%) 7/7 (100%) 30/30 (100%) 27/27 (100%) 46/54 (85.2%) 0.025
≥1% (n/N) (%) 0/7 (0%) 0/30 (0%) 0/27 (0%) 8/54 (14.8%)

POLEmut: polymerase epsilon mutated; MMRd: mismatch repair deficiency; p53 abn: p53 abnormal; NSMP: no specific molecular profile.

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J. Victoor, S.V. Borght, L. Spans et al.
0.016 for POLEmut and p = 0.006 for MMRd) and 1.1 for the NSMP sub-
group (p = 0.001 for POLEmut and p ≤ 0.001 for MMRd) (Fig. 2). Simi-
larly, a higher PD-L1 expression was observed in the tumor cells of EC
in the POLEmut and MMRd subgroups compared to the NSMP subgroup.
However, there was no difference between POLEmut and MMRd com-
pared to the p53 abn subgroup. On the contrary, between the p53 abn
and NSMP subgroups the difference was significant. The mean semi-
quantitative PD-L1 score was 1.6 for the POLEmut subgroup, compared
to 0.9 for the MMRd subgroup, 1 for the p53 abn subgroup and 0.4 for
the NSMP subgroup (p ≤ 0.001 for POLEmut, p = 0.004 for MMRd and
p ≤ 0.001 for p53 abn) (Fig. 2, Supplementary Fig. S1).
3.7. L1CAM
The mean percentage of L1CAM expression in tumor cells was signif-
icantly higher in the p53 abn subgroup compared to the POLEmut sub-
group, the MMRd subgroup and the NSMP subgroup (respectively
42.7%; 4.1%, p = 0.019; 1.9%, p ≤ 0.001; and 2.6%, p ≤ 0.001) (Fig. 3A, Sup-
plementary Fig. S2). A similar result was found for the number of
L1CAM-positive cases, i.e. cases with L1CAM expression in ≥10% of
tumor cells (respectively 62.7%; 14.3%, p = 0.035; 6.7%, p ≤ 0.001; and
5.6%; p ≤ 0.001) (Fig. 3B, C).
3.8. Beta-catenin
Beta-catenin-positive cases, i.e. cases with nuclear beta-catenin ex-
pression in ≥1% of tumor cells, were only seen in the NSMP subgroup
(n = 8/54, 14.8%) (Table 3, Supplementary Fig. S2). A significant associ-
ation between L1CAM and nuclear beta-catenin immunoreactivity
could not be demonstrated (p = 0.124) (Supplementary Table S5).
4. Discussion
For high-risk endometrial cancer, aberrant p53 expression is the
strongest unfavorable prognostic factor while the presence of POLE
pathogenic variants represents the strongest favorable factor. Still, iden-
tifying true POLEmut ECs remains challenging although recently a POLE-
scoring system has been developed that defines 6 additional likely path-
ogenic POLE variants apart from the 5 known hotspot variants [22]. In
our cohort of prospectively analyzed ECs, only 4 out of 8 cases with a
POLE exonuclease domain variant carried one of the 5 pathogenic
hotspot variants described so far, which is lower than expected [6,22].
As for the 4 non-hotspot POLE variants, one (p.L424V) was given a
CD3+/mm²
p = 0.002
3500
p = 0.005
3000 p = 0.038
2500
p = 0.004
2000
p < 0.001
1500
1000
500
0 0
POLEmut MMRd p53 abn
Fig. 1. Mean number (±SE) of CD3+ and CD8+ TILs per mm2 tumor, according to the different ProMisE subgroups (p ≤ 0.001).
Gynecologic Oncology 162 (2021) 694–701
POLE-score of 3 by León-Castillo et al. [22], meaning this variant should
be interpreted as VUS. Three more POLE extracellular domain variants,
p.D287E, p.L424P and p.S461T, were not encountered in TCGA ECs and
have not been studied by broad gene sequencing. In such case, it has
been proposed to use WES in order to determine pathogenicity of
novel POLE variants in the exonuclease domain using a POLE-scoring
system taking into account tumor mutational burden over 100 variants
per megabase, percentages of C > A, T > G, C > G, indel proportions and
recurrence of the variant [22,30]. However, WES is mostly not available
in routine daily practice and can be particularly challenging when ap-
plied on paraffin tissue. Nevertheless, comprehensive NGS panels such
as TSO500 are entering diagnostic practice and may be used to calculate
the proposed POLE-score. Indeed, TSO500 shows good concordance
with whole-genome sequencing for the detection of gene variants and
tumor mutational burden [31]. Using TSO500, we found that 3 POLE var-
iants (p.L424V, p.L424P, p.S461T) could be classified as pathogenic with
a POLE-score of 4 and high tumor mutational burden exceeding 100 var-
iants per megabase, while variant p.D287E was classified as VUS. The
latter POLE variant was found in the blood of the patient and most likely
represents a single nucleotide polymorphism. Moreover, the case carry-
ing this POLE p.D287E variant was characterized by serous histology and
diffuse aberrant p53 staining on IHC, low mutational load and low ex-
pression of CD3/CD8 and PD-L1, supporting the final classification of
this case as p53 abn. Nevertheless, accurate characterization of POLE
non-hotspot variants based on non-WES needs further validation as
one of our cases carrying the POLE variant p.L424V was reported to be
VUS [22] while TSO500 supported this variant as being pathogenic.
Still, given the high CD3/CD8 and PD-L1 expression on IHC, consistent
with the other known pathogenic POLE variants, we assume this variant
may indeed be pathogenic.
An enhanced immune response, as measured by IHC for CD3 and
CD8, was indeed obvious in all POLEmut cases, except for one case
(15) with a known hotspot p.V411L variant. The same applies to the
MMRd cases, albeit less pronounced. This observation is consistent
with the existing literature and adds to the assumption that tumor
cells of POLEmut and MMRd ECs exhibit more neoantigens, providing
a plausible explanation for the more extensive immune infiltration in
these groups and the major role TILs might play in a better prognosis
of EC [13–21]. However, a higher number of TILs seems to be compen-
sated by overexpression of PD-L1 in tumor-infiltrating immune cells.
It has been postulated that tumors are most likely to respond to immu-
notherapy when they show simultaneous presence of intratumoral
T-cell infiltration and PD-L1 expression [32,33]. With this in mind, our
CD8+/mm²
p = 0.002
3000
p = 0.008
2500 p = 0.035
2000
p < 0.001
1500
p = 0.015
1000
500
NSMP POLEmut MMRd p53 abn NSMP
698
J. Victoor, S.V. Borght, L. Spans et al. Gynecologic Oncology 162 (2021) 694–701

PD-L1 stromal PD-L1 epithelial

4.5 p = 0.001 2.5
p = 0.016 p < 0.001
4

3.5 p < 0.001 2

p = 0.004
p = 0.006
3
1.5 p < 0.001
2.5

2
1
1.5

1 0.5
0.5

0 0
POLEmut MMRd p53 abn NSMP POLEmut MMRd p53 abn NSMP

Fig. 2. Mean semiquantitative score (±SE) of PD-L1 expression for (stromal) tumor-infiltrating immune cells and (epithelial) tumor cells separately, according to the different ProMisE
subgroups (p ≤ 0.001).

A
L1CAM
80% p = 0.005
p = 0.044
70%
p = 0.019
60% p < 0.001
p < 0.001
50%

40%

30%

20%

10%

0%
POLEmut MMRd p53 abn NSMP

60 100%
p < 0.001
90%
50
80%
p = 0.035
70%
40
60%
p < 0.001
30 50%

40%
20
30%

20%
10
10%

0 0%
POLEmut MMRd p53 abn NSMP POLEmut MMRd p53 abn NSMP

B L1CAM+ L1CAM- C L1CAM+ L1CAM-

Fig. 3. Mean percentage (±SE) of L1CAM immunoreactivity in tumor cells, according to the different ProMisE subgroups (p ≤ 0.001) (A). L1CAM-positive cases (with immunoreactivity
≥10%) in the different ProMisE subgroups, represented as absolute numbers (B) and as the percentage per ProMisE subgroup (p ≤ 0.001) (C).

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J. Victoor, S.V. Borght, L. Spans et al. Gynecologic Oncology 162 (2021) 694–701

results and these of previous studies lead us to speculate that POLEmut CTNNB1 gene, we hypothesized that L1CAM expression is potentially in-
and MMRd ECs may indeed benefit from immune checkpoint inhibitors. duced by CTNNB1 variant and possibly linked with nuclear beta-catenin
In this regard, PD-L1 could be a potential biomarker for therapeutic pur- immunoreactivity, since CTNNB1 variants lead to activation of Wnt
poses. Additional determination of the number of CD3+/CD8+ TILs pathway and accumulation of beta-catenin within the nucleus [10,11].
could be important to predict therapy response and thus prognosis. In- However, we could not find such correlation in our study and thus
terestingly, Horeweg et al. [34] recently showed that image-based have no evidence for co-expression of L1CAM and nuclear beta-catenin.
quantification of intraepithelial CD8+ lymphocytes may improve mo- On the contrary, all L1CAM-positive cases were beta-catenin-negative
lecular EC classification and refine prognostication in early-stage and none of the cases with a CTNNB1 variant showed L1CAM overexpres-
endometrioid EC. Further prospective studies are required to evaluate sion. In our cohort, nuclear beta-catenin immunoreactivity could only be
and validate these potential biomarkers, and to further characterize im- demonstrated in the NSMP subgroup. The only beta-catenin-positive
mune response and elucidate tumor microenvironment. Concerning case included in the extended molecular analysis also contained a patho-
PD-L1 it is important to notice that, although overexpression is signifi- genic CTNNB1 variant. However, 4 out of 5 cases with a pathogenic
cantly higher in (stromal) tumor-infiltrating immune cells in POLEmut CTNNB1 variant did not show nuclear beta-catenin immunoreactivity.
and MMRd subgroups, our results for expression in (epithelial) tumor We conclude that beta-catenin immunostaining is not sensitive enough
cells are different. This finding is consistent with the observations of to detect a CTNNB1 variant.
Howitt et al. [14], Pasanen et al. [19] and Eggink et al. [20], who did Finally, we observed FGFR2 variants predominantly in the NSMP
not find significant correlations between PD-L1 expression in tumor subgroup. These variants have been shown to be associated with poor
cells and the different molecular subgroups. While most trials focus on prognosis in endometrioid ECs [40] and may represent a therapeutic
PD-L1 expression in tumor cells, a trial of Herbst et al. [35] interestingly target. Clinical trials evaluating the efficacy of FGFR inhibitors in FGFR2
showed that response to anti-PD-L1 agent MPDL3280A (atezolizumab) variant positive ECs are warranted.
correlated with PD-L1 expression in tumor-infiltrating immune cells
and not in tumor cells. This indicates that PD-L1, as a potential bio-
marker, should be evaluated for overexpression in both tumor- 5. Conclusions
infiltrating immune cells and tumor cells, both separately and com-
bined. Furthermore, since correlation between PD-L1 expression and In summary, our data show that comprehensive immunomolecular
immunotherapy efficacy may be tumor-specific, further studies are profiling adds relevant information to the molecular subgrouping of
needed to evaluate anti-PD-L1 response in EC (with PD-L1 overexpres- ECs, especially for those cases where individual markers are doubtful
sion) in particular. and inconclusive results need clarification. CIMP may also reveal ECs
Interestingly, we observed MMRd in 2 out of 7 POLEmut cases. The that are likely to benefit from immune checkpoint inhibition through
combination MMRd/POLEmut is rare in EC and its significance is cur- immunohistochemical determination of CD3, CD8 and PD-L1, and may
rently still unclear [22]. Notably, the MMRd encountered in those allow upfront identification of targetable alterations in poorly prognos-
cases was not attributed to the most commonly observed loss of tic ECs with HER2 amplification and FGFR2 variants being the most
MLH1/PMS2 expression accompanied by MLH1 promoter methylation, promising targets identified in respectively the p53 abn and NSMP ECs
but was due to loss of MSH6/MSH2 and isolated loss of PMS2 expres- in our series.
sion. A diagnosis of Lynch syndrome was confirmed in both cases with
the presence of a pathogenic MSH6 or PMS2 variant in the germline re-
Abbreviations
spectively. Further investigation is required in order to understand such
complex cases that might actually represent double classifiers. Still, as
the presence of a pathogenic POLE variant determines classification,
these cases would probably be classified as POLEmut in daily practice. ProMisE Proactive Molecular Risk Classifier for Endometrial Cancer
It has been shown that POLEmut tumors frequently carry TP53 vari- EC Endometrial carcinoma
NGS Next-generation sequencing
ants [36] as illustrated by our data as well: 4 out of 7 cases POLEmut
CIMP Comprehensive immunomolecular profiling
cases did show focal (3) or diffuse (1) aberrant p53 expression on IHC POLE DNA polymerase epsilon catalytic subunit
supported by the presence of underlying (likely) pathogenic TP53 vari- POLEmut POLE exonuclease domain mutated
ants. Recently, this type of double classifiers has been shown to behave MMRd Mismatch repair deficient
p53 abn p53 abnormal
as true POLEmut tumors with excellent prognosis and rather subclonal
NSMP No specific molecular profile
p53 expression [36]. TSO500 TruSight Oncology 500 NGS panel
In line with TCGA data, we observed ARID1A pathogenic variants in all TCGA the Cancer Genome Atlas
molecular subgroups except for the p53 abn subgroup. In contrast, copy FFPE Formalin-fixed paraffin-embedded
number variants were typically found in the p53 abn subgroup apart IHC Immunohistochemistry
L1CAM/CD171 L1 cell adhesion molecule
from pathogenic TP53 variants. Three p53 abn cases harbored HER2
TIL Tumor-infiltrating lymphocyte
amplifications, which has been shown to correlate with shorter overall H&E Hematoxylin and eosin
survival in EC [37]. However, recent trials have demonstrated benefit FIGO Fédération Internationale des Gynaecologistes et Obstetristes
from administration of trastuzumab in advanced or recurrent serous MSI Microsatellite instability
EC [38], making it important to detect HER2 amplifications in these tu- UHL96 In-house (University Hospitals Leuven) developed 96 cancer gene
next-generation sequencing panel
mors. We also observed a significant higher number of L1CAM-positive WES Whole-exome sequencing
cases in the p53 abn subgroup compared to the other subgroups. This TMA Tissue microarray
confirms the findings of Kommoss et al. [10], i.e. that L1CAM expression SE Standard error
seems to be related to p53 abn status. Since Kommoss et al. [10] were VUS Variant of unknown significance
able to demonstrate a prognostic unfavorable L1CAM-positive subgroup
within the NSMP subgroup and possibly the MMRd subgroup, and
Pasanen et al. [39] found a correlation between L1CAM expression and Ethics approval and consent to participate
poor survival in endometrioid ECs, further studies are required to evalu-
ate whether immunohistochemical determination of L1CAM could add This study was approved by the Ethics Committee Research Univer-
prognostic/therapeutic value in EC classification. Because L1CAM is sity Hospitals Leuven / Catholic University of Leuven, Leuven, Belgium
known as a target gene of beta-catenin, a protein that is encoded by the (S61501).

700
J. Victoor, S.V. Borght, L. Spans et al.
Data availability
The raw immunohistochemical data of the 120 studied cases are
available at the Department of Pathology, University Hospitals Leuven,
Leuven, Belgium on reasonable request.
The raw data of the 96 genes that were analyzed with a capture-
based targeted next generation sequencing method for these particular
cases are available at the Department of Human Genetics, University
Hospitals Leuven, Leuven, Belgium on reasonable request.
Funding
We are grateful for the received funding from the clinical research
and training council (KOOR), University Hospitals Leuven / Catholic
University of Leuven, Leuven, Belgium.
Author's contributions
All authors contributed in the writing of the manuscript and read
and approved the final manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
Not applicable.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ygyno.2021.06.030.
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