Prognostic Significance of

CD3+ Tumor-infiltrating Lymphocytes

in Patients with Endometrial
Carcinoma
. PETRA ČERMÁKOVÁ1,
. BOHUSLAV MELICHAR2,4⇑,
. MARKÉTA TOMŠOVÁ1,
. ZDENĚK ZOUL2,
. HANA KALÁBOVÁ4,
. JIŘÍ ŠPAČEK3 and
. MARTIN DOLEŽEL4
+Author Affiliations

The Fingerland Institute of Pathology, Charles University Medical School and Teaching Hospital,
1

Hradec Králové, Czech Republic

.
Department of Oncology and Radiotherapy, Charles University Medical School and Teaching
2

Hospital, Hradec Králové, Czech Republic

.
Department of Gynecology and Obstetrics, Charles University Medical School and Teaching
3

Hospital, Hradec Králové, Czech Republic

.
Department of Oncology, Palacký University Medical School and Teaching Hospital, Olomouc,
4

Czech Republic

.
. Correspondence to: Bohuslav Melichar, MD, Ph.D., Department of Oncology, Palacký
University Medical School, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic. Tel: +420 588444288,
Fax: +420 588442522, e-mail: [email protected]

Next Section
Abstract

Aim: The aim of the present study was to investigate tumor-infiltrating leukocytes in
patients with endometrial carcinoma. Patients and Methods: Cluster of differentiation
(CD)3+, CD8+ and C20+ tumor-infiltrating lymphocytes (TILs) and
CD68+ tumor-associated macrophages (TAMs) were evaluated retrospectively by
immunohistochemistry in tumor specimen from 124 patients with endometrial
carcinoma. Results: A significant decrease of CD3+ TILs and an increase of
CD68+ TAM count was associated with higher tumor stage. In patients with
early-stage, high-risk tumors, low intraepithelial CD3+ TIL counts were associated
with significantly inferior survival. In multivariate analysis of patients with
early-stage tumors, intraepithelial CD3+ TIL counts were an independent predictor of
survival. Conclusion: In patients with endometrial carcinoma a decrease of
intraepithelial CD3+ TIL counts is associated with advanced stage and high risk
group. Intraepithelial CD3+ TIL counts are an independent predictor of survival in
patients with early tumors.

 Biomarkers

 endometrial carcinoma
 tumor-associated macrophages
 tumor-infiltrating lymphocytes

Endometrial carcinoma is the most common gynecological cancer in developed

countries and the sixth most common cancer in women worldwide. This neoplasia
often produces symptoms such as vaginal bleeding at a relatively early stage.
Consequently, the disease is diagnosed and treated in a timely manner, resulting in
better prognosis compared to other gynecological malignancies. Multiple prognostic
factors have been defined for endometrial carcinoma, including stage of the disease,
histological type of the tumor, grade, myometrial invasion, angioinvasion, age at
diagnosis and presence of lymph node metastases (1). Endometrial carcinoma
comprises a histologically- and biologically-diverse group of neoplasms characterized
by different pathogeneses and tumor biology. Estrogen-dependent type (type I)
endometrial carcinoma characterized by endometroid histology encountered in
younger perimenopausal women is associated with a relatively good prognosis, while
estrogen non-dependent type (type II) endometrial carcinoma, including serous and
clear cell carcinoma, predominantly diagnosed in older women, is less related to
sustained estrogen stimulation and has, in general, a poor prognosis.

In recent years, considerable progress has been made in understanding the role of the
immune system in the progression and prognosis of malignant tumors. The presence
of tumor-infiltrating mononuclear cells consisting of T-lymphocytes (helper and
suppressor/cytotoxic), natural killer cells, B-lymphocytes and macrophages indicates
the existence of active immune response of the host that may be directed against the
tumor cells. It has been demonstrated across the spectrum of different primary tumors
that the presence of tumor-infiltrating lymphocytes (TIL) correlates with better
prognosis (2), and response to therapy (3). Among gynecological malignancies, the
prognostic significance of TILs has been extensively studied in epithelial ovarian
cancer (2, 4), and intraepithelial CD3+ TIL counts were shown to be an independent
prognostic biomarker in patients with epithelial ovarian cancer. On the other hand, the
data on the prognostic significance of TILs in endometrial carcinoma are more limited
(5-11). The aim of the present study was to analyze TILs and tumor-associated
macrophages (TAMs) detected using immunohistochemistry in patients with
endometrial carcinoma, including the impact of these parameters on overall survival.

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Patients and Methods

In the present study, tissue blocks of 124 consecutive patients aged (mean±standard
deviation) 66±9 years (range=45-85 years), who underwent primary surgery between
January 1999 and December 2004 at two departments performing gynecological
surgery in Hradec Králové, Czech Republic, were retrospectively evaluated. The
surgical specimens from these two hospitals were sent for histological examination
exclusively to the Fingerland Institute of Pathology, Charles University Medical
School Teaching Hospital in Hradec Králové, and the patients were referred for
adjuvant radiotherapy to the Department of Oncology and Radiotherapy of Charles
University Medical School Teaching Hospital in Hradec Králové. Abdominal
hysterectomy and bilateral salpingo-oophorectomy, and, in selected patients,
lymphadenectomy were performed. All patients were staged according to the
Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) staging system (1).

Survival was measured from the date of diagnosis to death. Surviving patients were
censored at the last follow-up. This study was part of a project approved by the
Ethical Committee of the Charles University Medical School Teaching Hospital,
Hradec Králové, Czech Republic.

The surgical specimens were fixed in 10% formalin, processed, embedded in paraffin,
and stained with hematoxylin-eosin. Hematoxylin-eosin-stained sections from each
histological specimen were reviewed by a single pathologist who confirmed the
histological diagnosis and the grade of the tumor according World Health
Organization and selected one representative paraffin block for immunohistochemical
analysis (2, 3).

For immunohistochemistry, 4-µm sections were prepared, and an indirect

immunohistochemical method was used. After de-paraffinization, sections were
immersed into pre-heated antigen-retrieval solution at pH 9.9 (S3308;
DakoCytomation, Glostrup, Denmark), incubated at 95°C for 40 min, and allowed to
cool to room temperature (before the detection of CD3 and CD68). Before the
detection of CD8 and CD20, sections were immersed in citrate buffer at pH 6 and
treated in a microwave oven (320 W for 10 min). For the detection of CD3+ TILs,
primary rabbit polyclonal antibody to human CD3 (DakoCytomation) was used at a
dilution of 1:100; for the detection of CD8+ TILs, antibody to human CD8 (CD8/144B;
DakoCytomation) was used at a dilution of 1:200; for the detection of CD20+ TILs,
antibody to human CD20 (L26; DakoCytomation) was used at a dilution of 1:300; and
for the detection of CD68+ TAMs, antibody to human CD68 (KP1; DakoCytomation)
was used at a dilution of 1:400. Visualization was achieved using the
EnVision+peroxidase system (DakoCytomation). Control specimens underwent a
similar staining procedure (2, 3).

Each tumor section was evaluated for TILs or TAMs by using a ×40 objective lens
(0.196 mm2), and six different areas with the most abundant TILs or TAMs were
selected (three cancer epithelium and three stromal areas). The numbers of TILs and
TAMs were then counted manually. The mean number of stromal or intraepithelial
TIL counts in an area of 1 mm2 of tumor for each patient was used for statistical
analysis. The immunohistochemically stained sections were evaluated by a single
pathologist without previous knowledge of the patient's clinical outcome.

Statistical analyses were performed using Number Cruncher Statistical System

software (NCSS, Kaysville, UT, USA). The survival was analyzed by the
Kaplan–Meier method. The prognostic significance of clinical, histological and
immunohistochemical parameters on overall survival of patients was analyzed by
log-rank test for univariate analysis and by Cox regression for multivariate analyses,
with the results expressed as hazard ratio (HR) with 95% confidence intervals (CI).
The values of non-categorical parameters were categorized according the median. The
differences between subgroups of patients were analyzed with Mann–Whitney U-test.
Correlations were investigated using the Spearman's rank correlation coefficient.
The p-value of 0.05 or less was considered statistically significant.

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Table I.
Tumor stage, histology and grade.

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Results

The pathological characteristics of the patients included in the present study are
summarized in Table I. The majority of patients had endometroid-type carcinoma,
stage I and grade 2. Lymphadenectomy was performed in 36 patients. Eighty-seven
patients underwent radiotherapy (external-beam radiation in 77 patients and vaginal
brachytherapy in 75 patients), seven patients had additional adjuvant chemotherapy
and four patients had chemotherapy in the palliative setting. Twenty-two patients
received subsequent hormonal therapy with gestagens.

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Table II.
Tumor-infiltrating leukocyte counts [(mean±SD)n/mm2] in patients with early- and
advanced-stage endometrial carcinomas.

The numbers of TILs were, in general, similar in patients with early-stage (IA-IIA)
and advanced-stage (IIB and higher) tumors (Table II), and among patients with
early-stage disease, patients with high-risk (defined as stage IC, IIA or grade 3 or
clear cell or papillary serous histology) or low-risk tumors (Table III). In patients with
advanced disease, intraepithelial CD3+ TIL counts (Figure 1), and
intraepithelial:stromal CD8+ TIL and CD68+ TAM ratios were significantly lower, and
the numbers of stromal CD68+ TAMs and stromal CD3+:CD8+ TIL ratio were
significantly higher. In patients with low-risk early tumors, stromal CD3+ TIL and
stromal CD20+ TIL counts were significantly lower compared to those with high-risk
early tumors.

When stage was expressed on a numerical scale (stage IA=1, stage IB=2, stage IC=3,
stage IIA=4, stage IIB=5, stage IIIA=6, stage IIIB=7, stage IIIC=8, stage IVA=9 and
stage IVB=10), a significant correlation was observed between stage and grade
(rs=0.443; p<0.00001), stage and stromal CD68+ TAM counts (rs=0.245; p=0.007), grade
and stromal CD20+ TIL counts (rs=0.208; p=0.024), grade and stromal CD8+ TIL
counts (rs=0.192; p=0.037), grade and stromal CD68+ TAM counts (rs=0.322; p=0.0004),
and grade and epithelial CD68+ TAM counts (rs=0.301; p=0.0009).

Out of the 124 patients examined, 45 (36%) died during the observation period. One
patient lost to follow-up was censored. The median survival in the whole cohort was
not reached, and 61% of patients were alive after 92 months, with a 5-year survival
rate of 72%. The median duration of follow-up of surviving patients was 102 months
(range=64-145 months). None of the TIL populations was predictive of survival when
the whole cohort was evaluated. In patients with early-stage, high-risk tumors,
intraepithelial CD3+ TIL counts below 17 per mm2 were associated with significantly
inferior prognosis (median survival=52.7 months vs. not reached, p=0.01; Figure 2). In
multivariate analysis of patients with early tumors that included the TIL populations
investigated and risk groups (high risk vs. low risk), intraepithelial CD3+ TIL counts
below 17 per mm2 were associated with inferior prognosis (HR=4.56, 95%
CI=1.77-11.75, p=0.002), while intraepithelial CD8+ TIL counts below 9 per mm2 were
associated with improved prognosis (HR=0.28, 95% CI=0.10-0.76, p=0.01). High-risk
classification itself was not a significant predictor of survival in this model. In patients
with early-stage, high-risk tumors, CD3+ TIL counts below 17 per mm2 were
associated with inferior prognosis in multivariate analysis (HR=3.79, 95%
CI=1.34-10.71; p=0.01).

Previous SectionNext Section

Discussion

The present study adds to the growing body of evidence indicating that the immune
system plays an important role in the progression and prognosis of endometrial
carcinoma. A significant trend of increasing CD68+ TAM and decreasing CD3+ TIL
counts associated with higher stage or grade was recorded. Compared to patients with
early tumors, stromal CD8+ TIL counts were increased relative to intraepithelial
CD8+ TIL and stromal CD3+ TIL counts in patients with advanced disease. Moreover,
a significant difference was observed in patients with high-risk and low-risk
early-stage tumors in stromal CD3+ TIL and CD20+ TIL counts. Most importantly,
although none of the TIL populations studied was associated with prognosis when the
whole cohort was examined, among patients with early-stage, high-risk tumors,
intraepithelial CD3+ TIL counts below median were predictive of poor prognosis. The
Kaplan–Meier overall survival curves started to diverge at three years after diagnosis,
and later, eight to nine years after diagnosis, both curves seemed to reach a plateau,
with the difference in long-term survival rates of patients with high versus low
intraepithelial CD3+ TIL counts approaching almost 40%. In multivariate analysis of
patients with early tumors, high intraepithelial CD3+ TIL counts and low
intraepithelial CD8+TIL counts were significant prognostic biomarkers that surpassed
the prognostic significance of the risk classification itself, which was not a significant
predictor of outcome in multivariate analysis. Interestingly, high intraepithelial
CD3+ and CD8+ TIL counts had opposing effects on prognosis. In the sub-group of
patients with high-risk early tumors, CD3+ TIL counts were also an independent
prognostic factor.

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Table III.
Tumor-infiltrating leukocyte counts [(mean±SD) n/mm2] in patients with early-stage
tumors according to risk.

Endometrial cancer usually manifests early by vaginal bleeding, resulting in timely

diagnosis and curative surgical therapy. In contrast to other gynecological
malignancies, e.g.epithelial ovarian carcinoma, most patients with endometrial
carcinoma are cured by surgery-alone or in combination with external-beam radiation
with or without vaginal brachytherapy. Consequently, compared to epithelial ovarian
carcinoma, patients with advanced/metastatic disease are rare such that metastatic
endometrial carcinoma represents an almost ‘orphan’ disease, and the information on
the systemic treatments and utilization of biomarkers in endometrial cancer is limited
(12). There is an unmet medical need for new treatments and biomarkers that would
allow the prediction of prognosis and response to therapy. Currently, tumor stage,
depth of myometrial invasion, and tumor grade are commonly used to predict clinical
outcome and to plan treatment modalities in patients with endometrial carcinoma (13).
Precise histological assessment of myometrial invasion and cervical involvement is
crucial in the staging of early disease. Patients with more than 50% myometrial
invasion are at increased risk for extrauterine metastasis and may require more
aggressive surgical staging. Vascular and lymphatic vessel invasion are encountered
in approximately one-fifth of cases of endometrial carcinoma and correlate
significantly with the extension of the primary tumor, depth of myometrial invasion
and histological grade. The presence of vascular invasion has been shown to be a
powerful independent indicator for increased risk of recurrence and diminished
survival in patients with clinical stage I endometrial carcinoma. The grading system
relies firstly on the architectural pattern of the glands and secondarily on the
cytological and nuclear appearances, but grading itself, as well as the subtyping of
high-grade endometrial carcinoma, was found to be frequently subjective and
poorly-reproducible. Among parameters that can be assessed using
immunohistochemistry, estrogen receptors and progesterone receptors expression play
a major role. Approximately 60-70% of endometrial carcinomas express both
receptors, which seem to characterize clinically less aggressive tumors, with a better
chance of response to endocrine therapy. Data have also been reported on the
significance of other molecular alterations, such as p53 mutation which is more
frequently detected in serous endometrial tumors, microsatellite instability, mutation
of phosphatase and tensin homologue on chromosome ten, β-catenin, or Kirsten
retrovirus-associated DNA sequences genes that characterize the pathogenesis of
endometrial tumors (13). Potential prognostic significance has also been attributed to
markers of proliferation of tumor cells, such as Ki-67.

Considerable progress has been made in the past few years in understanding the role
of the immune system and host–tumor interactions in tumor progression (14). The
presence of TILs within the tumor microenvironment is considered to represent a
biomarker of the host immune response against the tumor. T-Cells and macrophages
constitute major components of tumor-infiltrating leukocytes in epithelial
malignancies. Depending on the function and phenotype, T-cells may either act as
effectors in tumor cell destruction, or induce immune tolerance. Macrophages may
also play a more complex role in tumor progression and metastasis. Depending on the
context of the tumor microenvironment, macrophages may represent potent effectors
of the host antitumor response and a crucial component of the stroma contributing to
tumor cell proliferation and angiogenesis (15-17). In different reports examining
patients with endometrial carcinoma, the presence of TILs was associated with
microsatellite instability (11), expression of B7-H7, a membrane protein that
negatively regulates T-cell response (10), and the expression of
indoleamine-2,3-dioxygenase, the enzyme responsible for tryptophan depletion (6).
The reported results on the prognostic significance of the presence of TILs in patients
with endometrial carcinoma are inconsistent, or even conflicting because of
differences in size of the cohorts examined, number of events or differences in
methodology, including differences in lymphocyte populations analyzed or methods
for the detection of lymphocytic infiltration. In an early report, the presence of higher
numbers of intraepithelial CD8+ TILs at the invasive border was an independent
predictor of improved overall survival (7). The association of higher CD8+ TIL counts
with prolonged time to recurrence and improved overall survival was also reported in
a large cohort of 368 patients with endometrial carcinoma (8). A positive association
of overall survival with TILs that exhibit CD45RO+ (memory) T-cell phenotype has
also been noted (8). In some studies, the prognostic significance of forkhead box P3
(FOXP3)+CD4+ TILs was examined (5, 8, 9), and an association of high
FOXP3+CD4+ TIL counts with disease-free survival (5) and overall survival (8) has
been reported in patients with endometrial carcinoma. The lack of prognostic
significance of FOXP3+CD4+ TILs reported in another study may be attributed to an
insufficient number of events (9). The presence of FOXP3+CD4+ TILs was also
demonstrated to correlate with clinical and pathological characteristics associated
with high risk, including vascular density, stage, differentiation, and lymphovascular
space invasion (5, 9). In a study that demonstrated the association between
indoleamine-2,3-dioxygenase expression and tumor lymphocyte infiltration, high
stromal CD3+ TIL counts were reported to independently predict better relapse-free
survival (6). Less is known about the significance of TAMs in patients with
endometrial carcinoma. In one study, CD68+ TAM counts at the invasive margin were
associated with tumor stage, grade, myometrial invasion, lymph node metastasis and
vascular space invasion (18). Intratumoral CD68+ TAM counts were positively
associated with Ki-67 expression and microvascular density. High CD68+ TAM counts
at the invasive margin were associated with poor prognosis in univariate, but not
multivariate analyses (18).

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Figure 1.
Intraepithelial and stromal tumor-infiltrating lymphocytes.
Immunohistochemically-stained cluster of differentiation (CD3)+ lymphocytes in
endometrial carcinoma are shown. Both stromal and intraepithelial
CD3+ lymphocytes are evident (original magnification, ×200).

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Figure 2.
Survival of patients with early-stage, high-risk endometrial carcinoma according the
intraepithelial cluster of differentiation (CD3)+ tumor-infiltrating lymphocyte (TIL)
counts. The Kaplan–Meier survival curves of patients with intraepithelial CD3+ TIL
counts <17/mm2 and ≥17/mm2 in patients with early-stage, high-risk tumors are shown
(median survival of 52.7 months versus not reached, log-rank test, p=0.01).

Adjuvant radiation prevents only locoregional relapse, and the benefit of adjuvant
chemotherapy in endometrial carcinoma is controversial. In fact in most prospective
trials, the benefit of adjuvant chemotherapy was not evident. The lack of
demonstrable benefit of adjuvant systemic treatment could be due to the fact that
subsequent distant metastases occur infrequently in patients with early-stage disease.
A prognostic biomarker would first allow identification of a high-risk patient
population to be enrolled into prospective studies of adjuvant systemic therapy and,
subsequently, serve as a biomarker for treatment decisions. Future systemic therapies
may include not only cytotoxic chemotherapy regimens, but also targeted-therapy.
Conceptually, targeted treatments aim at one or more hallmarks of cancer. Hallmarks
of cancer are defined as pathological features that enable tumor growth and escape
from control, and one of the eight hallmarks currently recognized is the escape of
tumor from destruction by the immune system (14). CD3+ TIL counts could serve as a
biomarker for targeted therapies that aim at manipulating the immune
system, e.g. ipilimumab or nivolumab.

In the present study, no prognostic significance of intraepithelial CD3+ TIL counts

was detected when the whole cohort of patients was examined; an association with
prognosis was found only in patients with early-stage tumors. However, patients with
advanced endometrial cancer represent a subgroup of patients that is different from a
point of view of prognosis and biology. The outcome of patients with advanced
endometrial carcinoma is generally poor, and other factors, including therapy, age or
comorbidity, may modify the effect of the immune response on patient prognosis.
Moreover, patients with advanced disease constituted only 20% of patients in the
present study. Despite the fact that the prognostic significance of CD3+ TIL counts
was not evident in the whole cohort, CD3+TIL counts were independent predictors of
prognosis in patients with early-stage tumors, i.e. patients for whom the decision on
the choice of therapy is the most urgent and controversial. Of note, patients with
early-stage tumors in the present cohort were almost equally split between the low-
and high-risk groups. In fact, the identification of patients with high risk is most
crucial in patients with early disease, some of whom would have excellent prognosis
without any adjuvant treatment, while others will eventually experience relapse.

As in other reports (2, 3), the presence of CD3+ TILs in the present study was more
relevant in the tumor epithelium compared to the tumor stroma. In general,
lymphocytic infiltration is more abundant in the stroma compared to the tumor
epithelium, but intraepithelial lymphocytes are more likely to be associated with the
immune response to the tumor than lymphocytes encountered in the tumor stroma.
The finding of prognostic significance for the intraepithelial CD3+ TIL count is in
contrast with a report demonstrating prognostic significance of stromal CD3+ TIL
counts that, however, analyzed progression-free survival based on a smaller number
of events (6). Interestingly, high CD8+ TIL counts were associated with adverse
prognosis in the present cohort, in contrast with studies reporting positive prognostic
significance of high CD8+ TIL counts in patients with endometrial carcinoma (7, 8).

The current study has obvious limitations that are inherent to its retrospective nature.
Because of the retrospective nature of the study, overall survival rather than
relapse-free survival or progression-free survival was analyzed in the present cohort.
For obvious reasons, overall survival represents the most reliable end-point in a study
that attempts to identify prognostic biomarkers. From the current perspective, surgical
management of the patients in the present cohort may appear suboptimal. On the other
hand, the strong association of intraepithelial CD3+ TIL counts with overall survival
observed in the present study indicates the activity of the immune system determines
the natural history of this cancer.

In conclusion, the present data demonstrate that in patients with endometrial

carcinoma, a decrease of intraepithelial CD3+ TIL count is associated with advancing
stage and risk group. The intraepithelial CD3+ TIL count is also an independent
predictor of survival. Future studies should address the role of the intraepithelial
CD3+ TIL count as a prognostic and predictive biomarker in adjuvant therapy of
endometrial carcinoma.

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Acknowledgements

This study was supported by grants of the Czech Ministry of Health NT13566-4/2012
and NT13564-4/2012, program PRVOUK P37/11, Research Project LM2010004 and the
Research Project Biomedreg CZ.1.05/2.1.00/01.0030.

 Received May 28, 2014.

 Revision received July 15, 2014.
 Accepted July 16, 2014.
 Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios),
All rights reserved
Previous Section

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