VOLUME 27 NUMBER 2 JANUARY
JOURNAL OF CLINICAL ONCOLOGY
From the School of Surgery and Biosta-
tistics Unit, Cancer Trials, Western
Australian Institute for Medical
Research, University of Western
Australia; Department of Radiation
Oncology, Sir Charles Gairdner Hospital,
Nedlands; and Colorectal Cancer Unit,
St John of God Hospital, Subiaco,
Australia.
Submitted June 18, 2008; accepted
September 11, 2008; published online
ahead of print at www.jco.org on
December 8, 2008.
Supported in part by a Russell Walter
Gibbon Medical Research Award from
the University of Western Australia and
by a grant from the Colorectal Surgical
Society of Australia and New Zealand
Foundation (P.S.).
Terms in blue are defined in the glos-
sary, found at the end of this article
and online at www.jco.org.
Authors disclosures of potential con-
flicts of interest and author contribu-
tions are found at the end of this
article.
Corresponding author: Barry Iacopetta,
PhD, School of Surgery M507, Univer-
sity of Western Australia, 35 Stirling
Hwy, Nedlands 6009, Australia; e-mail:
barry.iacopetta@uwa.edu.au.
The Appendix is included in the
full-text version of this article,
available online at www.jco.org.
It is not included in the PDF version
(via Adobe Reader).
2008 by American Society of Clinical
Oncology
0732-183X/09/2702-186/$20.00
DOI: 10.1200/JCO.2008.18.7229
186 2008 by American Society of Clinical Oncology
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10 2009
O R I G I N A L R E P O R T
Tumor-Infiltrating FOXP3 T Regulatory Cells Show
Strong Prognostic Significance in Colorectal Cancer
Paul Salama, Michael Phillips, Fabienne Grieu, Melinda Morris, Nik Zeps, David Joseph,
Cameron Platell, and Barry Iacopetta
A B S T R A C T
Purpose
To determine the prognostic significance of FOXP3 lymphocyte (Treg) density in colorectal cancer
compared with conventional histopathologic features and with CD8 and CD45RO lympho-
cyte densities.
Patients and Methods
Tissue microarrays and immunohistochemistry were used to assess the densities of CD8,
CD45RO, and FOXP3 lymphocytes in tumor tissue and normal colonic mucosa from 967 stage
II and stage III colorectal cancers. These were evaluated for associations with histopathologic
features and patient survival.
Results
FOXP3 Treg density was higher in tumor tissue compared with normal colonic mucosa, whereas
CD8 and CD45RO cell densities were lower. FOXP3 Tregs were not associated with any
histopathologic features, with the exception of tumor stage. Multivariate analysis showed that
stage, vascular invasion, and FOXP3 Treg density in normal and tumor tissue were independent
prognostic indicators, but not CD8 and CD45RO. High FOXP3 Treg density in normal mucosa
was associated with worse prognosis (hazard ratio [HR] 1.51; 95% CI, 1.07 to 2.13; P .019).
In contrast, a high density of FOXP3 Tregs in tumor tissue was associated with improved survival
(HR 0.54; 95% CI, 0.38 to 0.77; P .001).
Conclusion
FOXP3 Treg density in normal and tumor tissue had stronger prognostic significance in colorectal
cancer compared with CD8 and CD45RO lymphocytes. The finding of improved survival
associated with a high density of tumor-infiltrating FOXP3 Tregs in colorectal cancer contrasts
with several other solid cancer types. The inclusion of FOXP3 Treg density may help to improve
the prognostication of early-stage colorectal cancer.
J Clin Oncol 27:186-192. 2008 by American Society of Clinical Oncology
cently proposed as having stronger prognostic
INTRODUCTION
significance than conventional TNM staging.14
It has been known for many years that lymphocytic Another T-cell subtype shown to have prog-
infiltrate surrounding primary colorectal cancer nostic significance in CRC was CD45RO.12,15
(CRC) is associated with improved prognosis.1-6 Al- These cells include both CD4 and CD8 lympho-
though the mechanism remains unclear, the cytes that have been exposed to antigen. Oberg et
adaptive immune system is thought to play an al15 reported that a high density of CD45RO T
important role in suppressing the progression of cells in lymph node metastases of CRC was asso-
this disease. Naito et al7 were the first to demon- ciated with improved prognosis. Pages et al12 sub-
strate that infiltrating CD8 cytotoxic T cells were sequently demonstrated that a high density of
a prognostic factor in CRC. These findings have CD45RO cells within the tumor was associated
since been supported by the work of other with decreased invasiveness, lower stage, and im-
groups.8-11 A high density of CD8 T cells has proved survival. The above findings provide clear
been associated with the absence of tumor inva- evidence that the host immune response plays an
sion, earlier stage, and improved patient surviv- important role in determining the outcome
al.12,13 Using CD3 as a universal marker of T cells, from CRC.
the ratio of T-cell density at the advancing tumor Regulatory T cells (Tregs) were initially charac-
margin compared with the central core was re- terized by the CD4CD25 phenotype and are
 T Regulatory Cells in Colorectal Cancer
thought to modulate the antitumor immune response.16,17 Tregs can
suppress the activity of cytotoxic T cells through direct cell-to-cell
contact or via the release of cytokines, especially transforming growth
factor . Depletion of intratumoral Tregs enhances antitumor immu-
nity and tumor rejection in mouse models.18 Similarly, depletion of
Tregs in the peripheral blood of patients with CRC was recently shown
to unmask CD4 T-cell responses to tumor antigens.19 The most
specific Treg cell marker identified to date is the nuclear transcription
factor known as FOXP3.20,21 A high density of tumor-infiltrating
FOXP3 Tregs has been associated with poor outcome in various
solid tumors, including ovarian,22,23 pancreatic,24 and hepatocellu-
lar carcinoma.25,26
Two groups have investigated infiltrating Tregs in CRC using
FOXP3 staining. In a study of 40 patients, Loddenkemper et al27
reported that Treg density was lower in node-positive disease but was
not associated with survival. Ling et al28 found no significant differ-
ence in Treg density between advanced and early-stage disease, but did
not evaluate the association with patient survival. The aim of the
present study was therefore to compare the prognostic value of
FOXP3 Treg cell density with that of the established T-cell markers
CD8 and CD45RO in a large cohort of patients with stage II and III
CRC with long follow-up information.
PATIENTS AND METHODS
Patients
Patients were diagnosed with CRC during the period from 1990 to 1999
at the Sir Charles Gairdner Hospital, Western Australia. Information on pa-
tient demographics (sex and age) and tumor features were obtained from
pathology records. Tumor site was classified as proximal to and including, or
distal, to the splenic flexure. A total of 593 patients with American Joint
Committee on Cancer (AJCC) stage II and 374 patients with AJCC stage III
CRC were studied (N 967). Information on T stage, anatomic site, histologic
grade, vascular invasion, lymphatic invasion, perineural invasion, lymphocytic
response, and microsatellite instability (MSI) determined using the BAT-26
marker was available for 100%, 96%, 65%, 76%, 72%, 70%, 91%, and 95% of
cases, respectively. Information on disease-specific survival was obtained from
the Cancer Registry of Western Australia. The median follow-up time was 69.7
months for patients with AJCC stage II disease and 52.4 months for patients
with stage III disease. Information on the use of adjuvant chemotherapy with
fluorouracil/leucovorin-based regimens was obtained from hospital medical
records. Seven percent of stage II and 37% of stage III patients received
fluorouracil. At the end of the study period, 31% of patients had died of disease
recurrence and 25% had died from other causes. Ethics approval for the
project was obtained from the Sir Charles Gairdner Hospital Research Eth-
ics Committee.
CRC Tissue Microarray
Construction of the tissue microarrays (TMAs) used in this study was
described previously.29 They contained 967 consecutive cases of surgically
Table 1. Median Density of the T-Cell Markers CD8, CD45RO, and FOXP3 in Normal Colonic Mucosa and Colorectal Tumor Tissues
Normal
Marker Density (cells/mm2) No.
CD8 322 704
CD45RO 1,287 686
FOXP3 44 644
Abbreviation: T/N, tumor/normal ratio.
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resected stage II and III CRC within 13 TMA blocks. For each case, two cores of
1 mm diameter were taken at random from the tumor, and an additional
1-mm diameter core was taken from histologically normal colonic mucosa.
The location of the tumor cores in relation to the invading margin or the center
of the tumor was not recorded.
Immunohistochemistry for T-Cell Markers
Sections of 5 m thickness were cut from the TMA blocks, mounted on
silanated slides, and subsequently dewaxed and rehydrated using xylene and
graded alcohol washes. Antigen retrieval was performed at 121C for 4 minutes
(DakoCytomation pressure cooker; Dako, Copenhagen, Denmark) with slides
placed in Dako target retrieval solution or citrate buffer, depending on the
antibody. Endogenous peroxidase activity was blocked (Real Peroxidase
Block; Dako) followed by Tris-buffered saline wash. Protein block solution
was applied for 10 minutes to stop nonspecific antibody binding (Dako).
Primary antibodies were used according to manufacturers instructions (CD8,
DakoCytomation clone C8/144B ready to use; CD45RO, DakoCytomation
clone UCHL1 ready to use; FOXP3, Abcam, ab20034, 1/100 dilution). After
incubation with primary antibody, slides were washed in two changes of TBS
before incubation with labeled polymer horseradish peroxidase rabbit/mouse
antibody for 15 minutes (Envision Plus Detection System; Dako). Sections
were subsequently incubated with Dako-Chromogen solution and washed in
deionized water. Background staining was performed with Mayers hematox-
ylin and sections then dehydrated through ascending alcohols to xylene
and mounted.
Measurement of T-Cell Density
After staining for the T-cell markers, slides were scanned with a high-
resolution scanner (ScanScope XT; Aperio) at 40 magnification. Image
analysis software (TMA lab v8.0, Aperio) was used to evaluate the density of
stained cells (cells per square millimeter). Evaluation of T-cell marker density
was carried out blinded to clinicopathologic information. Individual cores
were examined by one observer (P.S.) and annotated to ensure that only
normal colonic epithelium or viable tumor tissue was included in the area of
analysis. No attempt was made to evaluate the various tumor compartments
separately (eg, stroma, tumor cell nests). Results for cell density were exported
into an Excel file, and individual cores were matched to corresponding clini-
copathologic data.
Statistical Analysis
The normality and log-normality of the distributions of continuous
variables were examined using the Shapiro-Wilks test. Variables with a log-
normal distribution were transformed using a natural logarithm. Analysis of
the association between variables was conducted using the Pearson correlation
coefficient, t test, and one-way analysis of variance for transformed variables
where appropriate. The family-wise error rate was controlled by use of the
Holm adjustment for multiple comparisons. T-cell densities were classified as
high or low in relation to the median value. Associations with survival were
explored using the Cox proportional hazards regression model. Multivariate
models were constructed according to methods described by Harrell and
assessed using Harrells concordance statistic C.30 The analysis was conducted
using the STATA (version 9) statistical package (STATA Corp, College Sta-
tion, TX).
Tumor
Density (cells/mm2) No. T/N P
147 910 0.46 .0001
935 912 0.73 .0001
116 905 2.64 .0001
2008 by American Society of Clinical Oncology 187
 Salama et al

marker. The densities of CD8 and CD45RO cells in tumor tissue

Table 2. Associations Between T-Cell Marker Densities in Normal and
Tumor Tissue From Patients With CRC (CD8T and CD45ROT) were lower compared with those of normal
tissue (CD8N and CD45RON), whereas the FOXP3 cell density
Correlation
Feature Coefficient (r) P was higher in tumor tissue (Table 1). This was observed for both the
Normal tissue median and geometric mean.
CD8N v CD45RON 0.510 .0001 Correlations between the T-cell markers are listed in Table 2. The
CD8N v FOXP3N 0.273 .0001 densities of markers were strongly associated with each other in nor-
CD45RON v FOXP3N 0.446 .0001 mal and in tumor tissues. The densities of CD8T and CD45ROT
Tumor tissue were also correlated with those of CD8N and CD45RON from the
CD8T v CD45ROT 0.516 .0001
same patient. However, only a weak correlation was observed between
CD8T v FOXP3T 0.446 .0001
CD45ROT v FOXP3T 0.439 .0001
FOXP3T and FOXP3N (r 0.041).
Normal v tumor Associations between the density of tumor-infiltrating T cells and
CD8N v CD8T 0.306 .0001 pathologic features are listed in Table 3. Tumors with higher AJCC or
CD45RON v CD45ROT 0.164 .0001 T stage were found to have lower densities of all three markers. The
FOXP3N v FOXP3T 0.041 .011 density of CD45ROT was significantly lower in tumors showing early
Abbreviations: CRC, colorectal cancer; N, normal tissue; T, tumor tissue. signs of metastasis (vascular and perineural invasion); however, this
was not observed for CD8T or FOXP3T. As expected, CD8T and
CD45ROT cell densities were higher in tumors reported as showing a
lymphocytic response or MSI. In contrast, FOXP3T was not associ-
ated with either of these features. No significant associations were
observed between the density of T-cell markers in CRC and patient age
RESULTS
or sex (results not shown).
A total of 6,202 images of normal and tumor tissue cores were used for Table 4 shows the results of univariate survival analysis for the
analysis of T-cell marker density in 593 stage II and 374 stage III CRCs. major pathologic features and for T-cell marker density in normal
A log-normal distribution was observed for the density of each and tumor tissue. Higher stage and the presence of vascular or

Table 3. Univariate Analysis for Associations Between High Density of Tumor-Infiltrating T-Cell Types and Pathologic Features of CRC
CD8T CD45ROT FOXP3T

Feature OR P OR P OR P
AJCC stage
II 1.00 1.00 1.00
III 0.60 .0001 0.57 .0001 0.86 .0004
T stage
12 1.00 1.00 1.00
34 0.80 NS 0.61 .002 0.51 .007
Tumor site
Proximal 1.00 1.00 1.00
Distal 0.86 .017 0.84 .031 1.07 NS
Histologic grade
Well/moderate 1 1 1
Poor 1.06 NS 1.21 NS 0.87 NS
Vascular invasion
Absent 1.00 1.00 1.00
Present 1.01 NS 0.77 .009 0.94 NS
Lymphatic invasion
Absent 1.00 1.00 1.00
Present 0.96 NS 0.93 NS 1.13 NS
Perineural invasion
Absent 1.00 1.00 1.00
Present 0.80 NS 0.67 .013 0.81 NS
Lymphocytic response
Absent 1.00 1.00 1.00
Present 1.42 .0001 1.24 NS 1.10 NS
Microsatellite instability
Absent 1.00 1.00 1.00
Present 1.99 .0001 2.52 .0001 1.10 NS

NOTE. T-cell densities were classified as high or low in relation to the median value.
Abbreviations: CRC, colorectal cancer; T, tumor; OR, odds ratio; AJCC, American Joint Committee on Cancer; NS, not significant.

188 2008 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

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 T Regulatory Cells in Colorectal Cancer

Accurate prognostic markers are of most importance clinically

Table 4. Univariate Survival Analysis for Pathologic Features and for T-Cell
Density in Normal and Malignant Colorectal Tissue From Patients
for the stratification of stage II CRC. A multivariate model was there-
With Stage II and III CRC fore developed to test for independent prognostic significance of
Feature HR 95% CI P pathologic features and of T-cell densities in stage II cases (Table 6). In
Tumor features
the first model (A), only the major pathologic features were included,
AJCC, III v II 3.07 2.44 to 3.88 .0001 and this revealed that vascular invasion and perineural invasion had
Site, distal v proximal 1.04 0.82 to 1.31 NS the strongest prognostic values. The second model (B) included these
Grade, poor v well/moderate 1.73 1.17 to 2.57 .006 two features together with the densities of each T-cell marker in
Vascular invasion, yes v no 2.49 1.90 to 3.26 .0001 normal and tumor tissues except CD45RON, which had shown no
Lymphatic invasion, yes v no 2.39 1.50 to 3.86 .0009
prognostic value in univariate analysis (Table 4). This model revealed
Perineural invasion, yes v no 1.99 1.32 to 3.00 .001
Lymphocytic response, yes v no 0.64 0.44 to 0.95 .026
that vascular invasion, perineural invasion, FOXP3N, and FOXP3T
Microsatellite instability, yes v no 0.60 0.38 to 0.94 .027 were the strongest prognostic features. When these four features were
T-cell density, high v low included in a third model (C), all were found to have independent
CD8N 0.81 0.71 to 0.91 .001 prognostic value in stage II CRC. As with the overall patient cohort,
CD45RON 1.01 0.83 to 1.22 NS high FOXP3N and FOXP3T cell densities showed opposite associ-
FOXP3N 1.19 1.05 to 1.36 .007 ations with cancer-specific patient survival.
CD8T 0.74 0.67 to 0.82 .0001
CD45ROT 0.74 0.65 to 0.84 .0001
FOXP3T 0.78 0.70 to 0.87 .0001
DISCUSSION
NOTE. Univariate analysis. Cox proportional hazards regression.
Abbreviations: CRC, colorectal cancer; HR, hazard ratio; AJCC, American Joint
Committee on Cancer; NS, not significant; N, normal tissue; T, tumor tissue. The CD8 and CD45RO markers of cytotoxic immune response
have previously been associated with improved prognosis in patients
with CRC.1-7 In agreement with these earlier reports, the current study
confirmed the importance of CD8T and CD45ROT cell density as
perineural invasion were strongly associated with worse patient
prognostic factors. The novelty of this study was that FOXP3 was
outcome. A high density of CD8N was associated with better
examined in parallel with CD8 and CD45RO in both normal and
survival, whereas a high density of FOXP3N was associated with
malignant tissues. Tregs are generally considered to be immunosup-
significantly worse outcome. High densities for each of the T-cell
pressive and have been linked to poor outcome in several types of solid
markers in tumor tissue were associated with good patient out-
tumor.22-26 The major original findings of the present study were that
come, including that of FOXP3T. In exploratory subgroup anal-
FOXP3T Tregs are associated with better survival and show stronger
ysis of stage III patients, the good prognosis associated with high
prognostic significance than CD8T and CD45ROT. Furthermore,
T-cell marker density in tumor tissue seemed to be limited to
FOXP3N Tregs were associated with worse prognosis.
patients treated with surgery alone.
A multivariate model was used to identify independent prognos-
tic factors in the combined stage II and III CRC cohort (Table 5). The
model included all histopathologic variables and T-cell markers found
to have significant prognostic value in univariate analysis (Table 4). Table 6. Multivariate Analysis for Prognostic Significance of Pathologic
Features and T-Cell Marker Density in Stage II CRC
This analysis revealed that tumor stage, vascular invasion, FOXP3N,
and FOXP3T were the only markers to show independent prognostic Prognostic Feature HR 95% CI P

significance. It also confirmed that high densities of FOXP3N and Model A, n 360

FOXP3T were associated with opposite effects on patient outcome. Vascular invasion 1.88 0.77 to 4.63 .17
Lymphatic invasion 0.82 0.29 to 2.32 .70
FOXP3:CD8 and FOXP3:CD45RO ratios in normal and tumor Perineural invasion 2.73 1.13 to 6.56 .02
tissues were also examined and found not to be significant in multi- Lymphocytic response 0.94 0.47 to 1.87 .86
variate analysis. Microsatellite instability 0.92 0.37 to 2.27 .85
Model B, n 337
Vascular invasion 2.08 0.83 to 5.25 .12
Perineural invasion 2.54 0.71 to 9.08 .15
CD8N 0.82 0.53 to 1.28 .38
CD8T 1.04 0.63 to 1.71 .88
Table 5. Multivariate Analysis Showing the Significant Prognostic CD45ROT 0.95 0.50 to 1.84 .89
Indicators in Stage II and Stage III CRC (n 445)
FOXP3N 1.41 1.00 to 2.01 .05
Feature HR 95% CI P FOXP3T 0.74 0.44 to 1.24 .25
AJCC stage, III v II 3.29 2.25 to 4.81 .001 Model C, n 381
Vascular invasion, yes v no 1.98 1.39 to 2.83 .001 Vascular invasion 2.16 1.03 to 4.51 .041
FOXP3N, high v low 1.51 1.07 to 2.13 .019 Perineural invasion 3.53 1.34 to 9.33 .011
FOXP3T, high v low 0.54 0.38 to 0.77 .001 FOXP3N 1.42 1.05 to 1.92 .023
FOXP3T 0.65 0.48 to 0.89 .007
NOTE. Cox proportional hazards regression model.
Abbreviations: CRC, colorectal cancer; HR, hazard ratio; AJCC, American Abbreviations: CRC, colorectal cancer; HR, hazard ratio; N, normal tissue; T,
Joint Committee on Cancer; N, normal tissue; T, tumor tissue. tumor tissue.

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 Salama et al
T-cell markers were assessed objectively and quantitatively using
digitized, high-resolution images and specialized software, thus limit-
ing observer bias. The finding of a higher FOXP3T Treg density
compared with FOXP3N (Table 1) is in agreement with previous
studies.27,28 The tumor/normal ratio for Treg density observed here
(2.64) was also similar to that reported by Ling et al.28 In contrast to
FOXP3, the CD8 and CD45RO cell densities were lower in tumor
compared with normal colonic mucosa, suggesting that these T-cell
types play a different role to Tregs. Supporting this notion, the corre-
lation between FOXP3N and FOXP3T densities was weaker than
that observed for CD8 or CD45RO (Table 2).
The finding of lower CD8T and CD45ROT densities in more
advanced tumors (Table 3) agrees with the findings of several other
groups.1-6 Previously reported associations between high CD45ROT
cell density and signs of early metastasis (vascular and perineural
invasion)12 were also confirmed in the present study. As expected,
tumors reported to show a lymphocytic response or MSI also had
higher densities of CD8T and CD45ROT. In keeping with the sug-
gestion of a different role for Tregs, FOXP3T cell density was not
associated with early signs of metastasis, lymphocytic response, or
MSI. Although not as pronounced as for CD8T and CD45ROT, the
FOXP3T Treg density was lower in stage III tumors, confirming a
recent report by Loddenkemper et al.27
Univariate survival analysis confirmed the poor prognosis asso-
ciated with the conventional histopathologic markers of adverse out-
come (Table 4). To our knowledge, the present study is the first to
investigate the prognostic significance of T-cell markers in normal
colonic mucosa from patients with CRC. Similar to CD8T, CD8N
could be expected to have antitumor reactivity, thus explaining their
association with better prognosis. The better prognosis associated with
high densities of CD8T and CD45ROT (Table 4) agrees with earlier
studies.12,15 High densities of these cells have been linked to the sup-
pression of metastasis.12,14 Alternatively, they could also signal the
existence of a more antigenic tumor phenotype that has yet to acquire
the ability to evade immunosurveillance.
One of the original and intriguing findings of this study was the
opposite prognostic significance observed for high densities of
FOXP3T and FOXP3N Tregs (Tables 4 and 5). The worse outcome
observed for patients with CRC with high FOXP3N might be ex-
plained by the proposed role for these cells in suppressing antitumor
immunity.16 However, the observation of better survival for patients
with a high density of FOXP3T Tregs is counter-intuitive and con-
trasts with what has been reported for other solid tumor types, includ-
ing melanoma31 and breast,32 ovarian,22 hepatocellular,25,26 and
pancreatic24 cancers. Functional studies of FOXP3T and FOXP3N
Tregs may shed more light on their role in the antitumor response and
help to explain the observed associations with prognosis.
Current recommendations for the treatment of CRC are that
patients with stage III disease receive adjuvant chemotherapy. The
discovery and validation of novel prognostic indicators are therefore
of greatest importance for the management of stage II disease. Of
the three T-cell markers investigated in this study, only FOXP3T
and FOXP3N Tregs showed independent prognostic value in a
multivariate model of stage II CRC (Table 6). The other significant
prognostic factors in this model were vascular and perineural
invasion, both of which are indicators of early metastasis and have
been reported previously.33,34 As discussed earlier, the CD45ROT
190 2008 by American Society of Clinical Oncology
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Copyright 2009 American Society of Clinical Oncology. All rights reserved.
cell density was inversely related to the presence of vascular and
perineural invasion, whereas FOXP3T Tregs showed no associa-
tion (Table 3). This is likely to explain why CD45ROT failed to
show independent prognostic value in a multivariate model that
included these pathologic features. The Harrells concordance sta-
tistic C30 using stage together with vascular invasion was 69. This
improved to 74 with the addition of FOXP3T and FOXP3N Treg
densities. The present results indicate that FOXP3T and
FOXP3N Treg densities, in combination with vascular and peri-
neural invasion, could provide clinically useful prognostic stratifi-
cation for early-stage CRC.
One of the limitations of this study was that much of the his-
topathologic information was obtained from a period that predated
the introduction of synoptic reporting. The presence of vascular and
perineural invasion are therefore likely to have been underreported at
the initial diagnosis.35 Another limitation was the evaluation of T-cell
marker density in 1-mm diameter cores from tissue arrays. Although
tissue arrays allow for large cohorts to be assessed quickly, the relatively
small area investigated represents only a small proportion of the total
tumor volume. Furthermore, the cores were taken at random from
within the tumor block face, and their location relative to the tumor
margin was not recorded. In combination with T-cell type and den-
sity, the location of tumor-infiltrating lymphocytes relative to the
invading margin and central tumor area has recently been reported to
have prognostic value.14 To address the above limitations, further
studies are underway in stage II CRC that use full block-face tissue
sections to assess FOXP3T and FOXP3N Treg densities and where
the presence of vascular and perineural invasion are reviewed by
a pathologist.
Although there have been several publications using FOXP3 to
identify tumor-infiltrating Tregs,26-28,32,36 some researchers have
questioned the validity of this marker for defining Tregs in humans.37
FOXP3 Tregs were identified in the current study by immunohisto-
chemistry using the monoclonal antibody clone 236A/E7. This anti-
body has previously undergone extensive evaluation by Roncador et
al.38 On the basis of this study and other supporting evidence in the
literature,19,39,40 the vast majority of FOXP3 cells identified by mAb
236A/E7 are CD4CD25 Tregs. Although a small proportion of
FOXP3 cells may also be CD8 or CD25,38 it remains that FOXP3
lymphocyte density showed strong and independent prognostic sig-
nificance in CRC (Table 6).
In conclusion, the present study is the first to report on the
prognostic significance of FOXP3T and FOXP3N Treg densities in
patients with CRC. Multivariate models showed these markers had
stronger prognostic value than CD8T or CD45ROT. Although fur-
ther studies are required before changes in clinical practice can be
recommended, the present results suggest that assessment of
FOXP3T and FOXP3N Treg densities in combination with vas-
cular and perineural invasion should improve the prognostic stratifi-
cation of early-stage CRC. The better survival associated with a high
density of FOXP3T Tregs in CRC is in marked contrast to observa-
tions in other solid tumor types.
AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
The author(s) indicated no potential conflicts of interest.
JOURNAL OF CLINICAL ONCOLOGY

AUTHOR CONTRIBUTIONS
Conception and design: Paul Salama, Melinda Morris, Cameron Platell,
Barry Iacopetta
Financial support: Cameron Platell
Provision of study materials or patients: Fabienne Grieu, Nik Zeps,
David Joseph
REFERENCES
1. House AK, Watt AG: Survival and the immune
response in patients with carcinoma of the colorec-
tum. Gut 20:868-874, 1979
2. Svennevig JL, Lunde OC, Holter J, et al:
Lymphoid infiltration and prognosis in colorectal
carcinoma. Br J Cancer 49:375-377, 1984
3. Jass JR: Lymphocytic infiltration and survival
in rectal cancer. J Clin Pathol 39:585-589, 1986
4. Halvorsen TB, Seim E: Association between
invasiveness, inflammatory reaction, desmoplasia
and survival in colorectal cancer. J Clin Pathol 42:
162-166, 1989
5. Murphy J, OSullivan GC, Lee G, et al: The
inflammatory response within Dukes B colorectal
cancers: Implications for progression of microme-
tastases and patient survival. Am J Gastroenterol
95:3607-3614, 2000
6. Ohtani H: Focus on TILs: Prognostic signifi-
cance of tumor infiltrating lymphocytes in human
colorectal cancer. Cancer Immun 7:4, 2007
7. Naito Y, Saito K, Shiiba K, et al: CD8 T cells
infiltrated within cancer cell nests as a prognostic
factor in human colorectal cancer. Cancer Res 58:
3491-3494, 1998
8. Nagtegaal ID, Marijnen CA, Kranenbarg EK, et
al: Local and distant recurrences in rectal cancer
patients are predicted by the nonspecific immune
response; specific immune response has only a
systemic effecta histopathological and immunohis-
tochemical study. BMC Cancer 1:7, 2001
9. Menon AG, Janssen-van Rhijn CM, Morreau
H, et al: Immune system and prognosis in colorectal
cancer: A detailed immunohistochemical analysis.
Lab Invest 84:493-501, 2004
10. Prall F, Duhrkop T, Weirich V, et al: Prognostic
role of CD8 tumor-infiltrating lymphocytes in stage
III colorectal cancer with and without microsatellite
instability. Hum Pathol 35:808-816, 2004
11. Chiba T, Ohtani H, Mizoi T, et al: Intraepithelial
CD8 T-cell-count becomes a prognostic factor
after a longer follow-up period in human colorectal
carcinoma: Possible association with suppression of
micrometastasis. Br J Cancer 91:1711-1717, 2004
12. Pages F, Berger A, Camus M, et al: Effector
memory T cells, early metastasis, and survival in
colorectal cancer. N Engl J Med 353:2654-2666, 2005
13. Koch M, Beckhove P, Op den Winkel J, et al:
Tumor infiltrating T lymphocytes in colorectal can-
cer: Tumor-selective activation and cytotoxic activity
in situ. Ann Surg 244:986-992, 2006
www.jco.org
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T Regulatory Cells in Colorectal Cancer
Collection and assembly of data: Paul Salama, Fabienne Grieu, Melinda
Morris, Nik Zeps
Data analysis and interpretation: Paul Salama, Michael Phillips,
Fabienne Grieu, Cameron Platell, Barry Iacopetta
Manuscript writing: Paul Salama, Michael Phillips, Cameron Platell,
Barry Iacopetta
Final approval of manuscript: Paul Salama, Michael Phillips, Fabienne Grieu,
Melinda Morris, Nik Zeps, David Joseph, Cameron Platell, Barry Iacopetta
14. Galon J, Costes A, Sanchez-Cabo F, et al:
Type, density, and location of immune cells within
human colorectal tumors predict clinical outcome.
Science 313:1960-1964, 2006
15. Oberg A, Samii S, Stenling R, et al: Different
occurrence of CD8, CD45R0, and CD68 im-
mune cells in regional lymph node metastases from
colorectal cancer as potential prognostic predictors.
Int J Colorectal Dis 17:25-29, 2002
16. Curiel TJ: Tregs and rethinking cancer immu-
notherapy. J Clin Invest 117:1167-1174, 2007
17. Zou W: Regulatory T cells, tumour immunity
and immunotherapy. Nat Rev Immunol 6:295-307,
2006
18. Needham DJ, Lee JX, Beilharz MW: Intra-
tumoural regulatory T cells: A potential new target in
cancer immunotherapy. Biochem Biophys Res Com-
mun 343:684-691, 2006
19. Clarke SL, Betts GJ, Plant A, et al:
CD4CD25FOXP3 regulatory T cells suppress
anti-tumor immune responses in patients with colo-
rectal cancer. PLoS ONE 1:e129, 2006
20. Fontenot JD, Gavin MA, Rudensky AY: Foxp3
programs the development and function of
CD4CD25 regulatory T cells. Nat Immunol
4:330-336, 2003
21. Hori S, Nomura T, Sakaguchi S: Control of
regulatory T cell development by the transcription
factor Foxp3. Science 299:1057-1061, 2003
22. Curiel TJ, Coukos G, Zou L, et al: Specific
recruitment of regulatory T cells in ovarian carci-
noma fosters immune privilege and predicts re-
duced survival. Nat Med 10:942-949, 2004
23. Sato E, Olson SH, Ahn J, et al: Intraepithelial
CD8 tumor-infiltrating lymphocytes and a high
CD8/regulatory T cell ratio are associated with
favorable prognosis in ovarian cancer. Proc Natl
Acad Sci U S A 102:18538-18543, 2005
24. Hiraoka N, Onozato K, Kosuge T, et al: Preva-
lence of FOXP3 regulatory T cells increases during
the progression of pancreatic ductal adenocarci-
noma and its premalignant lesions. Clin Cancer Res
12:5423-5434, 2006
25. Kobayashi N, Hiraoka N, Yamagami W, et al:
FOXP3 regulatory T cells affect the development
and progression of hepatocarcinogenesis. Clin Can-
cer Res 13:902-911, 2007
26. Gao Q, Qiu SJ, Fan J, et al: Intratumoral
balance of regulatory and cytotoxic T cells is asso-
ciated with prognosis of hepatocellular carcinoma
after resection. J Clin Oncol 25:2586-2593, 2007
27. Loddenkemper C, Schernus M, Noutsias M,
et al: In situ analysis of FOXP3 regulatory T cells in
human colorectal cancer. J Transl Med 4:52, 2006
28. Ling KL, Pratap SE, Bates GJ, et al: Increased
frequency of regulatory T cells in peripheral blood
and tumour infiltrating lymphocytes in colorectal
cancer patients. Cancer Immun 7:7, 2007
29. Chai SM, Zeps N, Shearwood AM, et al:
Screening for defective DNA mismatch repair in
stage II and III colorectal cancer patients. Clin Gas-
troenterol Hepatol 2:1017-1025, 2004
30. Harrell FE Jr, Lee KL, Mark DB: Multivariable
prognostic models: Issues in developing models,
evaluating assumptions and adequacy, and measur-
ing and reducing errors. Stat Med 15:361-387, 1996
31. Miracco C, Mourmouras V, Biagioli M, et al:
Utility of tumour-infiltrating CD25FOXP3 regula-
tory T cell evaluation in predicting local recurrence in
vertical growth phase cutaneous melanoma. Oncol
Rep 18:1115-1122, 2007
32. Bates GJ, Fox SB, Han C, et al: Quantification
of regulatory T cells enables the identification of
high-risk breast cancer patients and those at risk of
late relapse. J Clin Oncol 24:5373-5380, 2006
33. Quirke P, Morris E: Reporting colorectal can-
cer. Histopathology 50:103-112, 2007
34. Morris M, Platell C, de Boer B, et al:
Population-based study of prognostic factors in
stage II colonic cancer. Br J Surg 93:866-871, 2006
35. Stewart CJ, Morris M, de Boer B, et al:
Identification of serosal invasion and extramural
venous invasion on review of Dukes stage B colonic
carcinomas and correlation with survival. Histopa-
thology 51:372-378, 2007
36. Alvaro T, Lejeune M, Salvado MT, et al: Im-
munohistochemical patterns of reactive microenvi-
ronment are associated with clinicobiologic behavior
in follicular lymphoma patients. J Clin Oncol 24:
5350-5357, 2006
37. Roncarolo MG, Gregori S: Is FOXP3 a bona
fide marker for human regulatory T cells? Eur J Im-
munol 38:925-927, 2008
38. Roncador G, Brown PJ, Maestre L, et al:
Analysis of FOXP3 protein expression in human
CD4CD25 regulatory T cells at the single-cell
level. Eur J Immunol 35:1681-1691, 2005
39. Walker MR, Carson BD, Nepom GT, et al: De
novo generation of antigen-specific CD4CD25
regulatory T cells from human CD4CD25- cells.
Proc Natl Acad Sci U S A 102:4103-4108, 2005
40. Wang J, Ioan-Facsinay A, van der Voort E, et
al: Transient expression of FOXP3 in human acti-
vated nonregulatory CD4 T cells. Eur J Immunol
37:129-138, 2007
2008 by American Society of Clinical Oncology 191
 Salama et al

Glossary Terms

CD45RO T cells: A memory/activation surface marker
glycoprotein commonly expressed by T cells infiltrating into
tumors, CD45 belongs to the family of leukocyte common
antigens found on the surface of the majority of human leuko-
cytes. CD45 has tyrosine phosphatase activity and augments
signaling through the T-cell and B-cell receptor. CD45RO is
suggestive of mature memory T cell and is different from
CD45RA, which is present on immature T cells.
Tissue microarray: Used to analyze the expression of genes of inter-
est simultaneously in multiple tissue samples, tissue microarrays consist of
hundreds of individual tissue samples placed on slides ranging from 2 to 3
mm in diameter. Using conventional histochemical and molecular detec-
tion techniques, tissue microarrays are powerful tools to evaluate the ex-
pression of genes of interest in tissue samples. In cancer research, tissue
microarrays are used to analyze the frequency of a molecular alteration in
different tumor type, to evaluate prognostic markers, and to test potential
diagnostic markers.

Immunohistochemistry: The application of antigen-antibody inter-

CD8 T cells: Cytotoxic T cells that are the primary effec-
tor cells of the immune system. They are characterized by the
presence of the CD8 cell-surface marker.
FoxP3: The forkhead transcriptional factor that is specifi-
cally expressed in CD4CD25 regulator T cells.
Prognostic factor: A measurable patient characteristic
that is associated with the subsequent course of disease
(whether or not therapy is administered). The identification of
a prognostic factor does not necessarily imply a cause-and-
effect relationship. However, within a suitable outcome
model, the measurement of a prognostic factor contributes to
an estimate of an outcome probability (eg, the probability of
disease-free survival within a given time interval).
actions to histochemical techniques. Typically, a tissue section is mounted
on a slide and is incubated with antibodies (polyclonal or monoclonal) spe-
cific to the antigen (primary reaction). The antigen-antibody signal is then
amplified using a second antibody conjugated to a complex of peroxidase-
antiperoxidase (PAP), avidin-biotin-peroxidase (ABC) or avidin-biotin
alkaline phosphatase. In the presence of substrate and chromogen, the en-
zyme forms a colored deposit at the sites of antibody-antigen binding. Im-
munofluorescence is an alternate approach to visualize antigens. In this
technique, the primary antigen-antibody signal is amplified using a second
antibody conjugated to a fluorochrome. On UV light absorption, the fluo-
rochrome emits its own light at a longer wavelength (fluorescence), thus
allowing localization of antibody-antigen complexes.
Microsatellite instability: Microsatellites are repeating units in DNA
of 1-5 basepairs that are ubiquitous, abundant, and repeated several times in
eukaryotic genomes. The presence of microsatellites is associated with
genomic instability, giving rise to mutations that involve the addition or
subtraction of one or two repeat units.

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