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JCO 2009 Salama 186 92
JCO 2009 Salama 186 92
thought to modulate the antitumor immune response.16,17 Tregs can resected stage II and III CRC within 13 TMA blocks. For each case, two cores of
suppress the activity of cytotoxic T cells through direct cell-to-cell 1 mm diameter were taken at random from the tumor, and an additional
contact or via the release of cytokines, especially transforming growth 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
factor . Depletion of intratumoral Tregs enhances antitumor immu-
of the tumor was not recorded.
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 Immunohistochemistry for T-Cell Markers
Sections of 5 m thickness were cut from the TMA blocks, mounted on
specific Treg cell marker identified to date is the nuclear transcription
silanated slides, and subsequently dewaxed and rehydrated using xylene and
factor known as FOXP3.20,21 A high density of tumor-infiltrating graded alcohol washes. Antigen retrieval was performed at 121C for 4 minutes
FOXP3 Tregs has been associated with poor outcome in various (DakoCytomation pressure cooker; Dako, Copenhagen, Denmark) with slides
solid tumors, including ovarian,22,23 pancreatic,24 and hepatocellu- placed in Dako target retrieval solution or citrate buffer, depending on the
lar carcinoma.25,26 antibody. Endogenous peroxidase activity was blocked (Real Peroxidase
Two groups have investigated infiltrating Tregs in CRC using Block; Dako) followed by Tris-buffered saline wash. Protein block solution
FOXP3 staining. In a study of 40 patients, Loddenkemper et al27 was applied for 10 minutes to stop nonspecific antibody binding (Dako).
Primary antibodies were used according to manufacturers instructions (CD8,
reported that Treg density was lower in node-positive disease but was DakoCytomation clone C8/144B ready to use; CD45RO, DakoCytomation
not associated with survival. Ling et al28 found no significant differ- clone UCHL1 ready to use; FOXP3, Abcam, ab20034, 1/100 dilution). After
ence in Treg density between advanced and early-stage disease, but did incubation with primary antibody, slides were washed in two changes of TBS
not evaluate the association with patient survival. The aim of the before incubation with labeled polymer horseradish peroxidase rabbit/mouse
present study was therefore to compare the prognostic value of antibody for 15 minutes (Envision Plus Detection System; Dako). Sections
FOXP3 Treg cell density with that of the established T-cell markers were subsequently incubated with Dako-Chromogen solution and washed in
deionized water. Background staining was performed with Mayers hematox-
CD8 and CD45RO in a large cohort of patients with stage II and III
ylin and sections then dehydrated through ascending alcohols to xylene
CRC with long follow-up information. and mounted.
Table 1. Median Density of the T-Cell Markers CD8, CD45RO, and FOXP3 in Normal Colonic Mucosa and Colorectal Tumor Tissues
Normal Tumor
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.
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.
T-cell markers were assessed objectively and quantitatively using cell density was inversely related to the presence of vascular and
digitized, high-resolution images and specialized software, thus limit- perineural invasion, whereas FOXP3T Tregs showed no associa-
ing observer bias. The finding of a higher FOXP3T Treg density tion (Table 3). This is likely to explain why CD45ROT failed to
compared with FOXP3N (Table 1) is in agreement with previous show independent prognostic value in a multivariate model that
studies.27,28 The tumor/normal ratio for Treg density observed here included these pathologic features. The Harrells concordance sta-
(2.64) was also similar to that reported by Ling et al.28 In contrast to tistic C30 using stage together with vascular invasion was 69. This
FOXP3, the CD8 and CD45RO cell densities were lower in tumor improved to 74 with the addition of FOXP3T and FOXP3N Treg
compared with normal colonic mucosa, suggesting that these T-cell densities. The present results indicate that FOXP3T and
types play a different role to Tregs. Supporting this notion, the corre- FOXP3N Treg densities, in combination with vascular and peri-
lation between FOXP3N and FOXP3T densities was weaker than neural invasion, could provide clinically useful prognostic stratifi-
that observed for CD8 or CD45RO (Table 2). cation for early-stage CRC.
The finding of lower CD8T and CD45ROT densities in more One of the limitations of this study was that much of the his-
advanced tumors (Table 3) agrees with the findings of several other topathologic information was obtained from a period that predated
groups.1-6 Previously reported associations between high CD45ROT the introduction of synoptic reporting. The presence of vascular and
cell density and signs of early metastasis (vascular and perineural perineural invasion are therefore likely to have been underreported at
invasion)12 were also confirmed in the present study. As expected, the initial diagnosis.35 Another limitation was the evaluation of T-cell
tumors reported to show a lymphocytic response or MSI also had marker density in 1-mm diameter cores from tissue arrays. Although
higher densities of CD8T and CD45ROT. In keeping with the sug- tissue arrays allow for large cohorts to be assessed quickly, the relatively
gestion of a different role for Tregs, FOXP3T cell density was not small area investigated represents only a small proportion of the total
associated with early signs of metastasis, lymphocytic response, or tumor volume. Furthermore, the cores were taken at random from
MSI. Although not as pronounced as for CD8T and CD45ROT, the within the tumor block face, and their location relative to the tumor
FOXP3T Treg density was lower in stage III tumors, confirming a margin was not recorded. In combination with T-cell type and den-
recent report by Loddenkemper et al.27 sity, the location of tumor-infiltrating lymphocytes relative to the
Univariate survival analysis confirmed the poor prognosis asso- invading margin and central tumor area has recently been reported to
ciated with the conventional histopathologic markers of adverse out- have prognostic value.14 To address the above limitations, further
come (Table 4). To our knowledge, the present study is the first to studies are underway in stage II CRC that use full block-face tissue
investigate the prognostic significance of T-cell markers in normal sections to assess FOXP3T and FOXP3N Treg densities and where
colonic mucosa from patients with CRC. Similar to CD8T, CD8N the presence of vascular and perineural invasion are reviewed by
could be expected to have antitumor reactivity, thus explaining their a pathologist.
association with better prognosis. The better prognosis associated with Although there have been several publications using FOXP3 to
high densities of CD8T and CD45ROT (Table 4) agrees with earlier identify tumor-infiltrating Tregs,26-28,32,36 some researchers have
studies.12,15 High densities of these cells have been linked to the sup- questioned the validity of this marker for defining Tregs in humans.37
pression of metastasis.12,14 Alternatively, they could also signal the FOXP3 Tregs were identified in the current study by immunohisto-
existence of a more antigenic tumor phenotype that has yet to acquire chemistry using the monoclonal antibody clone 236A/E7. This anti-
the ability to evade immunosurveillance. body has previously undergone extensive evaluation by Roncador et
One of the original and intriguing findings of this study was the al.38 On the basis of this study and other supporting evidence in the
opposite prognostic significance observed for high densities of literature,19,39,40 the vast majority of FOXP3 cells identified by mAb
FOXP3T and FOXP3N Tregs (Tables 4 and 5). The worse outcome 236A/E7 are CD4CD25 Tregs. Although a small proportion of
observed for patients with CRC with high FOXP3N might be ex- FOXP3 cells may also be CD8 or CD25,38 it remains that FOXP3
plained by the proposed role for these cells in suppressing antitumor lymphocyte density showed strong and independent prognostic sig-
immunity.16 However, the observation of better survival for patients nificance in CRC (Table 6).
with a high density of FOXP3T Tregs is counter-intuitive and con- In conclusion, the present study is the first to report on the
trasts with what has been reported for other solid tumor types, includ- prognostic significance of FOXP3T and FOXP3N Treg densities in
ing melanoma31 and breast,32 ovarian,22 hepatocellular,25,26 and patients with CRC. Multivariate models showed these markers had
pancreatic24 cancers. Functional studies of FOXP3T and FOXP3N stronger prognostic value than CD8T or CD45ROT. Although fur-
Tregs may shed more light on their role in the antitumor response and ther studies are required before changes in clinical practice can be
help to explain the observed associations with prognosis. recommended, the present results suggest that assessment of
Current recommendations for the treatment of CRC are that FOXP3T and FOXP3N Treg densities in combination with vas-
patients with stage III disease receive adjuvant chemotherapy. The cular and perineural invasion should improve the prognostic stratifi-
discovery and validation of novel prognostic indicators are therefore cation of early-stage CRC. The better survival associated with a high
of greatest importance for the management of stage II disease. Of density of FOXP3T Tregs in CRC is in marked contrast to observa-
the three T-cell markers investigated in this study, only FOXP3T tions in other solid tumor types.
and FOXP3N Tregs showed independent prognostic value in a
multivariate model of stage II CRC (Table 6). The other significant AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS
prognostic factors in this model were vascular and perineural OF INTEREST
invasion, both of which are indicators of early metastasis and have
been reported previously.33,34 As discussed earlier, the CD45ROT The author(s) indicated no potential conflicts of interest.
14. Galon J, Costes A, Sanchez-Cabo F, et al: 28. Ling KL, Pratap SE, Bates GJ, et al: Increased
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Glossary Terms
CD45RO T cells: A memory/activation surface marker Tissue microarray: Used to analyze the expression of genes of inter-
glycoprotein commonly expressed by T cells infiltrating into est simultaneously in multiple tissue samples, tissue microarrays consist of
tumors, CD45 belongs to the family of leukocyte common hundreds of individual tissue samples placed on slides ranging from 2 to 3
antigens found on the surface of the majority of human leuko- mm in diameter. Using conventional histochemical and molecular detec-
cytes. CD45 has tyrosine phosphatase activity and augments tion techniques, tissue microarrays are powerful tools to evaluate the ex-
signaling through the T-cell and B-cell receptor. CD45RO is pression of genes of interest in tissue samples. In cancer research, tissue
suggestive of mature memory T cell and is different from microarrays are used to analyze the frequency of a molecular alteration in
CD45RA, which is present on immature T cells. different tumor type, to evaluate prognostic markers, and to test potential
diagnostic markers.