J Cancer Res Clin Oncol (2013) 139:1845–1852 DOI 10.1007/s00432-013-1500-7
ORIGINAL PAPER
The appearance of Tregs in cancer nest is a promising independent risk factor in colon cancer Wei Xu • Hao Liu • Jun Song • Hai-Xiao Fu Lei Qiu • Bao-Fu Zhang • Hui-Zhong Li • Jin Bai • Jun-Nian Zheng
•
Received: 2 May 2013 / Accepted: 21 August 2013 / Published online: 5 September 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose To investigate the prognostic value of tumorinfiltrating regulatory T cells (Tregs) in the distribution of cancer nest, cancer stroma and normal mucosa and FOXP3-positive cancer cells in colon cancer patients after resection. Methods Paraffin blocks of operation resection of primary adenocarcinoma of colon were obtained from ninety patients. The distribution of tumor-infiltrating Tregs was detected by tissue microarray and immunohistochemistry staining technique to evaluate the prognostic effects by Kaplan–Meier and Cox regression analysis using median values as cutoff. Results The intratumoral Tregs counts were significantly higher than that in corresponding normal mucosa tissues (P \ 0.001); the Tregs counts in cancer nest were significantly lower than that in corresponding cancer stroma tissues (P \ 0.001); the increased intratumoral Tregs counts were associated with favorable prognosis (P \ 0.05); the presence of Tregs in cancer nest was associated with unfavorable prognosis and was an independent prognostic factor for overall survival (P \ 0.05). The appearance of
Wei Xu and Hao Liu have contributed equally to this paper. W. Xu H. Liu J. Song H.-X. Fu L. Qiu Department of Surgery, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China B.-F. Zhang H.-Z. Li J. Bai (&) J.-N. Zheng (&) Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou 221002, Jiangsu, China e-mail:
[email protected] J.-N. Zheng e-mail:
[email protected]
FOXP3-positive cancer cells was associated with worse prognosis (P \ 0.05). In addition, the frequency of the presence of FOXP3-positive cancer cells was higher in patients with lymphatic invasion (P \ 0.001) and lower in patients with early TNM stage (P \ 0.01). Conclusions The higher tumor-infiltrating Tregs counts are closely associated with the improved prognostic effects of colon carcinoma. Tregs play different roles in cancer nest and cancer stroma. And the appearance of Tregs in cancer nest is a promising independent risk factor for overall survival in colon carcinoma. FOXP3-positive cancer cells may also be a risk factor for overall survival in colon carcinoma. Keywords Colon carcinoma Tregs FOXP3 Prognostic Tissue microarray
Introduction Colorectal cancer is one of the most common malignant tumors in the world. Despite improved progress that has been made in basic and clinical research, the overall survival of colorectal cancer did not improve significantly (Mitry et al. 2008). Previous studies, which focused their key point on the tumors characteristics, tried to change gene and phenotype of cancer cells to inhibit the progress of the tumors. Recently, tumor immune microenvironment was reported to play an important role in the development of the tumor (Galon et al. 2007). In particular the regulatory T cells (Tregs), these cells were first reported by (Sakaguchi et al. 1995) in both mice and humans as a key contributor to the maintenance of immunological tolerance and the prevention of autoimmunity. In tumor tissues, Tregs could migrate to tumor locality and inhibit anti-
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from anti-tumor immunity because of the ability to block the T cell-mediated anti-tumor immune response in colorectal cancer (Clarke et al. 2006). Recent studies found that FOXP3 was not only expressed in Tregs, but also in several types of malignant tumor cells, including pancreatic (Hinz et al. 2007), breast (Zuo et al. 2007), lung (Tao et al. 2012) and prostate (Wang et al. 2009) cancers. And FOXP3-positive cancer cells may have ability to escape immune surveillance in pancreatic cancer, which likes the function of Tregs (Hinz et al. 2007). But in breast cancer, this type of cells is reported as favorable prognostic factor (Ladoire et al. 2011a, b). In colorectal cancer, high FOXP3 expression of the cancer cells was associated with poor prognosis compared to patients with low FOXP3 expression (Kim et al. 2013). However, how to evaluate the ability of inhibiting antitumor immunological response that Tregs have in the microenvironment of colorectal cancer is less clear. In this study, the prognostic value of Tregs in the distribution of cancer nest, cancer stroma and normal mucosa and FOXP3positive cancer cells was investigated. We found that Tregs
tumor immune response by influencing the effect of CD4? and CD8? T cells, NK cells and myeloid cells, through different mechanisms (Sakaguchi et al. 2010). And a series of studies have revealed that the high tumor-infiltrating Tregs counts often associate with a poor prognosis in breast (Bates et al. 2006), ovarian (Curiel et al. 2004), lung (Petersen et al. 2006), hepatocellular (Gao et al. 2007), renal cell (Li et al. 2009), pancreatic (Hiraoka et al. 2006), gastric (Perrone et al. 2008) and cervical (Jordanova et al. 2008) cancers. In these studies, FOXP3, which belongs to the family of forkhead box transcription factors, has been a specific surface marker of Tregs (Hori et al. 2003). Surprisingly, seven clinical studies have showed that (Salama et al. 2009; Yoon et al. 2012), in patients bearing colorectal carcinoma, a high tumor-infiltrating Tregs counts are associated with an improved prognosis. And they tried to explain this paradox by a hypothesis that Tregs can indirectly inhibit cancer growth by restraining T cellmediated inflammatory anti-microbial response that can promote tumor growth in colorectal cancer, although Tregs are considered as a major cause of protecting cancer cells Fig. 1 Representative images of FOXP3 immunohistochemical staining in human colon cancer. a Tregs in cancer tissue were detected with positive nuclear staining of FOXP3 (red and black arrow), and tumorinfiltrating Tregs were detected, respectively, in cancer nest (red arrow) and in cancer stroma (black arrow); b Tregs in normal mucosa tissue were also detected with positive nuclear staining of FOXP3 (red arrow); c FOXP3 positive staining in the cytoplasm of colon cancer cells; d FOXP3 negative staining in the cytoplasm of colon cancer cells. Magnification 9400
Table 1 The distribution of Tregs of 90 colon cancer patients in intratumoral, normal mucosa, cancer stroma and cancer nest Location
N
Intratumoral (total)
90
Normal mucosa Cancer stroma Cancer nest
123
90
Minimum
Maximum
Mean
Median
Treg count (per HPF)
t
P
3.95
\0.001
6.019
\0.001
0
78.0
16.1
11.0
16.08 ± 16.70
0
112.0
7.0
3.0
6.97 ± 14.67
0
78.0
13.4
7.0
13.39 ± 16.17
0
18.0
2.7
1.0
2.67 ± 4.54
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Immunohistochemistry of TMA
play different roles in cancer nest and cancer stroma, that is the first report by now, and the appearance of Tregs in cancer nest is a promising independent risk factor in colon carcinoma.
TMA slides were deparaffinized and rehydrated using xylene and graded alcohol washes. After heat-mediated antigen retrieval via the microwave method and a series of blocking with 3 % H2O2 and normal goat serum, the tissue microarray was incubated with primary mouse monoclonal to FOXP3 with 1:100 dilution (Abcam, Cambridge, MA) overnight at 4 °C. And then, biotinylated secondary antibody followed by horseradish peroxidase (HRP)-conjugated streptavidin and DAB-chromogen was applied according to the manufacturer’s instructions and counterstained with hematoxylin. Negative control was made using PBS to take the place of the primary antibodies in another serial section. Two independent investigators reviewed the tissue microarray independently for a random selection of 5 high-power fields (HPF, 4009) each point. The nuclear staining was considered as the Tregs in cancer nest, cancer stroma and normal mucosa, and the cytoplasmic staining which was more than 20 % of examined fields in cancer nest was considered as the FOXP3-positive cancer cell (Tao et al. 2012). The average count of Tregs of 5 HPF was recorded for further analysis.
Materials and methods Patients and samples The tissue microarray that includes 90 patients of primary colon adenocarcinoma was obtained from Shanghai Outdo Biotech (Shanghai, China). And each patient enrolled has two cores, one is the cancer tissue, the other is the normal mucosa tissue ([5 cm from the tumor margin). All of the patients, who underwent operative resection for colon cancer between July 2006 and May 2007, did not accept any neoadjuvant therapy or postoperative radiotherapy and chemotherapy. This study contains 9 cases of TNM stage I, 50 cases of TNM stage II, 29 cases of TNM stage III and 2 cases of TNM stage IV. Histologic type was determined according to the WHO classification, and the tumor staging was determined according to the TNM classification system of American Joint Committee on Cancer and International Union against Cancer (the seventh edition). Followup was completed in August 2012. The median follow-up was 65 months (63–73 months). This study was approved by the research ethics committee and agreed by each patient with written consent. Table 2 Association of Tregs count with clinicopathologic features of 90 colon cancer patients
Group
Statistical analysis Statistic analysis was performed with SPSS 16.0 software (SPSS, Chicago, IL). Means and standard deviations
N
The count of Tregs
t
P
-0.344
0.732
0.155
0.877
Age (years) \70
43
15.44 ± 18.16
C70
47
16.66 ± 15.41
Male
47
16.34 ± 18.62
Female
43
15.79 ± 14.52
56 34
18.98 ± 19.40 11.29 ± 9.32
2.525
0.013
\35
45
16.56 ± 17.45
0.27
0.788
C35
45
15.60 ± 16.09 -0.539
0.591
2.606
0.011
2.801
0.006
Gender
Pathological grading I ? I - II ? III II - III ? III Tumor size (cm3)
The number of tumor 1
82
15.78 ± 16.76
2
8
19.13 ± 16.82
No
53
19.38 ± 20.02
Yes
37
11.35 ± 8.44
I ? II
58
18.98 ± 19.38
III ? IV
32
10.81 ± 8.05
Lymphatic invasion
TNM stage
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( x S) were used for the descriptive statistics of enumeration data. For the comparison of two groups of enumeration data, two independent samples t test, two paired samples t test, v2 test or Fisher’s exact test were adopted appropriately. Kaplan–Meier method and log-rank test were used for survival analysis. Univariate and multivariate analyses were based on the Cox regression. Two-tailed P \ 0.05 was considered to be statistically significant.
53.3 % (48/90) and 46.7 % (42/90). The intratumoral Tregs counts were significantly higher than that in corresponding normal mucosa tissues (P \ 0.001); the Tregs counts in cancer nest were significantly lower than that in corresponding cancer stroma tissues (P \ 0.001) (Table 1). These suggested that Tregs may play a key role in the development of colon cancer. In addition, FOXP3-positive cancer cells, which were detected by cytoplasmic staining in the cancer nest (Fig. 1c, d), were found in 15 of 90 patients.
Results Analysis on the distribution of Tregs and the expression status of FOXP3
Association of the distribution of Tregs and the expression status of FOXP3 with clinicopathologic features
The counts of the Tregs which were detected by positive nuclear staining of FOXP3 (Fig. 1a, b) ranged from 0 to 78.0 (mean 16.1, median 11.0) per HPF in tumor tissue, from 0 to 18.0 (mean 2.7, median 1.0) per HPF in cancer nest. Of the 90 patients analyzed, low expression levels and high expression levels of intratumoral Tregs were 76.7 % (69/90) and 23.3 % (21/90). Cancer cells expression FOXP3-negative and expression FOXP3-positive were
According to the cutoff value of the count of Tregs in cancer nest, cancer stroma and normal mucosa, 90 patients were divided into two groups separately. The count of Tregs in cancer tissue did not correlate with age, gender, tumor size or the number of tumors. Patients with higher pathological differentiation, no lymphatic invasion and earlier tumor-node-metastasis (TNM) stage showed higher tumor-infiltrating Tregs (Table 2). In addition, the presence
Table 3 Association of FOXP3 expression in cancer cells with clinicopathologic features of 90 colon cancer patients
Group
Expression of FOXP3 in cancer cells FOXP3 (?)
FOXP3 (-)
\70
6 (14.0 %)
37 (86.0 %)
C70
9 (19.1 %)
38 (80.9 %)
v2
P
Age (years) 0.436
0.509
2.574
0.109
Gender Male
5 (10.6 %)
42 (89.4 %)
10 (23.3 %)
33 (76.7 %)
6 (10.7 %) 9 (26.5 %)
50 (89.3 %) 25 (73.5 %)
3.782
0.052
\35
8 (17.8 %)
37 (82.2 %)
0.08
0.777
C35
7 (15.6 %)
38 (84.4 %)
1
13 (15.9 %)
69 (84.1 %)
0.439
0.508
2
2 (25.0 %)
6 (75.0 %) 11.244
\0.001
7.603
0.006
1.006
0.316
Female Pathological grading I ? I - II ? II II - III ? III Tumor size (cm3)
The number of tumor
Lymphatic invasion Yes
12 (32.4 %)
25 (67.6 %)
No
3 (5.7 %)
50 (94.3 %)
TNM stage I ? II
5 (8.6 %)
53 (91.4 %)
10 (31.2 %)
22 (68.8 %)
High
2 (9.5 %)
19 (90.5 %)
Low
13 (18.8 %)
56 (81.2 %)
III ? IV Tumor-infiltrating Tregs
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of FOXP3-positive cancer cells did not correlate with age, gender, tumor size, the number of tumor, pathological differentiation or the count of tumor-infiltrating Tregs. The frequency of the presence of FOXP3-positive cancer cells was higher in patients with lymphatic invasion or lower in patients with early TNM stage (Table 3).
Tregs play different roles in cancer nest and cancer stroma by Kaplan–Meier survival analyses of 90 colon cancer patients A total of 90 patients had completed follow-up and clinical information. To evaluate the prognosis, Kaplan–Meier survival analyses were used. The results revealed that the increased intratumoral Tregs counts were associated with favorable prognosis (P \ 0.05) (Fig. 2a); the presence of Tregs in cancer nest was associated with unfavorable prognosis (P \ 0.05) (Fig. 2b); the increased Tregs counts in cancer stroma were seemingly associated with favorable prognosis but no statistical significance (P = 0.103) (Fig. 2c); the reduced Tregs counts in normal mucosa were seemingly associated with favorable prognosis but also no statistical significance (P = 0.206) (Fig. 2d); the expression of FOXP3 in cancer cells was associated with a poor prognosis (P \ 0.05) (Fig. 2e).
Fig. 2 Kaplan–Meier survival analyses of 90 colon cancer patients. a High intratumoral Tregs counts correlate with an improved overall survival (P = 0.020, log-rank test). b The presence of Tregs in cancer nest was associated with unfavorable prognosis (P = 0.011, log-rank test). c High Tregs counts in cancer stroma were seemingly associated with favorable prognosis but no statistical significance (P = 0.103,
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The presence of Tregs in cancer nest is an independent risk factor for overall survival We also used univariate Cox proportional hazards regression model to estimate the crude hazard ratios (HRs) of Tregs or each clinicopathological variable on patient survival. The log-rank test and univariate Cox regression analyses revealed that pathological grading, tumor size, the number of tumor, lymphatic invasion, TNM staging, tumor-infiltrating Tregs in cancer tissue, the presence of Tregs in cancer nest and the expression of FOXP3 in cancer cells were significantly associated with overall survival in all 90 patients (Table 4). To further validate the prognostic value of Tregs and FOXP3 expression in colon cancer cells, multivariate analysis was performed, where pathological grading, tumor size, the number of tumor, lymphatic invasion, TNM stage, tumor-infiltrating Tregs, Tregs in cancer nest and expression of FOXP3 in cancer cells were included. And the results showed that pathological grading (HR 2.238; 95 % confidential interval (CI) 1.075–4.659; P = 0.031), tumor size (HR 3.146; 95 % CI 1.528–6.479; P = 0.002), the number of tumor (HR 2.914; 95 % CI 1.192–7.123; P = 0.019) and the presence of Tregs in cancer nest (HR 2.179; 95 % CI 1.091–4.349; P = 0.027) were significant factors (Table 5). The Cox regression model indicated that
log-rank test). d Low Tregs counts in normal mucosa were seemingly associated with favorable prognosis but also no statistical significance (P = 0.206, log-rank test). e The expression of FOXP3 in cancer cells was associated with a poor prognosis (P = 0.035, log-rank test). Cum cumulative
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1850 Table 4 Univariate Cox proportional regression analysis on 5-year overall survival of 90 colon cancer patients
Table 5 Multivariate Cox regression analysis on 5-year overall survival of 90 colon cancer patients
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Group
Hazard ratio
95 % CI
P
Age (\70 years vs. C70 years)
1.594
0.855–2.972
Gender (male vs. female)
0.917
0.499–1.684
0.78
Pathological grading (I ? I - II ? II vs. II - III ? III)
1.936
1.056–3.551
0.033
Tumor size (\35 cm3 vs. C35 cm3)
2.145
1.148–4.009
0.017
The number of tumor (1 vs. 2)
2.854
1.261–6.459
0.012
Lymphatic invasion (no vs. yes)
3.849
2.034–7.282
\0.001
0.143
TNM stage (I ? II vs. III ? IV)
3.049
1.651–5.630
\0.001
Tumor-infiltrating Tregs (low vs. high)
0.348
0.137–0.886
0.027
Tregs in cancer nest (yes vs. no)
2.193
1.174–4.096
0.014
Tregs in cancer stroma (low vs. high)
0.603
0.326–1.117
0.108
Tregs in normal mucosa (low vs. high) Expression of FOXP3 in cancer cells (no vs. yes)
1.533 2.102
0.784–2.995 1.033–4.280
0.212 0.04
Group
Overall survival Hazard ratio
95 % CI
P
Pathological grading (I ? I - II ? II vs. II - III ? III)
2.238
1.075–4.659
0.031
Tumor size (\35 cm3 vs. C35 cm3)
3.146
1.528–6.479
0.002
The number of tumor (1 vs. 2)
2.914
1.192–7.123
0.019
Lymphatic invasion (no vs. yes)
2.600
0.816–8.288
0.106
TNM stage (I ? II vs. III ? IV)
1.284
0.380–4.340
0.687
Tumor-infiltrating Tregs (low vs. high)
0.581
0.205–1.641
0.305
Tregs in cancer nest (yes vs. no)
2.179
1.091–4.349
0.027
Expression of FOXP3 in cancer cells (no vs. yes)
1.284
0.562–2.935
0.553
the presence of Tregs in cancer nest is an independent risk factor for overall survival.
Discussion Tregs have the ability to inhibit the immune response against tumors mediated by T cells in colorectal cancer (Clarke et al. 2006), which can protect the growth behavior of tumor. This finding seems to be consistent with other outcome studies in various human carcinomas (Curiel et al. 2004; Gao et al. 2007; Hiraoka et al. 2006; Jordanova et al. 2008; Li et al. 2009; Perrone et al. 2008; Petersen et al. 2006), in which the high accumulation of Tregs often results in a poor prognosis. But conclusions contrary to the previous reports in other cancers were established by recent studies in which high count of tumor-infiltration Tregs was associated with improved survival in colorectal cancer (Frey et al. 2010; Pages et al. 2005; Salama et al. 2009). And 216 cases of colon carcinomas from participants in 5-FU-based adjuvant trials were analyzed to find that among CD8?low tumors, FOXP3?high cases had
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Overall survival
significantly improved overall survival compared to FOXP3?low cases, but Tregs counts in cancer epithelial did not significantly influence overall survival (Yoon et al. 2012). In our study, through the precise location of Tregs in the microenvironment of colon cancers which were not treated with neoadjuvant radiochemotherapy or postoperative radiochemotherapy, we found that the higher tumor-infiltrating Tregs counts are closely associated with the improved prognostic effects. The reason of this discrepancy may be attributed to the existence of microbiological flora in colonic lumen which can infiltrate the tumor through the increased permeability of the physical structure of the colonic mucosal barrier (Ladoire et al. 2011a, b). The tumor-infiltrating microbe can cause the inflammatory anti-microbial response, which involves Th17 cells, mediated by T cells. This can stimulate angiogenesis through VEGF production and induce inflammatory reaction triggered by cytokines like IL-17, IL-1, IL-6 and TNF-a, with subsequent proangiogenic (Numasaki et al. 2003) and tumor-enhancing effects (Bromberg and Wang 2009), thus contributing to the progress of tumor. This anti-microbial
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response can be attenuated by Tregs (Ladoire et al. 2011a, b), which may give Tregs the anti-tumorigenic effects indirectly in colon cancer and explain the relationship, unlike other carcinomas, between the higher tumor-infiltrating Tregs counts and improved prognosis. But more precise location of Tregs in the microenvironment of colon cancer, whereas the cancer nest and cancer stroma were investigated separately, showed that the appearance of Tregs in cancer nest was correlated with a poor outcome based on both univariate and multivariate analyses. We surmised that Tregs in cancer nest may inhibit anti-tumor immunity and help the cancer cells to escape from immune surveillance maybe through the direct contact with the cancer cells. We firstly report that, unlike other carcinomas, Tregs in colon cancer, whereas the tumor-infiltrating Tregs have specificity not only for tumor-specific antigens but also for the commensal microflora, play different roles in cancer nest and cancer stroma, mainly for inhibition of inflammatory anti-microbial response in cancer stroma and mainly for suppression of anti-tumor response in cancer nest. In addition, we also found that FOXP3 could also be expressed in colon cancer cells detected by cytoplasmic staining. Correlated with clinicopathological parameters and overall survival of patients, the frequency of the presence of FOXP3-positive cancer cells was higher in patients with lymphatic invasion or lower in patients with early TNM stage. The expression of FOXP3 in cancer cells was associated with a poor prognosis, which is similar to previous studies (Kim et al. 2013). In conclusion, our results have shown that the prognosis could be assessed on the basis of tumor-infiltrating Tregs and tumor cells FOXP3 expression status that may be predictors for overall survival in colon carcinoma. What is more, the appearance of Tregs in cancer nest is a promising independent risk factor for overall survival in colon carcinoma. Our findings may be beneficial to design a new immunotherapy targeting Tregs, especially in different locations, and FOXP3-positive cancer cells in colon cancer. Conflict of interest
We declare that we have no conflict of interest.
References Bates GJ, Fox SB, Han C, Leek RD, Garcia JF, Harris AL, Banham AH (2006) 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 Bromberg J, Wang TC (2009) Inflammation and cancer: IL-6 and STAT3 complete the link. Cancer Cell 15:79–80 Clarke SL, Betts GJ, Plant A, Wright KL, El-Shanawany TM, Harrop R, Torkington J, Rees BI, Williams GT, Gallimore AM, Godkin AJ (2006) CD4? CD25? FOXP3? regulatory T cells suppress
1851 anti-tumor immune responses in patients with colorectal cancer. PLoS One 1:e129 Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10:942–949 Frey DM, Droeser RA, Viehl CT, Zlobec I, Lugli A, Zingg U, Oertli D, Kettelhack C, Terracciano L, Tornillo L (2010) High frequency of tumor-infiltrating FOXP3(?) regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients. Int J Cancer 126:2635–2643 Galon J, Fridman WH, Pages F (2007) The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res 67:1883–1886 Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY (2007) Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol 25:2586–2593 Hinz S, Pagerols-Raluy L, Oberg HH, Ammerpohl O, Grussel S, Sipos B, Grutzmann R, Pilarsky C, Ungefroren H, Saeger HD, Kloppel G, Kabelitz D, Kalthoff H (2007) Foxp3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer. Cancer Res 67:8344–8350 Hiraoka N, Onozato K, Kosuge T, Hirohashi S (2006) Prevalence of FOXP3? regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions. Clin Cancer Res 12:5423–5434 Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061 Jordanova ES, Gorter A, Ayachi O, Prins F, Durrant LG, Kenter GG, van der Burg SH, Fleuren GJ (2008) Human leukocyte antigen class I, MHC class I chain-related molecule A, and CD8?/ regulatory T-cell ratio: which variable determines survival of cervical cancer patients? Clin Cancer Res 14:2028–2035 Kim M, Grimmig T, Grimm M, Lazariotou M, Meier E, Rosenwald A, Tsaur I, Blaheta R, Heemann U, Germer CT, Waaga-Gasser AM, Gasser M (2013) Expression of foxp3 in colorectal cancer but not in treg cells correlates with disease progression in patients with colorectal cancer. PLoS One 8:e53630 Ladoire S, Arnould L, Mignot G, Coudert B, Rebe C, Chalmin F, Vincent J, Bruchard M, Chauffert B, Martin F, Fumoleau P, Ghiringhelli F (2011a) Presence of Foxp3 expression in tumor cells predicts better survival in HER2-overexpressing breast cancer patients treated with neoadjuvant chemotherapy. Breast Cancer Res Treat 125:65–72 Ladoire S, Martin F, Ghiringhelli F (2011b) Prognostic role of FOXP3? regulatory T cells infiltrating human carcinomas: the paradox of colorectal cancer. Cancer Immunol Immunother 60:909–918 Li JF, Chu YW, Wang GM, Zhu TY, Rong RM, Hou J, Xu M (2009) The prognostic value of peritumoral regulatory T cells and its correlation with intratumoral cyclooxygenase-2 expression in clear cell renal cell carcinoma. BJU Int 103:399–405 Mitry E, Rachet B, Quinn MJ, Cooper N, Coleman MP (2008) Survival from cancer of the rectum in England and Wales up to 2001. Br J Cancer 99(1):S30–S32 Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo T, Robbins PD, Tahara H, Lotze MT (2003) Interleukin-17 promotes angiogenesis and tumor growth. Blood 101:2620–2627 Pages F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R, Mlecnik B, Kirilovsky A, Nilsson M, Damotte D, Meatchi T, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Galon J
123
1852 (2005) Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med 353:2654–2666 Perrone G, Ruffini PA, Catalano V, Spino C, Santini D, Muretto P, Spoto C, Zingaretti C, Sisti V, Alessandroni P, Giordani P, Cicetti A, D’Emidio S, Morini S, Ruzzo A, Magnani M, Tonini G, Rabitti C, Graziano F (2008) Intratumoural FOXP3-positive regulatory T cells are associated with adverse prognosis in radically resected gastric cancer. Eur J Cancer 44:1875–1882 Petersen RP, Campa MJ, Sperlazza J, Conlon D, Joshi MB, Harpole DJ, Patz EJ (2006) Tumor infiltrating Foxp3? regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 107:2866–2872 Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164 Sakaguchi S, Miyara M, Costantino CM, Hafler DA (2010) FOXP3? regulatory T cells in the human immune system. Nat Rev Immunol 10:490–500 Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, Platell C, Iacopetta B (2009) Tumor-infiltrating FOXP3? T regulatory
123
J Cancer Res Clin Oncol (2013) 139:1845–1852 cells show strong prognostic significance in colorectal cancer. J Clin Oncol 27:186–192 Tao H, Mimura Y, Aoe K, Kobayashi S, Yamamoto H, Matsuda E, Okabe K, Matsumoto T, Sugi K, Ueoka H (2012) Prognostic potential of FOXP3 expression in non-small cell lung cancer cells combined with tumor-infiltrating regulatory T cells. Lung Cancer 75:95–101 Wang L, Liu R, Li W, Chen C, Katoh H, Chen GY, McNally B, Lin L, Zhou P, Zuo T, Cooney KA, Liu Y, Zheng P (2009) Somatic single hits inactivate the X-linked tumor suppressor FOXP3 in the prostate. Cancer Cell 16:336–346 Yoon HH, Orrock JM, Foster NR, Sargent DJ, Smyrk TC, Sinicrope FA (2012) Prognostic impact of FoxP3? regulatory T cells in relation to CD8? T lymphocyte density in human colon carcinomas. PLoS One 7(8):e42274 Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W, Liu Y, Wang Y, Liu X, Chan MW, Liu JQ, Love R, Liu CG, Godfrey V, Shen R, Huang TH, Yang T, Park BK, Wang CY, Zheng P, Liu Y (2007) FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell 129:1275–1286
