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Intratumoral Balance of Regulatory and Cytotoxic T Cells Is Associated With Prognosis of Hepatocellular Carcinoma After Resection

Qiang Gao, Shuang-Jian Qiu, Jia Fan, Jian Zhou, Xiao-Ying Wang, Yong-Sheng Xiao, Yang Xu, Yi-Wei Li, Zhao-You Tang

From the Liver Cancer Institute, Zhong Shan Hospital and Shanghai Medical School, Fudan University, Key Laboratory for Carcinogenesis & Cancer Invasion, the Chinese Ministry of Education, Shanghai, People's Republic of China

Address reprint requests to Jia Fan, MD, PhD, Liver Cancer Institute, Fudan University, 136 Yi Xue Yuan Rd, Shanghai 200032, People's Republic of China; e-mail: jiafan99{at}yahoo.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Purpose To investigate the prognostic value of tumor-infiltrating lymphocytes (TILs), especially regulatory T cells (Tregs), in hepatocellular carcinoma (HCC) patients after resection.

Patients and Methods CD3⁺, CD4⁺, CD8⁺, Foxp3-positive, and granzyme B-positive TILs were assessed by immunohistochemistry in tissue microarrays containing HCC from 302 patients. Prognostic effects of low- or high-density TIL subsets were evaluated by Cox regression and Kaplan-Meier analysis using median values as cutoff.

Results CD3⁺, CD4⁺, CD8⁺ TILs were associated with neither overall survival (OS) nor disease-free survival (DFS). The presence of low intratumoral Tregs in combination with high intratumoral activated CD8⁺ cytotoxic cells (CTLs), a balance toward CTLs, was an independent prognostic factor for both improved DFS (P = .001) and OS (P < .0001). Five-year OS and DFS rates were only 24.1% and 19.8% for the group with intratumoral high Tregs and low activated CTLs, compared with 64.0% and 59.4% for the group with intratumoral low Tregs and high activated CTLs, respectively. Either intratumoral Tregs alone (P = .001) or intratumoral activated CTLs (P = .001) alone is also an independent predictor for OS. In addition, high Tregs density was associated with both absence of tumor encapsulation (P = .032) and presence of tumor vascular invasion (P = .031).

Conclusion Tregs are associated with HCC invasiveness, and intratumoral balance of regulatory and cytotoxic T cells is a promising independent predictor for recurrence and survival in HCC. A combination of depletion of Tregs and concomitant stimulation of effector T cells may be an effective immunotherapy to reduce recurrence and prolong survival after surgery.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
The overall survival of patients with hepatocellular carcinoma (HCC) remains poor despite improved diagnostic and treatment strategies, among which resection is one of the first priorities.¹ A high postoperative recurrence rate is a major obstacle, and thus many biomarkers for prediction and intervention, mainly HCC metastasis-related chromosomes/genes/proteins, have been tried.² Recently, it was reported that biologic behaviors of HCC are associated with a unique immune response signature of the liver microenvironment,³ and outcome is governed predominantly by immune responses at the primary tumor site,⁴ which suggest that precise evaluation of local immune responses could be useful for predicting prognosis.⁵

Tumor-infiltrating lymphocytes (TILs) are considered manifestations of host immune reactions against cancers. Patients with a prominent lymphocyte infiltrate, especially T lymphocytes, who underwent resection for HCC have reduced recurrence and better survival.⁶ However, tumor progression, which often also is seen in the presence of substantial lymphocytic infiltration, suggests that T cells are not capable of mounting effective immune responses to control tumor growth.⁷ Evidence has accumulated that TILs are functionally defective, incompletely activated, and include regulatory subtypes, which varied with the types of cancers.^8-11 Therefore, if distinctions were made with respect to lymphocyte types, location, functional state, and their interactions, a more profound impact on prognosis was observed compared with only the overall degree of lymphoid infiltration.⁴

CD4⁺CD25⁺ regulatory T cells (Tregs) are believed to dampen T-cell immunity and to be the main obstacle tempering immunotherapy.¹² Foxp3, a critical regulator of the development and function of Tregs, fulfills the criteria of a Treg-specific marker.^5,13,14 It was reported that high Foxp3 expression is associated not only with dismal prognosis in ovarian cancer but also represents an independent predictor for overall survival (OS) and progression-free survival.^5,15 However, discrepancies remain among different cancer types. No prognostic influence of tumor-infiltrating Tregs was found in anal squamous cell carcinoma¹⁴; in head and neck squamous cell carcinoma, Tregs correlated positively with locoregional control.¹⁶ Specifically, the balance between cytotoxic and regulatory T cells is believed to be of greater significance. The ratio of CD8⁺ CTLs and Tregs in ovarian cancer,⁵ as well as the concurrent low-Tregs density and high-CTLs density in Hodgkin's lymphoma,¹⁷ is reported to be more valuable than the single TILs subtypes.

However, the prognostic role of TILs, especially Tregs, in patients with HCC who underwent resection is less clear. In this study, the significance of various subtypes of TILs (including regulatory, cytotoxic, and their balance) and their relation to established markers of HCC prognosis such as vascular invasion¹⁸ were examined. We suggest that Tregs might have a role in the promotion of HCC invasiveness, and the intratumoral balance of Tregs and CTLs is a promising independent predictor for recurrence and survival.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Patients and Specimens
Archival specimens were obtained from 302 patients at Zhong Shan Hospital (Shanghai, People's Republic of China) between 1997 and 1999 after informed consent. All of the patients without distant metastasis or any prior anticancer treatment, underwent curative resection for HCC, defined as complete macroscopic removal of the tumor.¹⁹ Paraffin blocks were selected only on the basis of the availability of suitable formalin-fixed, paraffin-embedded tissue and complete clinicopathologic and follow-up data for the patients. The clinical typing of tumors was determined according to the TNM classification system of International Union Against Cancer (edition 6). The histologic grade of tumor differentiation was assigned by the Edmondson grading system. Liver function was assessed by Child-Pugh score system. Clinicopathologic characteristics are summarized in Appendix Table A1 (online only). Ethical approval was obtained from Zhong Shan Hospital research ethics committee.

Follow-Up and Postoperative Treatment
After surgery, patients with a high risk of recurrence, such as vascular invasion and spreading nodules, were treated with prophylactic transcatheter arterial chemoembolization (doxorubicin, cisplatin, fluorouracil, and iodized oil; one to three courses). Follow-up was completed March 15, 2006. The median follow-up was 58 months (range, 2 to 109 months). All patients were monitored prospectively by serum alpha-fetoprotein (AFP), abdomen ultrasonography, and chest x-ray every 1 to 6 months according to the postoperative time. For patients with test results suggestive of recurrence, computed tomography and/or magnetic resonance imaging were used to verify whether recurrence had occurred. A diagnosis of recurrence was based on typical imaging appearance in computed tomography and/or magnetic resonance imaging scan and an elevated AFP level. The treatment modality after relapse varied among individuals, and nine patients with recurrent disease and poor liver function received no treatment. Patients died as a result of recurrence, metastasis, or complicated liver cirrhosis. OS was defined as the interval between surgery and death or between surgery and the last observation for surviving patients. The data were censored at the last follow-up for living patients.

Tissue Microarray and Immunohistochemistry
Tissue microarrays were constructed as described previously.^4,14,17,20 All samples from HCC patients were reviewed histologically by hematoxylin and eosin staining, and representative areas with small round lymphocyte infiltrate were premarked in the paraffin blocks, away from necrotic and hemorrhagic materials. Duplicates of 1-mm-diameter cylinders from two different areas, tumor center and nearest noncancerous margin (designated as intratumor and peritumor, respectively; a total of four punches) were included in each case, along with different controls, to ensure reproducibility and homogenous staining of the slides (Shanghai Biochip Co Ltd, Shanghai, People's Republic of China). Thus, four different tissue microarray blocks were constructed, each containing 312 cylinders. Sections 4 µm thick were placed on slides coated with 3-aminopropyltriethoxysilane.

The mouse monoclonal antibodies used were antihuman CD3 (DAKO, Carpinteria, CA), CD4, CD8, granzyme B (Novocastra, Newcastle, United Kingdom), and Foxp3 (Biolegend, San Diego, CA). Immunohistochemistry of paraffin sections was carried out using a two-step protocol (Novolink Polymer Detection System, Novocastra) according to the manufacturer's instructions and as described previously.²¹ Briefly, paraffin sections were first deparaffinized and then hydrated. After microwave antigen retrieval, as required, endogenous peroxidase activity was blocked with incubation of the slides in 0.3% H₂O₂, and nonspecific binding sites were blocked with Protein Block (RE7102; Novocastra). After serial incubation with primary antibodies, Post Primary Block (RE7111; Novocastra), and secondary antibody (Novolink Polymer RE7112), the sections were developed in diaminobenzidine solution under a microscope and counterstained with hematoxylin. Negative control slides omitting the primary antibodies were included in all assays.

Evaluation of Immunohistochemical Variables
The number of TILs was counted using a computerized image analysis system composed of a Hitachi HV-C20A CCD camera (Hitachi, Tokyo, Japan), installed on a Leica DMLA light microscope (Leica Microsystems, Wetzlar, Germany) and attached to a personal computer. Under 400x magnification, there exists at least 12 independent and intact computerized microscopic fields for the duplicates of each patient sample. Five independent microscopic fields (400x), representing the densest lymphocytic infiltrates, were selected for each patient sample to ensure representativeness and homogeneity. The results were expressed as the mean (± SE) number cells for one computerized 400x microscopic field (0.0768 mm²/field). Granzyme B, constitutively expressed by natural-killer cells, is only expressed on activated CD8⁺ CTLs.^17,22 Granzyme B–positive cells with a sparsely granulated pattern were evaluated as activated CTLs, and densely positive natural-killer cells were excluded.^23,24

The evaluation of TILs was performed without knowledge of the clinicopathologic data by two independent investigators. Variations in the enumeration, within a range of 5%, were re-evaluated and a consensus decision was made.

Statistic Analysis
Statistical analyses were performed with SPSS 12.0 software (SPSS, Chicago, IL). Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. For all immunohistochemical markers, the cutoff for definition of subgroups was the median value.

A secondary analysis was performed to assess the relationship among lymphocytic variables and clinicopathologic characteristics. For the comparison of individual variables, ² tests, Fisher's exact tests, and Spearman coefficients tests were carried out as appropriate. Two-tailed P < .05 was judged to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Immunohistochemical Characteristics
By hematoxylin and eosin staining, immune cell infiltration was observed to be relatively homogenous within a tumor excluding necrotic, hemorrhagic, and fibrotic components. Lymphocytes infiltrated HCC tissue in a diffuse manner or in lymphoid aggregates, with more abundant cells in peritumoral areas, and the ratio of CD4⁺ and CD8⁺ cells varied substantially among samples. The numbers of most TIL subtypes correlated with each other (range of correlation coefficients, 0.21 to 0.76; P = 0.49 to < .0001 for significant correlations; Appendix Table A2, online only). The duplicate of spots for each tumor showed a good level of homogeneity for stained cell density. Granzyme B–positive lymphocytes display a granular cytoplasmic staining pattern, which reflects the granular localization of granzyme B. Foxp3-positive cells exhibited distinct nuclear staining, reflecting transcription function of Foxp3 (Fig 1). Representative images (Appendix Figs A1 and A2, online only) and statistics of immunohistochemical variables (Table 1) are shown.

View larger version (117K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Tumor-infiltrating Foxp3-positive and granzyme B–positive cells. Consecutive sections were used for immunohistochemical study on (A, C, E, and G) Foxp3-positive and (B, D, F, and H) granzyme B–positive cells: (A and B) both high; (C and D) both low; (E and F) high Foxp3-positive and low granzyme B–positive cells; (G and H) low Foxp3-positive and high granzyme B–positive cells. Positive lymphocytes were stained brown (400x magnification).

On univariate analysis, age, sex, hepatitis history, cirrhosis, AFP level, ALT level, Child-Pugh score, and tumor differentiation showed no prognostic significance for OS and DFS. For DFS, both TNM stage and vascular invasion were predictors. In addition to TNM stage and vascular invasion, tumor size, number, and encapsulation were also associated with OS.

No lymphocyte types infiltrating the peritumoral area showed any prognostic significance (Table 2). Intratumoral CD3⁺, CD4⁺, and CD8⁺ TILs were not associated with either OS or DFS (Table 2; Appendix Fig A3, online only). Activated CTLs (granzyme B positive), which were reported to be prognostic in various cancer types,^14,25,26 were only associated with improved OS (P = .026) and had no influence on DFS (P = .129; Figs 2C and 2D; Table 2). Tregs (Foxp3 positive) were found to be prognostic for both OS (P = .006) and DFS (P = .015; Figs 2A and 2B; Table 2). Patients with low intratumoral Tregs had longer OS (median, 70 months) and DFS (median, 69 months) than did those with high intratumoral Tregs (median, 51 and 34 months, respectively). Although the numbers of most TIL subtypes correlated with each other, only these two types showed prognostic value.

View larger version (26K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Kaplan-Meier analysis of disease-free survival and overall survival for (A and B) tumor-infiltrating regulatory T cells (Tregs), (C and D) activated CD8+ cytotoxic cells (CTLs), and (E and F) their balance. Low Tregs with concomitant high activated CTLs were associated with both prolonged survival and reduced recurrence.

Clinicopathologic features showing significance by univariate analysis were adopted as covariates when multivariate Cox proportional hazards analyses were performed. Vascular invasion, an established prognostic predictor for HCC,^18,19 remained associated with both DFS and OS. However, TNM stage was not an independent prognostic factor, which may result from patients’ heterogeneous distribution in TNM stage (I, n = 185; II, n = 52; III, n = 65), underlying cirrhosis,¹ and different treatment approaches (Tables 2 and 3; Appendix Table A3, online only) The presence of low intratumoral Tregs in combination with high intratumoral activated CTLs, with a balance toward CTLs, was an independent prognostic factor for both improved DFS (P = .001) and OS (P < .0001) (Figs 2E and 2F; Table 3). In another multivariate analysis, either intratumoral Tregs alone (P = .001) or intratumoral activated CTLs alone (P = .001) was an independent prognostic factor for OS, and patients with low intratumoral Tregs (P = .051) had a propensity of reduced recurrence (Appendix Table A3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Tregs have a crucial role in impeding immune surveillance against cancer and hampering the development of effective antitumor immunity.¹² It has been reported that the frequencies of Tregs are increased in both peripheral blood and the tumor microenvironment in HCC, and Tregs from HCC can suppress proliferation and perforin expression of autologous T cells in vitro.^11,27,28 After removal of the tumors, elevated levels of circulating Tregs subsided. Similar to other cancer types, TILs in HCC are also functionally defective and incompletely activated, in which Tregs are suggested to have an important role.¹¹ However, these studies include no prognostic information on Tregs, and additional study failed to provide any association between Tregs infiltration and recurrence in HCC patients who underwent transplantation.²⁹ In addition, Yang et al²⁸ recently reported that an inverse correlation exists between Tregs and CD8⁺ CTLs in the liver of HCC patients. In contrast, our data indicated a positive correlation between Tregs infiltration and CD8⁺ CTLs infiltration. Several aspects may contribute to this discrepancy. The first could be different sample sizes (25 v 302). Second, we used Foxp3-positive cells, a more specific marker than CD4⁺CD25⁺ cells, to assess tumor-infiltrating Tregs. Third, peritumoral regions contained part of the tumor in the study by Yang et al,²⁸ whereas the tumor center and noncancerous margin were investigated separately in our study. We found that lymphocytes in the noncancerous margin showed no influence on prognosis, which is different from their significant counterparts in tumor center. These differences attest to the value of TILs precise location. More importantly, even though the presence of intratumoral Tregs alone is an independent predictor for survival in HCC, our study demonstrates a relation of the balance of intratumoral Tregs and activated CTLs to both survival and recurrence. In patients with undesirable outcome, the balance is tipped in favor of Tregs (high Tregs and low activated CTLs), whereas in patients with relatively desirable outcome, the balance is tipped toward effector T cells (low Tregs and high activated CTLs). To our knowledge, this is the first report demonstrating prognostic values of tumor-infiltrating regulatory and cytotoxic T cells in human HCC.

These results are also in line with a series of recent reports. It is documented in mice sarcoma models that the ratio of regulatory to effector T cells, and not simply the presence or absence of Tregs, critically determined the in vivo growth behavior of the tumor.³⁰ Manipulating an increase in the intratumoral ratio of effector to regulatory T cells in poorly immunogenic mice melanoma can overcome Treg-mediated suppression and tip the balance toward tumor rejection.³¹ Therefore, a combination of depletion or attenuation of Tregs and concomitant stimulation of tumor-specific effector T cells, systemically or locally in tumors, may be a feasible immunotherapy for HCC; such as combination has been proved to be effective in some other cancer types.^32,33 Notably, even though a larger percentage of patients received prophylactic treatment in the high Tregs and low activated CTLs group than in other groups (IV, 70%; III, 64%; II, 52%; I, 50%; P = .071), their dismal outcome remained unchanged. These may indicate that prognosis is governed mainly by local immune responses.⁴ Thus, the development of strategies to tip the balance toward CTLs (encouraging the production of CTLs and depleting Tregs simultaneously) are urgently recommended.

Although tumor-infiltrating CD8⁺ T lymphocytes are believed to be front fighters against cancers, they have no prognostic role in many cancer types,^14,23 as proved in this study. Two major points are suggested to be responsible for the compromised tumor-specific killing by CD8⁺ CTLs: the dysfunction of cytolytic machineries and an increase in inhibitory molecules, in which both tumor and host play a part.^9,10 However, accumulating evidence has indicated that the activation state of CTLs, instead of just the existence of CTLs, were of great prognostic significance.^14,25,26 Our data showed that activated CTLs, unlike the total number of CD8⁺ CTLs, were positively correlated with survival, authenticating the importance of the functional state of TILs. Nevertheless, in patients with high levels of activated CTLs infiltration, prognosis was not indiscriminately favorable but was dichotomized by Tregs infiltration, indicating the significance of the modulating effector function in Tregs of primed CD8⁺ T cells and the key role of their balance. Although the widely used method of evaluating the distribution of TILs immunohistochemically was used, caution should be warranted in interpreting these results; the presence of specific T-cell subsets could also be influenced by other components in the tumor microenvironment. A prospective analysis on intratumoral expression profiles of groups of the immune-related genes using quantitative real-time reverse transcriptase polymerase chain reaction is being conducted to determine the local immune response in HCC more precisely.

We also found that high-density Tregs correlated positively with vascular invasion and correlated negatively with tumor encapsulation. Tumor encapsulation and vascular invasion are two relatively putative clinicopathologic markers of HCC invasiveness,^29,34 which were both found to be prognostic in this study. These results sustain the hypothesis that Tregs could modify HCC cells in ways that potentiate their invasiveness.

In conclusion, our results demonstrate that intratumoral Tregs are associated with HCC invasiveness and that the concurrence of intratumoral low Tregs with high activated CTLs, with a balance toward CTLs, is associated with improved survival and reduced recurrence in HCC after resection. These results may provide a novel independent predictor for prognosis, and suggest that the combination of depletion of Tregs and concomitant stimulation of effector T cells may be an effective strategy for HCC to reduce recurrence and prolong survival.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Conception and design: Qiang Gao, Shuang-Jian Qiu, Jia Fan, Jian Zhou, Xiao-Ying Wang, Yong-Sheng Xiao

Financial support: Shuang-Jian Qiu, Jia Fan, Zhao-You Tang

Administrative support: Shuang-Jian Qiu, Jia Fan, Jian Zhou, Zhao-You Tang

Provision of study materials or patients: Shuang-Jian Qiu, Jia Fan, Jian Zhou, Xiao-Ying Wang, Yong-Sheng Xiao, Yang Xu

Collection and assembly of data: Qiang Gao, Xiao-Ying Wang, Yong-Sheng Xiao, Yang Xu, Yi-Wei Li

Data analysis and interpretation: Qiang Gao, Shuang-Jian Qiu, Jia Fan, Jian Zhou, Xiao-Ying Wang, Yong-Sheng Xiao, Yang Xu, Yi-Wei Li

Manuscript writing: Qiang Gao, Shuang-Jian Qiu, Jia Fan

Final approval of manuscript: Qiang Gao, Shuang-Jian Qiu, Jia Fan

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 

View larger version (131K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A1. Peritumoral tumor-infiltrating lymphocytes (TILs) outnumbered intratumoral TILs. (A) A typical tissue disk. (B) Peritumoral lymphocytes form an aggregate. In a representative case, (C) in the peritumoral noncancerous area, lymphocytic infiltration was more abundant than that of (D) the intratumoral area. In each case, positive lymphocytes were stained brown (B, x200; C and D, x400).

View larger version (129K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A2. Representative pictures of tumor-infiltrating lymphocytes (TILs) in hepatocellular carcinoma. (A) CD3+ T cells were abundant along the tumor margin (tumor-host interface). Upper left, tumor; lower right, noncancerous cirrhotic liver; (B) intratumoral CD3+ TILs form a lymphoid aggregate; (C) intratumoral CD3+ TILs dispersed; (D) CD4+ TILs; (E) CD8+ TILs. In each case positive lymphocytes were stained brown (A and B, x200; C to E, x400).

View larger version (21K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A3. Prognostic significance of intratumoral CD3+, CD4+, and CD8+ lymphocytes in surgical hepatocellular carcinoma patients. Cumulative survival time were calculated by the Kaplan-Meier method and analyzed by the log-rank test. Intratumoral (A and B) CD3+, (C and D) CD4+, and (E and F) CD8+ lymphocytes were not associated with either disease-free survival or overall survival.

	ACKNOWLEDGMENTS

 
We thank Yuan Ji, MD, PhD, and Hai-Ying Zeng for their expertise in immunohistochemistry, and Shanghai Biochip Co Ltd for help in tissue microarray construction.

	NOTES

 
Supported by grants from the National Natural Science Foundation of China (Grant No. 30200268) and the Foundation of China National "211" Project for Higher Education.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted October 15, 2006; accepted February 1, 2007.

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