Elsevier

European Journal of Cancer

Volume 45, Issue 12, August 2009, Pages 2123-2131
European Journal of Cancer

Accumulation of regulatory T cells in sentinel lymph nodes is a prognostic predictor in patients with node-negative breast cancer

https://doi.org/10.1016/j.ejca.2009.03.024Get rights and content

Abstract

It has been revealed that sentinel lymph nodes (SLNs) from patients with node-negative breast cancer involve RT-PCR detected micrometastases and isolated tumour cells. However, the prognostic significance of the pathologically undetectable micrometastases is still controversial. In this study, we evaluated Foxp3 positive regulatory T cells (Treg) in SLNs as host-side immune marker that has the potential to detect these micrometastases. In the analyses of training set (n = 30), elevated Treg was strongly associated with the pathologically undetectable micrometastases. In the analyses of validation set (n = 129) in patients with node-negative, relapse-free survival in patients with elevated Treg was significantly shorter than those with lower Treg (p = 0.005). Furthermore, in multivariate analyses, elevated Treg was correlated with relapse-free survival (p = 0.012). Our data indicate that Treg may increase in the microenvironment of SLNs along with pathologically undetectable micrometastases and is a prognostic predictor in patients with node-negative breast cancer.

Introduction

Invasion into axillary lymph nodes remains the most important prognostic factor for breast cancer.1, 2, 3 For patients with node-negative breast cancer, however, appropriate systemic treatment must be determined by comprehensive assessment of other prognostic factors.4, 5 Although the evaluation of malignant potency of breast cancer has improved with the advent of DNA microarray cluster analyses, treatment decisions for patients with node-negative breast cancer remain difficult. The emergence of sentinel lymph node biopsy (SLNB) as a sensitive screening technique has revealed that the sentinel lymph nodes (SLNs) can harbour a number of micrometastases currently only detectable by reverse transcription polymerase chain reaction (RT-PCR).6, 7, 8, 9 Although SLN analyses using multiple new RT-PCR markers are being investigated, the correlation between metastases detected by RT-PCR and prognosis in patients with node-negative breast cancer is still controversial.10, 11 Because of the clinical significance of these micrometastases, a reliable method for detecting or predicting them is needed.

SLNs are the nodes nearest to a primary tumour on the direct lymphatic drainage pathway of the breast and are the typical site of earliest metastasis.6, 7 In a recent immunological study of SLNs and downstream lymph nodes (called non-SLNs) from breast cancer patients, SLN was identified as an important site for anti-tumour immunity.12, 13, 14, 15, 16 Furthermore, in an immune profile of SLN and non-SLN based on T cells and dendritic cells, immunological changes were observed in these lymph nodes prior to pathological invasion.12 Although the immune profile of SLNs can be used to detect pathologically undetectable micrometastases, the relationship between the immune profile of SLNs using T cells and dendritic cells and the prognosis for a breast cancer patient has not yet been reported. Therefore, a new immune profile marker that can reliably detect pathologically undetectable micrometastases is needed.

Regulatory T cells (Treg) have important roles in maintaining immunological self-tolerance through their ability to suppress wide-ranging immunological responses, including tumour immunity.17 A previous study reported that CD4(+)CD25(+)Treg accumulate in the main tumours of various cancers.18, 19, 20, 21 Since the identification of the forkhead box p3 (Foxp3) gene as the master regulator of Treg,22 the relationship between Foxp3(+)Treg and tumour progression has been clarified.23, 24, 25, 26 In breast cancer, the number of Foxp3(+)Treg in a main tumour has been correlated with bad prognosis27; thus, Foxp3(+)Treg are under investigation as a new therapeutic target.

We hypothesise that the immune profile of SLNs using Foxp3(+)Treg, due to its specificity for tumours, may be useful for detecting the pathologically undetectable micrometastases of node-negative breast cancer and could become an important prognostic factor for node-negative breast cancer patients. The aims of this study were to evaluate the relationship between the immune profile of SLNs based on Foxp3(+)Treg and pathologically undetectable micrometastases, and to assess this new SLNs profile as a prognostic predictor in patients with node-negative breast cancer.

Section snippets

Patients and tissue samples

It the early part of this study, we examined 30 SLNs samples from patients with various clinical stages. We evaluated these samples for analyses of real time RT-PCR, immunohistochemical estimation of SLNs and obtaining the cut off of immunohistochemistry. We called these samples ‘training set’ in this study. Thirty patients in the training set were all cases who underwent initial surgical resection and sentinel node biopsy at the Department of General Surgery, Chiba University Graduate School

Patient characteristics and sentinel node biopsy

The characteristics of patients in the training set and the validation set are shown in Table 1. SLNs could be detected in all patients in the training set and the validation set who were node-negative according to the radio-guided method. In the training set patients, the average number of resected SLNs was 1.9; all patients were stained by blue-dye. In the validation set patients, the average number of resected SLNs was 2.1, and 126 of 129 (97.7%) patients were stained by blue-dye. In the

Discussion

To the best of our knowledge, this is the first report showing that high accumulation of Foxp3(+)Treg cells in SLNs is an independent prognostic predictor in patients with node-negative breast cancer. Although the classification of breast cancer patients has improved with the advent of DNA microarray cluster analysis, invasion into axillary lymph nodes, which is related to metastatic potential, remains the most important prognostic predictor in breast cancer.1, 2, 3 In patients with

Conflict of interest statement

None declared.

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    R. Nakamura and M. Sakakibara contributed equally to this paper.

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