Mahmoud Sma

Paish Ec

Powe Dg

Macmillan Rd

Lee Ahs

Ellis Io

Green Ar

Andrew R Green
email: andrew.green@nottingham.ac.uk

An evaluation of the Clinical Significance of FOXP3 + Infiltrating Cells in Human Breast Cancer

Keywords: FOXP3, Tissue microarray, Immunohistochemistry, breast cancer, T regulatory cells

Introduction Studies in mice have that shown thymic-derived CD4+ CD25+ regulatory T cells (T reg; FOXP3 + lymphocytes) inhibit an antitumour immune response. Additional studies have also reported that the T reg population increases in peripheral blood and tumour tissues from patients with cancer. However, the relationship between the T reg population and patient prognosis remains controversial. Our aim was to determine the prognostic value of T reg cell density in breast cancer using immunohistochemical assessment of FOXP3, which has been shown to be the optimal marker for T regs.

Patients and methods Tissue microarrays were used, and the density of FOXP3 + cells was determined in a series of 1445 cases of well-characterised primary invasive breast carcinoma cases with long-term follow up.

Results FOXP3 + cell numbers were counted in tumour nests, in tumour-adjacent stroma, and in distant stroma. The total number of FOXP3 + cells significantly correlated with higher tumour grade (r s = 0.37, p< 0.001) and ER negativity (Mann-Whitney U test, p< 0.001). In addition, FOXP3 infiltration positively correlated with HER2 expression and basal phenotype subclass. On univariate analysis, FOXP3 + cells were associated with a worse prognosis (P= 0.012, log rank= 6.36). This association was found for intratumoural FOXP3 + and for tumour-adjacent stromal FOXP3 + -cells (tumour-cell associated FOXP3, p= 0.001 and log rank 10.35). However, the number of FOXP3 + cells was not found to be an independent prognostic factor in multivariate analysis.

Conclusion

We therefore conclude that FOXP3 + infiltrating cells do not have a dominant role in breast cancer prognosis and suggest that other inflammatory cell subsets may be more critical variables.

Introduction

Breast cancer is a heterogeneous disease with different known biological sub classes some of which are associated with immune cell infiltration [START_REF] Callagy | Molecular classification of breast carcinomas using tissue microarrays[END_REF]. The existing literature is not consistent on the importance of inflammatory cell infiltration in breast cancer development [START_REF] Stewart | Immunological enhancement of breast cancer[END_REF]. The presence of inflammatory cells may represent an immune response to the tumour, but could also potentially exhibit pro-tumour effects including stimulation of tumour invasion or angiogenesis. Some studies have reported that a dense lymphohistiocytic infiltrate is associated with a favourable prognosis [START_REF] Lee | Different patterns of inflammation and prognosis in invasive carcinoma of the breast[END_REF][START_REF] Rilke | Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors[END_REF], however, other reports have shown an association between prominent inflammation and a poor patient outcome [START_REF] Carlomagno | Prognostic significance of necrosis, elastosis, fibrosis and inflammatory cell reaction in operable breast cancer[END_REF]. A detailed understanding of the functional peculiarities and phenotype of individual T lymphocyte subtypes may explain this paradox [START_REF] Yu | Tumor-infiltrating T lymphocytes: friends or foes?[END_REF].

An important mechanism of defence against autoimmunity is the central tolerance which involves deletion of self-reactive T lymphocytes in the thymus at an early stage of development [START_REF] Sprent | Knowing one's self: central tolerance revisited[END_REF]. However, autoreactive T cells that migrate into the tumour periphery are controlled by a number of peripheral mechanisms including the induction of regulatory/suppressor T cells [START_REF] Curiel | Tregs and rethinking cancer immunotherapy[END_REF]. CD4 + T regulatory (T reg) cells include at least four populations that differ in phenotype, cytokine secretion profile, and suppressive mechanisms [START_REF] Buckner | Regulating the immune system: the induction of regulatory T cells in the periphery[END_REF][START_REF] Kosmaczewska | The significance of Treg cells in defective tumor immunity[END_REF]. One prevailing phenotype is the naturally occurring T reg cells that originate in the thymus and are involved in protection from autoimmune responses. These natural T reg cells have the ability to suppress the activation of conventional T cells in a cell-contact-dependent, IL-10-independent and TGF-β-independent manner [START_REF] Buckner | Regulating the immune system: the induction of regulatory T cells in the periphery[END_REF][START_REF] Ramsdell | Foxp3 and natural regulatory T cells: key to a cell lineage?[END_REF].

Naturally occurring CD4 + T reg cells are characterised by the expression of the IL-2 receptor α-chain (CD25) and constitute 5-10% of peripheral CD4 + in healthy individuals [START_REF] Dieckmann | Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood[END_REF]. These cells naturally arise in the thymus and after differentiation, the naïve CD4 + CD25 + CD45RA + T cells are exported to the periphery where they suppress the activation of T cells potentially reactive to the self [START_REF] Kosmaczewska | The significance of Treg cells in defective tumor immunity[END_REF]. In addition to CD4 and CD25 markers, T reg also constitutively express CD45RO and CD152 (CTLA-4) [START_REF] Liyanage | Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma[END_REF]. Ex vivo studies on these cells revealed a poorly proliferative cell population that secretes inhibitory cytokines such as TGF-β and IL-10 [START_REF] Dieckmann | Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood[END_REF]. They are implicated in many autoimmune diseases such as multiple sclerosis, diabetes, thyroiditis, and inflammatory bowel diseases. Besides their role in autoimmunity, Treg cells participate in transplantation tolerance and tumour immunity [START_REF] Sakaguchi | Regulatory T cells: key controllers of immunologic selftolerance[END_REF].

Although the expression of CD25 on T cells has been described as a marker of T reg cells, its expression is not restricted to T reg cell function as it is also expressed by activated non-regulatory effector lymphocytes [START_REF] Fontenot | Regulatory T cell lineage specification by the forkhead transcription factor foxp3[END_REF]. Previous studies revealed that the most specific marker to identify T reg cells is FOXP3, a me mber of the forkhead or winged helix family of transcription factors encoded on the X chromosome [START_REF] Hori | Control of regulatory T cell development by the transcription factor Foxp3[END_REF][START_REF] Roncador | Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level[END_REF].

The nuclear expression of FOXP3 protein is highly restricted to discrete T reg cell populations and it is therefore considered to be a specific marker to detect T reg cells in vivo [START_REF] Roncador | Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level[END_REF].

Studies in mice showed that T reg inhibit the antitumour response [START_REF] Onizuka | Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody[END_REF][START_REF] Shimizu | Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity[END_REF] and that their depletion can result in effective antitumour immune response [START_REF] Sutmuller | Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses[END_REF][START_REF] Tanaka | Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes[END_REF], implicating these cells in immune evasion by cancer cells. Additional studies have reported that the T reg population increases in peripheral blood and tumour tissues in patients with several types of human cancer including breast cancer [START_REF] Liyanage | Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma[END_REF][START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF][START_REF] Curiel | Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[END_REF][START_REF] Wolf | Increase of regulatory T cells in the peripheral blood of cancer patients[END_REF]. A high density of tumour-infiltrating FOXP3 + cells has been associated with poor outcome in various solid tumours including ovarian [START_REF] Curiel | Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[END_REF], pancreatic [START_REF] Hiraoka | Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions[END_REF], lung [START_REF] Petersen | Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients[END_REF], and hepatocellular carcinoma [START_REF] Gao | Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection[END_REF]. In contrast, improved patient survival has been shown to be associated with a high density of tumour infiltrating FOXP3 + T regs in oesophageal squamous cell carcinoma [START_REF] Yoshioka | Infiltrating regulatory T cell numbers is not a factor to predict patient's survival in oesophageal squamous cell carcinoma[END_REF] and colorectal cancer [START_REF] Salama | Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer[END_REF].

A few studies have recently examined the role of FOXP3 + cells in breast cancer and have utilised relatively small patient cohorts [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF][START_REF] Ladoire | Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating foxp3+ regulatory T cells[END_REF]. In the largest study, Bates et al [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF] studied 222 invasive breast carcinomas and focused on the prognostic value of FOXP3 + cells in ER-positive tumours, while Ladoire et al [START_REF] Ladoire | Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating foxp3+ regulatory T cells[END_REF] concentrated on the pathologic response to neoadjuvant chemotherapy. There is still limited information delineating the mechanisms behind T reg accumulation within the breast cancer microenvironment and their expansion locoregionally. Thus, it is important to evaluate the localisation of infiltrating T regs in relation to clinical outcome. The aim of this study was therefore to determine the prognostic value of FOXP3 + cell density and localisation in a large well-characterised series of invasive breast cancer with long follow-up. Furthermore, we have correlated the number of FOXP3 + cells infiltrating breast carcinomas with other recognised prognostic factors.

Patients and methods

Study population

This retrospective study is based on a consecutive unselected series of 1902 patients from the Nottingham Tenovus Primary Breast Carcinoma series who presented during the period 1987 to 1998. This is a well-characterised series of primary operable invasive breast carcinomas patients less than 71 years of age, treated in a single institution, which has been previously investigated for a wide range of prognostic factors [START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] El-Rehim | Expression of luminal and basal cytokeratins in human breast carcinoma[END_REF][START_REF] Rakha | Basal phenotype identifies a poor prognostic subgroup of breast cancer of clinical importance[END_REF][START_REF] Zhang | The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer[END_REF]. Data on a range of patient, pathology and biomarkers were available.

These include histologic grade [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF], histologic tumour type [START_REF] Ellis | Pathological prognostic factors in breast cancer. II. Histological type. Relationship with survival in a large study with long-term follow-up[END_REF], vascular invasion (VI) [START_REF] Pinder | Pathological prognostic factors in breast cancer. III. Vascular invasion: relationship with recurrence and survival in a large study with long-term follow-up[END_REF], primary tumour size, and lymph node (LN) stage, and Nottingham prognostic index (NPI) [START_REF] Galea | The Nottingham Prognostic Index in primary breast cancer[END_REF] which were routinely assessed and recorded in the database as well as patients' information such as age, family history, and menopausal status.

All patients received standard surgical treatment of either mastectomy or wide local excision with radiotherapy. Prior to 1988, patients did not routinely receive systemic adjuvant treatment. From 1988, patients were offered systemic adjuvant treatment based on NPI score and hormone receptor status [START_REF] Green | The prognostic significance of steroid receptor co-regulators in breast cancer: co-repressor NCOR2/SMRT is an independent indicator of poor outcome[END_REF]. Patients with an NPI score ≤ 3.4 received no adjuvant therapy, those with a NPI score >3.4 received Tamoxifen if oestrogen receptor (ER) positive (± Zoladex if pre-menopausal) or classical cyclophosphamide, methotrexate and 5-fluorouracil if ER negative and fit enough to tolerate chemotherapy. Information on local, regional and distant recurrence and survival was maintained on a prospective basis. Patients were followed up at 3-month intervals initially, then every 6 months, then annually for a median follow-up period of 128 months (range 4-243). Breast cancer specific survival (BCSS) was defined as the time (in months) from the date of the primary surgical treatment to the time of death from breast cancer. Patients who died from other causes or were still alive were censored at the time of last follow-up. Disease-free interval (DFI) was calculated from the date of first operation until the first recurrence (local, regional, or distant). The number of deaths due to breast cancer was 398 while the number of relapses was 581.

The clinicopathological characteristics of the series are presented in Table 1.

Reporting Recommendations for Tumour Marker Prognostic Studies ( REMARK) criteria were followed throughout, as recommended by McShane et al [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies[END_REF].

This study was approved by Nottingham Research Ethics Committee 2 under the title of "Development of a molecular genetic classification of breast cancer".

Tissue microarrays and immunohistochemistry

Breast cancer tissue microarrays (TMA) and whole breast cancer tissue sections were prepared and immunohistochemistry performed employing the standard streptavidinbiotin complex method as previously described [START_REF] Rakha | Expression of BRCA1 protein in breast cancer and its prognostic significance[END_REF]. In short, tissue sections were deparaffinised with xylene then rehydrated through 3 changes of alcohol. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 10 minutes. Antigen retrieval was performed by microwave treatment in 0.01M TRIS-EDTA buffer (pH 8) for 20 min. Slides were incubated for 1 hour with monoclonal mouse anti-human FOXP3 (clone 236A/E7, Abcam, Cambridge, UK, 1:100 dilution). After washing with Tris-buffered saline (TBS, pH 7.6), sections were incubated with biotinylated secondary antibody (1:100 dilution) for 30 minutes, followed by streptavidin coupled to biotinylated horse radish peroxidase (1:100 dilution) for a further 55 minutes using streptABC kit (streptABComplex/HRP Duet, biotinylated goat antimouse/rabbit immunoglobulin kit, Dako Corporation, Denmark, K0492). Antigen-antibody reaction was visualised with 3, 3' Diaminobenzidine tetrahydrochloride (Dako, K3468). After counterstaining with Mayer's haematoxylin, sections were dehydrated through ascending alcohols to xylene and mounted. Positive staining controls were performed in parallel with paraffin sections of normal human tonsil. Negative control was performed by omitting the primary antibody.

To assess the topographical distribution and heterogeneity of FOXP3 + cell distribution, immunoreactivity was also determined in 30 cases (included in the TMA) using full-face tissue sections.

Quantification of FOXP3 + cells

The number of FOXP3 + cells was counted in each tumour core (0.6 mm diameter) using an eyepiece graticule. The scorer was blinded to the patients' clinical information (S.M.). Scores were audited by a second assessor (I.O.E) and a very good correlation was found between the two investigators' scores (κ value = 0.9). FOXP3 + cells were counted in three compartments in each tumour; intratumoural compartment (within the tumour cell nests); within distant stroma not touching tumour cells (defined as the distance between positive FOXP3 cell and tumour nest is more than the size of one tumour cell); and within adjacent stroma and touching tumour cells (the distance between positive FOXP3 cell and tumour nest is equal or less than the size of one tumour cell) (Fig. 1 C andD). The total number of FOXP3 + cells was determined by adding the counts for the three compartments.

Statistical analyses

Analysis of the correlation between the number of FOXP3 + cells and clinicopathological characteristics was conducted using Spearman rank order correlation or Mann-Whitney U test where appropriate. Concordance between scores from both observers was obtained using Kappa (κ) measure of agreement test. All tests were 2-tailed. X-tile software program (version 3.4.7, Robert L Camp, 2005) was utilised in order to define the single optimal cut-off point for FOXP3 + cell count with corrected pvalue and relative risk against BCSS in each tumour compartment [START_REF] Camp | X-tile: a new bioinformatics tool for biomarker assessment and outcome-based cut-point optimization[END_REF]. X-tile software provides a method to determine the optimal divisions of a continuous population and then does statistical testing using training set/validation set method for p-value estimation when no foreknowledge about how a marker parses a population into subsets. In addition, the software can perform standard Monte Carlo crossvalidation to produce corrected p-values to assess statistical significance of data assessed by multiple cut-points [START_REF] Camp | X-tile: a new bioinformatics tool for biomarker assessment and outcome-based cut-point optimization[END_REF][START_REF] Dolled-Filhart | Quantitative in situ analysis of beta-catenin expression in breast cancer shows decreased expression is associated with poor outcome[END_REF].

Survival rates were determined using Kaplan-Meier method and compared by the logrank test. Multivariate analysis using Cox proportional hazards regression model determined the influence of T regs, when adjusted to other variables, on BCSS. All analyses were conducted using Statistical Package for the Social Sciences (SPSS Inc, version 15, Chicago, IL, USA) software for windows. P-value < 0.05 was identified as statistically significant.

Cut-off values for ERα, PgR, and HER2 included in this study were chosen before statistical analyses as previously described for this patients' series [START_REF] El-Rehim | Expression of luminal and basal cytokeratins in human breast carcinoma[END_REF][START_REF] Rakha | Prognostic markers in triple-negative breast cancer[END_REF]. Basal phenotype positivity was defined as detection of expression in 10 % or more of invasive malignant cells for CK5/6 and/or CK14 [START_REF] Rakha | Basal phenotype identifies a poor prognostic subgroup of breast cancer of clinical importance[END_REF].

Results

The frequency and localisation pattern of FOXP3 + cells in breast cancer

The evaluation of full size tissue sections demonstrated random distribution of the FOXP3-positive cells indicating suitability for use of TMAs. In addition, both full sections and arrays showed similarity in intensity of staining and area of positive staining. FOXP3-positive cells exhibited distinct nuclear staining, consistent with the expected transcription function role of FOXP3. In the tonsil positive control section, FOXP3 + cells were distributed mainly in interfollicular (T-cell) areas, although occasionally low numbers of FOXP3 + cells were found within follicles (Fig. 1 A andB). None of the carcinoma cells or mesenchymal cells exhibited staining.

A total of 1445 tumours were examined after exclusion of the uninformative cores.

FOXP3 + cells were distributed among the three defined tumour compartments with higher numbers of positive cells in the stroma. Figure 1 shows representative photomicrographs of immunohistochemical staining of Treg using FOXP3 antibody. There were more FOXP3 + cells in the distant stroma compared to FOXP3 + cells within the tumour and adjacent stroma (Table 2).

Correlation with other prognostic factors

There was a moderate positive correlation between the total number of FOXP3 + cells and tumour grade (Table 1). Furthermore, there was a significant correlation between total number of FOXP3 + cells and both ER and PgR negativity (Mann-Whitney U test). On the other hand, total number of FOXP3 + cells was positively correlated with HER2 expression and basal phenotype subclasses. FOXP3 expression was weakly inversely correlated with patient age (Table 1).

Survival analysis

Using X-tile software, the optimal cutpoints identified for positivity and negativity for the total number of FOXP3 + cells and distant stromal FOXP3 + cells was ≥ 3 positive cells and for intratumoural and adjacent stromal FOXP3 + cells was ≥ 1 positive cells.

In univariate analysis, patients with positive FOXP3 expression, irrespective of locality, had a significantly shorter BCSS than those tumours negative for FOXP3 + cells (P= 0.012, log rank= 6.36, Figure 2A), with hazard ratio of 1.32 (95% CI, 1.06 to 1.63). This effect on survival was most evident during the first 180 months of follow-up. Similarly, an association between intratumoural FOXP3 cell number positivity and shorter BCSS was observed (P= 0.037, log rank= 4.34, Figure 2B), with hazard ratio of 1.26 (95% CI, 1.01 to 1.56). Again, the effect on survival was most apparent before 180 months of follow-up. In addition, adjacent stromal FOXP3 cell number positivity was associated with shorter BCSS (P= 0.001, log rank= 10.60, Figure 2D), with hazard ratio of 1.39 (95% CI, 1.14 to 1.70). However, there was no significant difference in survival between patients with positive distant stromal FOXP3 cell numbers and those with negative distant stromal FOXP3 cell numbers (P= 0.235, log rank= 1.41, Figure 2C).

In multivariate Cox regression model including well-recognised parameters related to patient outcome (histological grade, tumour size, LN stage, and vascular invasion), total FOXP3 cell number did not retain significance (Table 3). Likewise, neither intratumoural FOXP3 nor adjacent stromal FOXP3 cell numbers kept significance in the same multivariate model (HR = 0.97, 95% CI = 0.78-1.22, p= 0.81; HR = 1.10., 95% CI = 0.90-1.36, p= 0.35, respectively).

With regard to DFI, total FOXP3 + cell number positivity was associated with decline in DFI however that did not reach a significant level (P= 0.068, log rank= 3.33).

Neither intratumoural nor distant stromal FOXP3 + cell numbers had a significant prognostic effect (P= 0.06, log rank= 3.61; P= 0.58, log rank= 0.31 respectively).

Conversely, patients who were positive for adjacent stromal FOXP3 + cell numbers had a significantly reduced DFI (P= 0.013, log rank= 6.23).

Relationship between tumour-cell associated FOXP3 and patient outcome

To further assess the role of FOXP3 + cell localisation in this patient group, we combined intratumoural FOXP3 + cells and adjacent stromal FOXP3 + cells as an inclusive tumour cell associated FOXP3 + populations. We defined, using X-tile software, the cut-off value for the new variable as ≥ 1 and categorised patients based on this cut-off point into tumour-cell associated FOXP3 + cell number positive and negative patients. Tumour-cell associated FOXP3 + cell number positivity was accompanied with significantly shorter BCSS (P= 0.001, log rank= 10.35, Figure 3), with hazard ratio of 1.38 (95% CI, 1.13 to 1.68). In multivariate analysis, after adjustment for histological grade, tumour size, LN stage, and vascular invasion, tumour-cell associated FOXP3 + cell numbers lost significance and was not therefore an independent prognostic factor for patient survival (HR = 1.063, 95% CI = 0.865-1.306, p= 0.562). Of note, after exclusion of tumour grade from the multivariate model, tumour-cell associated FOXP3 expression was an independent predictor of patient survival (HR = 1.27, 95% CI = 1.04-1.55, p= 0.019). In the same way, patients with tumours positive for tumour-cell associated FOXP3 + cells had a significantly shorter DFI (P= 0.003, log rank= 9.01). Nevertheless, that was not significant in multivariate analysis (HR = 1.13, 95% CI = 0.95-1.34, p= 0.17).

Survival in estrogen receptor-positive patients

Bates et al [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF] found that FOXP3 + cells were an independent prognostic factor in patients with ER-positive tumours, but not in patients with ER-negative tumours. We therefore investigated the prognostic significance of T regs in both ER-positive andnegative tumours. Within the ER-positive group total FOXP3 + cell number positivity was significantly associated with shorter BCSS on univariate analysis (P= 0.012, log rank = 6.32, Figure 4A), with hazard ratio of 1.39 (95% CI, 1.07 to 1.81), but was not independent of other prognostic factors in multivariate analysis. Also, tumour-cell associated FOXP3 + cell number positivity was significantly associated with worse outcome in ER-positive patients (P= 0.004, log rank = 8.48, Figure 4B), with hazard ratio of 1.45 (95% CI, 1.13 to 1.86), however, was no longer significant in multivariate analysis (data not shown). In contrast, FOXP3 + cell numbers had no significant influence on BCSS within the ER-negative group on univariate analysis (P= 0.79, log rank = 0.07, for total FOXP3 and P= 0.88, log rank = 0.02, for tumourcell associated FOXP3, Figure 4 C and D, respectively).

Prognostic value of FOXP3 + cells in different adjuvant therapy groups

The patients were categorised according to the systemic adjuvant treatment received.

The numbers of FOXP3 + cells were not of prognostic significance in either the group that received chemotherapy (n=223) or the group that received endocrine therapy (n= 441) on univariate analysis. FOXP3+ cell number positivity was associated with a worse prognosis in univariate analysis of the group no t receiving systemic adjuvant treatment (n=491), but not in multivariate analysis. There were too few patients receiving both treatments for analysis. No patients received trastuzumab.

Discussion

Naturally occurring T regulatory cells (T regs) have functionally suppressive actions on other effector T cells and have been characterised as a CD4 + CD25 + population among CD4 + T lymphocytes [START_REF] Dieckmann | Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood[END_REF][START_REF] Sakaguchi | Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases[END_REF]. Consequently, an inhibitory effect of T regs on anti-tumour immunity has been proposed implicating them in impeding immunosurveillance against cancer [START_REF] Zou | Regulatory T cells, tumour immunity and immunotherapy[END_REF]. In agreement with this hypothesis, higher numbers of tumour-infiltrating T reg were shown to be associated with poor outcome in pancreatic [START_REF] Hiraoka | Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions[END_REF] and hepatocellular [START_REF] Gao | Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection[END_REF] carcinomas. In follicular lymphoma, head and neck, and ovarian cancer the number of infiltrating FOXP3 + immune cells has shown to be an independent prognostic factor [START_REF] Badoual | Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers[END_REF][START_REF] Carreras | High numbers of tumor-infiltrating FOXP3-positive regulatory T cells are associated with improved overall survival in follic ular lymphoma[END_REF][START_REF] Wolf | The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer[END_REF]. However, studies of other tumours have led to conflicting results. No prognostic effect of T reg was observed in a study of anal carcinomas [START_REF] Grabenbauer | Tumorinfiltrating cytotoxic T cells but not regulatory T cells predict outcome in anal squamous cell carcinoma[END_REF] whereas in colorectal and oesophageal carcinomas, tumour-infiltrating T reg were associated with favourable prognosis [START_REF] Yoshioka | Infiltrating regulatory T cell numbers is not a factor to predict patient's survival in oesophageal squamous cell carcinoma[END_REF][START_REF] Salama | Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer[END_REF]. Limited data are available about T reg in breast cancer [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF][START_REF] Ladoire | Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating foxp3+ regulatory T cells[END_REF][START_REF] Bohling | Immunosuppressive regulatory T cells are associated with aggressive breast cancer phenotypes: a potential therapeutic target[END_REF][START_REF] Ghebeh | FOXP3+ Tregs and B7-H1+/PD-1+ T lymphocytes coinfiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy[END_REF]. Furthermore, no data has been previously published on the localisation patterns of FOXP3 + cells in breast cancer. The present study is, to the best of our knowledge, the first report describing the density and localisation of FOXP3+ infiltrating cells in relation to breast prognosis in a large series of patients.

It has been recently reported that FOXP3 is the most reliable marker of T regs [START_REF] Fontenot | Regulatory T cell lineage specification by the forkhead transcription factor foxp3[END_REF]. Moreover, It was confirmed that the 236A/E7 antibody clone specifically recognises FOXP3 and not other FOXP proteins [START_REF] Roncador | Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level[END_REF]. In fact, this clone has been shown the best currently available to study FOXP3 expression by immunohistochemistry [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF]. More importantly, although the reactivity spectrum of FOXP3 is composed of T reg and may include small number of CD8 + cells, it has been recently shown that it is the most accurate single marker of T regs [START_REF] Ahmadzadeh | FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions[END_REF]. In this study, we observed FOXP3 + cell infiltration in most of the tumours. Recent studies have reported the expression of FOXP3 mRNA and protein in a limited number of pancreatic carcinoma cells, cell lines [START_REF] Hinz | Foxp3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer[END_REF], and other tumour cell lines of several origins, including breast cancer [START_REF] Karanikas | Foxp3 expression in human cancer cells[END_REF].

In contrast to these studies we did not detect any immunoreactive carcinoma cells in our series. Our data showed significant correlations between T reg infiltration and high tumour grade and ER negativity, consistent with previous reports in breast cancer [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF][START_REF] Ghebeh | FOXP3+ Tregs and B7-H1+/PD-1+ T lymphocytes coinfiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy[END_REF]. In accord with previously published data in breast cancer, we have demonstrated an association between elevated numbers of FOXP3 + infiltrating cells and HER2 positivity. Furthermore, we found a similar relationship in basal phenotype tumours. The association between HER-2 and FOXP3 + cell infiltration can probably be explained by the association of both with histological grade. Eighty-five percent of HER-2 positive tumours were grade 3 and high FOXP3 + cell counts were also associated with higher grade. There is no correlation between HER-2 expression and FOXP3 + cell counts in grade 3 carcinomas (data not shown). By contrast although 73% of basal carcinomas were grade 3, FOXP3 + cell counts in grade 3 carcinomas were higher in basal tumours. This suggests that factors other than grade are important in the correlation between basal types and FOXP3 + cell counts.

We found that higher numbers of FOXP3+ infiltrating cells were associated with a worse prognosis on univariate analysis. This association was found for intratumoural FOXP3 + cells and for adjacent stromal FOXP3 + cells. However, the number of FOXP3 + cells was not identified as an independent prognostic factor in multivariate analysis. This is probably because of the correlation with histological grade, which is a well recognised and powerful prognostic factor in breast cancer. In the only previous prognostic study, Bates et al [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF], in a much smaller series (222 pateints), found that FOXP3 + cell infiltration was associated with a shorter relapse free survival (p= 0.04) independent of other prognostic factors, but this was not seen for overall survival (p= 0.07).

Bates et al [START_REF] Bates | Quantification of regulatory T cells enables the identification of highrisk breast cancer patients and those at risk of late relapse[END_REF] found that FOXP3+ infiltrating cells were an independent prognostic factor in patients with ER-positive tumours, but not in patients with ER-negative tumours. We found no such distinction, FOXP3 + cells were not identified as an independent prognostic factor in either group in our series. The number of events (deaths/relapses) in the ER-positive group was small in the Bates study and their results must be interpreted with caution.

In summary, in the present large study we found that FOXP3+ infiltrating cells in human breast cancer are associated with histological grade, but are not an independent prognostic factor. There are numerous other infiltrating inflammatory cell types observed in breast cancer and the association between T regs and other cells is not clear. Our data suggest that predicting clinical outcome is not possible using T regs alone. We are currently investigating other cell types.

Figure 1 :

 1 Figure 1

Figure 2 :

 2 Figure 2: Kaplan-Meier curves for breast cancer specific survival (BCSS) stratified by the predefined cut-off points (for the whole series). (A) Survival curve for total FOXP3. (B) Survival curve for intratumoural FOXP3. (C) Survival curve for distant stromal FOXP3. (D) Survival curve for adjacent stromal FOXP3.

Figure 3 :

 3 Figure 3: Kaplan-Meier curve showing significant association between positive tumour-cell associated FOXP3 expression and shorter breast cancer specific survival (BCSS). Tumour-cell associated FOXP3 consists of the sum of intratumoural FOXP3 + cells and adjacent stromal FOXP3 + cells.

Figure 4 :

 4 Figure 4: Kaplan-Meier curves for breast cancer specific survival (BCSS) of FOXP3 + cell expression in ER-positive tumours (A and B) and ER-negative tumours (C and D). (A) Kaplan-Meier blot shows the significance of total number of FOXP3 + cell expression in ER-positive patient outcome. (B) Kaplan-Meier blot depicts the significance of tumour-cell associated FOXP3 + cell expression in ER-positive patient outcome. Neither total number nor tumour-cell associated FOXP3 + cell expression had an impact on BCSS in ER-negative group (C and D respectively).

Table 1 :

 1 Correlation between presence of FOXP3 + cells and clinicopathological and some immunohistochemical markers in invasive breast cancer.

	Pathological/clinical parameter	FOXP3 n (%) positive	FOXP3 n (%) negative	Significance
	Age, years
	<40	81 (72)	31 (28)
	40-50	283 (68)	134 (32)	r s = -0.11
	51-60	292 (61)	188 (39)	p <0.001
	>60	268 (62)	167 (38)
	Tumour size ≤2cm >2cm	563 (62) 355 (67)	341 (38) 177 (33)	r s = 0.08 p =0.002
	Nodal status 1 (negative) 2 (1-3 LN)	562 (63) 268 (64)	333 (37) 151 (36)	r s = 0.06 p =0.015
	3 (>3 LN)	90 (73)	34 (27)
	Grade
	1 2 3	115 (47) 241 (51) 562 (79)	131 (53) 233 (49) 154 (22)	r s = 0.37 p <0.001
	Vascular invasion
	Absent Present	612 (63) 300 (67)	366 (37) 148 (33)	P =0.106 *
	Distant metastases
	Absent Present	617 (62) 298 (67)	372 (38) 145 (33)	P =0.121 *
	Any recurrence
	Absent Present	387 (67) 514 (62)	195 (33) 311 (38)	P =0.516 *
	ER alpha status
	Negative Positive	573 (58) 297 (82)	409 (42) 67 (18)	P <0.001 *
	PgR status
	Negative Positive	401 (74) 461 (58)	138 (26) 333 (42)	P <0.001 *
	HER 2 status
	Negative Positive	731 (61) 151 (86)	473 (39) 24 (14)	P <0.001 *
	Basal phenotype
	Negative Positive	648 (61) 221 (78)	412 (39) 63 (22)	P <0.001

*

Significant p-values are in bold; * Mann-Whitney U test.

FOXP3 positive is ≥ 3 cells/core.

Table 2 :

 2 Expression of FOXP3 + cells in the three compartments per tumour core (0.28 mm 2 s.a.) in breast cancer.

	Location	Mean	Median	Interquartile range
	Total No. of FOXP3+ cells *	12.7	6	1 -17
	Intratumoural FOXP3+ cells *	2.3	0	0 -1
	Distant stromal FOXP3+ cells *	8.8	3	0 -11
	Adjacent stromal FOXP3+ cells *	1.6	0	0 -1

* Number of patients= 1445.

Acknowledgement

Sahar M. A. Mahmoud would like to thank the Egyptian Ministry of Higher Education for funding her during this work.


Julien Vinit
email: julien.vinit@chu-dijon.fr

Géraldine Muller
email: geraldine.muller@dijon.fr

Pierre Pfitzenmeyer
email: pierre.pfitzenmyer@chu-dijon.fr

Philippe Bonniaud
email: philippe.bonniaud@chu-dijon.fr

Bernard Lorcerie
email: bernard.lorcerie@chu-dijon.fr

Jean-François Besancenot
email: jean-francois.besancenot@chu-dijon.fr

M D Pr

Churg-Strauss syndrome: retrospective study in Burgundian population in France in past ten years

Keywords: Vasculitis, Churg-Strauss Syndrome, Epidemiology, Prevalence, Incidence

Churg-Strauss Syndrome (SCS) is a systemic vasculitis associated with asthma and eosinophilia. The aim of our work is to describe this pathology in the Burgundian population in France.

Methods:

We counted from the hospitalization data processing summaries, the whole of the SCS hospitalized in Burgundy between 1998 and 2008. During the follow-up, the clinical and paraclinical characteristics of every patient were collected.

Results:

The average prevalence is of 11.3 per million inhabitants and the incidence is of 1.2 new cases per million inhabitants and per annum. There exists however, a great prevalence disparity and incidence amongst the various departments of the area. The patient's average follow-up is of 7.7 years. In 23% of the cases one finds a starting factor for vasculitis. The delay between the first signs and the diagnostic is of an average of 61 months. The ANCA are positive in 26% of cases and of anti-myeloperoxydase specificity in 83% of cases (p<0.001). The most profitable biopsies are essentially cutaneous and neuromuscular. At the diagnostic, two thirds of the patients have had a treatment adapted according to the current recommendations based on the Five Factor Score. The remission rate within a oneyear period is of 77%. The remission is strongly correlated to the therapeutic protocol associating corticoids and cyclophosphamide (p <0.05).

Conclusion: 4

The prevalence of SCS in our area is similar to that observed in other European regions. However, this vasculitis remains a difficult and often a tardive diagnostic pathology.

Words=245.

Introduction

The Churg-Strauss Syndrome (CSS), described since 1951 [START_REF] Churg | Allergic granulomatosis, allergic angiitis, and periarteritis nodosa[END_REF], is a systemic and pulmonary vasculitis, characterised by tardive asthma, a peripheral and tissular eosinophilia, and the formation of granulomas [START_REF] Guillevin | Churg-Strauss syndrome. Clinical study and long-term follow-up of 96 patients[END_REF]. This vasculitis which, preferentially affects small calibre vessels, is characterised by an intraparietal and perivascular Eosinophil-rich inflammation and may be complicated by serious visceral attacks, which can put at risk the vital and functional prognostic. This syndrome includes a significant clinical-biological and progressive heterogeneity. Darker prognosis visceral manifestations can incite to a therapeutic intensification. Burgundy is a historical and cultural area in Western Europe. It is a vast region of France (6% of metropolitan area). No data exists on the CSS in this population of over 1,631,000 people. In our work, we have studied the presentation of CSS. We have therefore listed all Churg-Strauss illnesses hospitalised in our area between 1998 and 2008 and have recorded their clinical-biological presentation mode.

Patients and methods

It is about a retrospective, descriptive work concerning all the patients who stayed, in complete hospitalization, at least once in a health establishment in the area of Burgundy, France, between January 1998 and May 2008 for the care of an CSS or one of its complications. The search for these patients was carried out starting from data-processing coding CIM10 (Classification International of the Diseases 10th version) from the hospitalisation summaries. All the medical computing departments from public health corporations of the four departments of the area (Côte d'Or, Nièvre, Yonne, and Saône-et-Loire) were interviewed with the code M30.1 "Allergic Angéite of Churg-Strauss" in principal diagnosis or associated during the period chosen. Many departments (pneumology, internal medicine …) were also asked to identify all patients. All doctors treating patients lost to follow-up were contacted to verify if patients have died and if they had relapses and then survival rate has been calculated.

Each file retained during this research has been consulted, diagnostic criteria ACR 1990 and residence in burgundy checked in order to retain the final CSS diagnostic. An exhaustive compilation of clinical, biological data as well as the imagery has been established during the various hospitalisations or follow-up consultations making it possible to present the clinical-biological profile during the diagnostic, in the previous months the diagnostic as well as the various relapses of the illness. Prevalence of the disease was estimated by comparing the number of patients seen in the region each year related to the overall population according to the demographics of the region (http://www.insee.fr). We have considered as a flare-up all new visceral attack related to Churg-Strauss vasculitis hitherto latent or asymptomatic as well as any known visceral attack requiring a rise of the corticosteroid therapy strictly of more than 50% or the addition of an immunosuppressive treatment. The FFS (Five Factor Score) evaluating the vasculitides prognostic at ANCA (Anti-cytoplasm antibodies of polynuclear neutrophils) [START_REF] Guillevin | Prognostic factors in polyarteritis nodosa and Churg-Strauss syndrome. A prospective study in 342 patients[END_REF] has been calculated during a diagnosis and compared to the initial treatment received by the patients and to the therapeutic recommendations based on this score [START_REF] Solans | Churg-Strauss syndrome: outcome and long-term follow-up of 32 patients[END_REF][START_REF] Seo | The antineutrophil cytoplasmic antibody-associated vasculitides[END_REF][START_REF] Ribi | Treatment of Churg-Strauss syndrome without poor-prognosis factors: a multicenter, prospective, randomized, open-label study of seventy-two patients[END_REF][START_REF] Cohen | Churg-Strauss syndrome with poor-prognosis factors: A prospective multicenter trial comparing glucocorticoids and six or twelve cyclophosphamide pulses in forty-eight patients[END_REF]. The remission rate (free from flare-up for over 6 months) to a one-year treatment has also been counted. For the statistical analysis, a first sort risk of 5% was chosen. The analysis of the qualitative variables was carried out using a nonparametric test: Fisher's exact test. As for the quantitative variables, they were analysed in parametric with the help of Student's Ttest and in nonparametric using the test of Wilcoxon.

Results

Over this 10-year period, in our area, we have counted 31 different patients being followed for CSS. The majority ( 21) are followed at the University Hospital complex (CHU) of Dijon primarily in internal medicine and pneumology, the others are followed in local hospitals. The sex man/woman ratio is of 1.38. At diagnosis 50% of the patients live in the Côte d'Or department, 37.5% in the Saône-et-Loire, 8.5% in Nièvre and 4% in Yonne (p=0.02). In 48 % the patients live in rural zone in and in 52% in urban zone (p=NS). Six patients died during the period studied i.e., a death rate of 0.6 /year (4 women and 2 men). The survival rate at 5 years is 93%. The average age of death was 82 years (extreme: 72-98 years). The average period between the first signs and death is 189 months (median: 192; extremes: 66-278) that is more than 15 years. The current average age of the living patients is 62.3 years (extreme 25-97; DS=25.37; women: 61.5 years; men: 62.7 years, p=NS)). The patients' follow-up is on average 7.7 years (5 years median, extremes 0.9 -22 years; DS=7.3). Between 1998 and 2008, the average prevalence is of 11.3 patients for one million inhabitants, with an increase in the annual prevalence between 2003 and 2006 (Figure1). As for the average incidence, it is of 1.2 new patients fo r one million inhabitants and per annum. There is a great disparity of prevalence and incidence between the various departments. Similarly, the average incidence is 1.9 new patients per million Twenty-eight patients out of 31 (90%; IC=80-100%) showed signs of a pulmonary attack evolving before the CSS diagnosis. Thirteen patients have had ENT history (nasal polyposis, chronic sinusitis, allergic rhinitis) before the diagnosis i.e., in 42% of them (IC=25-59%). The nasal polyposis -asthmaallergy to aspirin triad was thus noted in two patients (6%; IC=0-15%). Osteoarticular symptoms (arthralgia, myalgia) were present in 13 patients before the diagnosis i.e. in 42% of the cases (IC=25-59%). Nine patients have presented a digestive symptom (abdominal pain, gastrointestinal hemorrhage, eosinophilic cholecystitis, diarrhea) associated with the vasculitis of Churg-Strauss before the diagnosis (29%; IC=13-45%). General signs (fever, weight loss, asthenia) were also present in seven patients (23%; IC=8-37%). Four patients have shown cardiac abnormalities related to the CSS before the diagnosis was established, i.e. 13% of the cases (IC=1-25%). There was a neurological attack (peripheral neuropathy, damage to the central nervous system) in two patients (6%; IC=0-15%). No ophthalmologic, renal or dermatological attack was observed.

The average age at the diagnosis was 56 years (median: 57 years; extremes: 17-87 years; DS=20.2) without significant difference between the men (average 57 years; extremes: 21-77 years; DS=16.9) and the women (average 56 years; extremes: 17-87 years; DS=22.7). The average time between the first signs and the diagnosis is 61 months (median: 36; extremes: 2-232). A triggering factor was found in 23% (IC=8-37%). In three patients, it was about desensitization, an asthma following an intake of aspirin in one patient, a dose of naphazoline in another, in one case contact with plants (Lilac) and finally of an intestinal parasitosis. The median time between the first symptoms and the diagnosis is 36 months (61 months average; extreme:2-232; DS=63).

A pulmonary symptomatology was present in 97% of cases (IC=91-100%) (Figure2). General signs were present at the diagnosis in 61% (IC=44-78%) of cases.

Abnormalities in the neurological examination were found in 18 patients (58%; IC=41-75%). Signs of ENT were present in 15 patients when diagnosed (48%; IC=31-66%), osteoarticular pains in 11 patients (35%; IC=19-52%), and digestive signs in 8 patients (26%; IC=10-41%). Cardiac symptoms were reported during the diagnosis in 8 patients (26%; IC=10-41%). Cutaneous lesions were present in 5 patients (16%; IC=3-29). Only 3 patients out of 31 (10%; IC=0-20%) had an ophthalmologic attack when diagnosed and a renal attack (glomerulonephritis, renal failure, proteinuria) was observed in 3 patients (10%; IC=0-20%).

At the time of the diagnosis, the average rate of leucocytes was of 16342/mm 3 (median: 16000; extremes: 6770-29300) and that of eosinophilic of 4901/mm 3 (median: 2748; extremes: 262-15000). All the patients had an inflammatory syndrome (average rate of fibrinogen=5,4g/l (median = 5.2; extremes: 3.4-8.6), of CRP=106 mg/l (median: 85; extremes: 10-237) and VS=57 mm during the first hour (median: 53,5; extremes: 3-120)).

The creatinemia was on average at 100µmol/l (median: 85; extremes: 51-210). The ANCA were positive only in 26% of patients (IC=8-44%). It was about a perinuclear fluorescence in immunofluorescence with an anti-MPO activity in ELISA in 83% of cases (p=0.004), and without any specificity in 17% of cases. Twenty-three patients (73%) had benefited from the search for antinuclear antibodies (ANA) and 14 (45%) of the IgE dosage. The ANA were always negative or of a non-significant level (<1/160). The average IgE rate was 861 UI/ml (Median: 602; extremes: 10-3399). Abnormalities of the pulmonary radiography were observed in 21 of the 31 patients i.e. in 68% of cases (IC=51-84%). It was a cardiomegaly in 24% of cases, of a pleural effusion in one third of cases, radiological pulmonary infiltrates in 86% of the cases. The biopsies carried out for diagnostic purposes were cutaneous (21%), musculo-nervous (21%), bronchial (17%), digestive (17%), muscular (12%), arterial (4%), renal (4%) or osseous (4%). It was normal in 15% of the cases. A vasculitis was present in 70% of the samples, an extravascular tissular eosinophilia in 55% and a granuloma in 10%. All the cutaneous biopsies, neuromuscular, renal and osseous carried out were valuable, i.e. showed at least one of the histological characteristics of the CSS (granuloma, tissular eosinophilia and compatible vasculitis). It was the same for 67% of the muscular biopsies, 50% of the pulmonary or bronchial biopsies and 25% of the digestive biopsies. FFS score was zero in 8 patients (26%, IC=10-42%), of 1 in 15 patients (48%, IC=31-66%), of 2 in 5 patients (16%, IC=3-29%) and of 3 in 3 patients (10%, IC=0-20%). The neurological and digestive attacks were the criteria the most frequently present before cardiac and renal attacks.

The initial treatment rests on corticoids on its own in 52% of cases, on corticoids associated with cyclophosphamide in 39% of cases, on corticoids associated with azathioprine in 3% of cases, and corticoids associated to methotrexate in 6% of cases. Two patients with an FFS score of 0 have however benefited from an aggressive treatment using corticoids associated with cyclophosphamide; 6 patients with an FFS score of 1, and 2 patients with an FFS score of 2, have been treated initially with corticoids only. The maintenance treatment within a one-year period consists of corticoids on its own in 61% of cases, of corticoids plus azathioprine in 13%, of corticoids plus cyclophosphamide in 6%, corticoids plus mycophenolate mofetil in 3% and corticoids plus methotrexate in 3% of cases.

The remission rate within a one-year period was 77% (IC=63-92%); It was not significantly dependent on the FFS score. On the other hand, the treatment using corticoids and cyclophosphamide was significantly associated with a better remission rate in comparison with that of corticoids on its own (100% versus 63% p=0.02) (Figure3). The remission rate within one-year was not depended significantly on the type of maintenance treatment in our series.

We afterwards studied the clinical signs observed in the patients during the reexamination at the time of the various flare-ups, defined previously (Figure2). The average number of flare-up is 2.13 per patient (Median of 1.5; extremes 0-12; DS=2.57). One then noted a pulmonary attack in 61% of the cases (IC=50-73%) and a cardiac attack (30%; IC=19-41%). Similarly, there were some general signs in 30% of the cases (IC=19-41%).

In 19% of the flare-ups, there was an ENT attack (IC=10-29%). Digestive symptoms were observed in 18% of the flare-ups (IC=9-27%) and the patients presented a neurological flare-up in 13% of cases (IC=10-29%). Ten percent of the patients had renal symptoms during the flare-ups of the disease (IC=3-18%). An osteoarticular attack was observed in 9% of cases (IC=2-16%) and only 7% of patients had cutaneous lesions (IC=1-14%).

Abnormalities with pulmonary radiography were observed in 37% (IC=26-49%). It was about pulmonary infiltrates (76%), pleural effusion (32%) or about a cardiomegaly (24%).

On the whole, 100% of the patients suffering from Churg-Strauss presented at least once during the diagnosis or during the follow-up, a pulmonary attack (Figure2). Similarly, general signs were observed in 24 patients (77%; IC=63-92%), an ENT attack in 22 patients (71%; IC=55-87%), a neurological attack in 19 patients (61%; IC=44-78%), a digestive attack in 17 patients (55%; IC=37-72%), an osteoarticular attack in 16 patients (52%; IC=34-69%), a cardiac attack in 13 patients (42%; IC=25-52%), a cutaneous attack in 9 patients (29%; IC=13-45%), a renal attack in 6 patients (19%; IC=5-33%) and an ophthalmologic attack in 5 patients (16%; IC=3-29%).

Discussion

We have counted 31 files of Churg-Strauss vasculitis in the Burgundy area during a 10-year period. Within the limits of a retrospective work, it is about an exhaustive census.

However, there are limits to our work: the very census mode used makes it possible to enter only the patients who were hospitalised. Some minor forms have probably been followed only in a consultation or in outpatient clinic and are not recorded here. However, CSS is a rare pathology, multisystemic, seldom taken care of exclusively in outpatient clinic. It is difficult to establish whether patients living in Burgundy are followed outside of the region and particularly in the Paris region. Our average incidence of 1.2 new patient per million inhabitants and per annum and our average prevalence of 11.3 patients per million inhabitants in our series are in agreement with those found in other French epidemiological studies. Mahr et al.find in 2000 a prevalence of 10.7 per million inhabitants in the Seine Saint Denis [START_REF] Mahr | Prevalences of polyarteritis nodosa, microscopic polyangiitis, Wegener's granulomatosis, and Churg-Strauss syndrome in a French urban multiethnic population in 2000: a capture-recapture estimate[END_REF]. These results are also superimposable with other important European series (incidence: 1-4/million inhabitants/ annum within the general population [START_REF] Watts | Epidemiology of systemic vasculitis: changing incidence or definition?[END_REF][START_REF] Watts | Geoepidemiology of systemic vasculitis: comparison of the incidence in two regions of Europe[END_REF][START_REF] Reinhold-Keller | Stable incidence of primary systemic vasculitides over five years: results from the German vasculitis register[END_REF][START_REF] Haugeberg | Primary vasculitis in a Norwegian community hospital: a retrospective study[END_REF] and prevalence: 2-13/million inhabitants [START_REF] Haugeberg | Primary vasculitis in a Norwegian community hospital: a retrospective study[END_REF][START_REF] Reinhold-Keller | No difference in the incidences of vasculitides between north and south Germany: first results of the German vasculitis register[END_REF][START_REF] Martin | Prevalence of Churg-Strauss syndrome, vasculitis, eosinophilia and associated conditions: retrospective analysis of 58 prescriptionevent monitoring cohort studies[END_REF]), confirming the absence of North-South or East-West gradient within this pathology [START_REF] Reinhold-Keller | No difference in the incidences of vasculitides between north and south Germany: first results of the German vasculitis register[END_REF] or evolution in time [START_REF] Reinhold-Keller | Stable incidence of primary systemic vasculitides over five years: results from the German vasculitis register[END_REF] contrary to other vasculitis as the disease of Wegener whose incidence is currently in increase and who has a North-South decreasing gradient [START_REF] Mahr | Epidemiology of Wegener's granulomatosis: Lessons from descriptive studies and analyses of genetic and environmental risk determinants[END_REF]. However we have observed a great disparity of prevalence and incidence depending on the various departments' of the area. For these calculations, we have based ourselves on the department of the patients' residence and not on the department of the diagnosis or follow-up establishment thus avoiding the skew of recruitment of some important establishments such as the university hospital complex. Three explanations can be proposed: under diagnosis of the populations of the departments of the Nièvre and Yonne due to a medical density and particularly in terms of less significant specialist in these departments; under incidence of the illness within these departments thus suggesting a different environmental exposure; followed up and diagnosed by the health establishments of the bordering areas.

The majority of the demographic data as well as the proportion of each clinical manifestations are similar to those of the studies published to date (Figure4) [START_REF] Guillevin | Churg-Strauss syndrome. Clinical study and long-term follow-up of 96 patients[END_REF][START_REF] Solans | Churg-Strauss syndrome: outcome and long-term follow-up of 32 patients[END_REF][START_REF] Reid | Churg-Strauss syndrome in a district hospital[END_REF][START_REF] Rossa | Churg-Strauss syndrome: clinical and serological features of 19 patients from a single Italian centre[END_REF][START_REF] Keogh | Churg-Strauss syndrome: clinical presentation, antineutrophil cytoplasmic antibodies, and leukotriene receptor antagonists[END_REF][START_REF] Sinico | Prevalence and clinical significance of antineutrophil cytoplasmic antibodies in Churg-Strauss syndrome[END_REF][START_REF] Sable-Fourtassou | Antineutrophil cytoplasmic antibodies and the Churg-Strauss syndrome[END_REF].

Our series on the other hand, counts slightly more digestive symptoms (54%) and cardiac (49%) and less osteoarticular or dermatological manifestations. These were not always clearly advised in the files and did not always take advantage of biopsies making their record difficult. In our work, the initial manifestations are primarily pulmonary, ENT and osteoarticular and digestive; the diagnosis is readily brought up before pulmonary symptoms, neurological manifestations and general signs; at the time of the flare-ups, symptomatology is primarily pulmonary or cardiac associated with general signs. The neurological attack is thus very frequent at the time of the diagnosis but putting aside the potential after-effects, does not recur much during the follow-up. The average age at the start of the illness is 40 years and the ratio man/woman is 1.38 in our study, very close to that correlated in other publications [START_REF] Guillevin | Churg-Strauss syndrome. Clinical study and long-term follow-up of 96 patients[END_REF][START_REF] Lhote | Churg-Strauss syndrome[END_REF]. Average age however tends to be higher in recent publications (53.3 years) contrary to our study. The current clinical tables would be more often "frustre" than in the past. The currently frequent and " easy" use of corticoids by systemic way for asthmatic treatment could be responsible for an increase in the proportion of "frustre" or tardive forms of CSS [START_REF] Lhote | Churg-Strauss syndrome[END_REF].

As shown in our work, CSS still remains a difficult diagnosis with a significant latency time (on average 61 months) since the first signs. Even, if a better knowledge of this illness makes it possible for the clinician to evoke more frequently and more precociously this illness, its diagnosis can still prove to be difficult nowadays. The initial plain or pauci-visceral attack may not suggest the diagnosis for several months if not years.

The easy dosage of the ANCA in current practice has certainly made it possible to improve and facilitate our practice. But it is often because of the appearance of other visceral and in particular neurological complications that the diagnosis is brought up. Taking care of this pathology is thus often late. Few clinical data make it possible to evoke the illness precociously and thus make it possible to treat it before serious viscera l lesions.

In our series, we have found a positivity of the ANCA (generally anti-MPO pANCA) in 26% of cases which is slightly lower than in the other series [START_REF] Seo | The antineutrophil cytoplasmic antibody-associated vasculitides[END_REF][START_REF] Sinico | Prevalence and clinical significance of antineutrophil cytoplasmic antibodies in Churg-Strauss syndrome[END_REF][START_REF] Sable-Fourtassou | Antineutrophil cytoplasmic antibodies and the Churg-Strauss syndrome[END_REF]. The difference between clinical manifestations of o ur study and the others series like more heart manifestation can maybe explain this low rate of ANCA positivity [START_REF] Neumann | Cardiac involvement in churg-strauss syndrome: impact of endomyocarditis[END_REF]. The survival rate at 5 years is similar to other series [START_REF] Phillip | Mortality in systemic vasculitis: a systematic review[END_REF].

Thirteen patients out of 31 (42%; IC=25-59%) did not have the adequate initial treatment according to current recommendations' based on the FFS score [START_REF] Solans | Churg-Strauss syndrome: outcome and long-term follow-up of 32 patients[END_REF][START_REF] Seo | The antineutrophil cytoplasmic antibody-associated vasculitides[END_REF][START_REF] Ribi | Treatment of Churg-Strauss syndrome without poor-prognosis factors: a multicenter, prospective, randomized, open-label study of seventy-two patients[END_REF][START_REF] Cohen | Churg-Strauss syndrome with poor-prognosis factors: A prospective multicenter trial comparing glucocorticoids and six or twelve cyclophosphamide pulses in forty-eight patients[END_REF]. Globally, the proportion of inadequate treatment is in decline from 1982 to 2008: the patients are thus currently better treated. Besides, our work confirms the interest on the rate of remission within a one-year period of the association of cyclophosphamide to corticoids for the initial treatment of vasculitis of poor prognostic [START_REF] Guillevin | Prognostic factors in polyarteritis nodosa and Churg-Strauss syndrome. A prospective study in 342 patients[END_REF][START_REF] Cohen | Churg-Strauss syndrome with poor-prognosis factors: A prospective multicenter trial comparing glucocorticoids and six or twelve cyclophosphamide pulses in forty-eight patients[END_REF][START_REF] Bourgarit | Deaths occurring during the first year after treatment onset for polyarter itis nodosa, microscopic polyangiitis, and Churg-Strauss syndrome: a retrospective analysis of causes and factors predictive of mortality based on 595 patients[END_REF].

Words: 3000

Learning points:

  inhabitants and per annum in Côte d'Or, of 1.34 in Saône-et-Lo ire, of 0.75 in the Nièvre and 0.23 in Yonne (p=0.016). Likewise, the prevalence is on average of 17.85 patients per million inhabitants in Côte d'Or, of 12.6 in Saône-et-Loire, 7.04 in the Nièvre and 2.14 in Yonne (p=0.029). The patients present an atopy or allergy in 51.6% (IC=34-69%). First of all, we have studied the clinical symptoms observed in the patients before the diagnosis of Churg-Strauss and which have been clinically invalidating and/or have remained without any accurate explanation until the diagnosis (Figure2). The average age for the first signs was thus of 51.5 years (54 years median, extremes: 15-84, DS=21.25).

Incidence and prevalence of CSS in Burgundy, France, is similar to other area in Europe.

Diagnosis remains difficult and often tardive.

The cardiac manifestations appear more prevalent in our population and, conversely, ANCA positivity is lower.

Cyclophosphamide use is correlated with better prognosis.