Gianluigi Bajocchi

Rossana Terlizzi

Ilaria Chiarolanza

Pietro Cortelli

Primary and secondary Raynaud's phenomenon: does small neuropathy remain the same?

Primary and secondary Raynaud's phenomenon: does small-neuropathy remain the same? Dear Sir, We took great interest in reading Manek and Coll paper's [1], who hypothesize the presence of a small-fiber neuropathy in primary Raynaud's phenomenon (RP). Indeed, we reached similar results testing 12 patients with secondary RP in systemic sclerosis (SSc) when compared to a group of healthy controls [2]. In these patients, cardiovascular reflexes tested by tilt-test, Valsalva manoeuver, deep breathing and sustained handgrip were normal but the cold face test was abnormal in almost all patients. By this test we explored the function of afferent cutaneous C and A fibres instead of using the cold pressor test, which is painful and is difficult to perform in SSc patients with severe sclerodactyly. In healthy people a cold stimulus applied to the face during spontaneous breathing evokes bradycardia and peripheral vasoconstriction as measured by increased systolic and diastolic blood pressure [START_REF] Mathias | Investigation of autonomic disorders In[END_REF]. Our SSc patients with secondary RP showed blunted bardycardic and hypertensive response to cold-face test, suggesting that a dysfunction of epidermal small fibers (C-fibers) may be involved in SSc.

Manek observations as well as ours are very preliminary and the number of patients is too low to definitely prove a unique and characteristic small-fiber neuropathy in the RP. However, assuming there is a small-fiber neuropathy in both primary and secondary RP, the question arises as to whether the same fibers are involved or to the same extent in both conditions. There are several observations supporting the idea that this may not be the case. Autonomic c-fibers around sweet glands are deep, mostly located between dermis and subcutaneous tissue. Skin biopsy in SSc with secondary RP showed loss of sensory neuropeptidic fibers sprouting from dermal papillary layer into the epidermis, whereas a similar picture but with less severe loss of fibers was described in primary RP [START_REF] Bunker | Deficiency of calcitonin gene-related peptide in Raynaud's phenomenon[END_REF][START_REF] Terenghi | Image analysis quantification of peptidemmunoreactive nerves in the skin of patients with Raynaud's phenomenon and sistemic sclerosis[END_REF].

Peripheral neuropathy is intriguingly common evidence of very different diseases such erythromelalgia [START_REF] Davis | Erythromelalgia: vasculopathy, neuropathy, or both? A prospective study of vascular and neurophysiologic studies in erythromelalgia[END_REF], connective tissue diseases [2,5,7,8], and RP [1,2,4,5,9]. It seems unlikely that the same postganglionic neuropathy is involved in all these conditions, unless the small fiber neuropathy is considered as a sign of neuropeptidic dysfunction that may manifest in different pathophysiologic ways thereby leading to a different clinical presentation.

Further studies are needed to clarify this issue although the number of converging results not due to chance is increasing, possibly opening up new avenues of therapies for these diseases.


Hany Onsy Habashy

Desmond G Powe

Enrico Glaab

Graham Ball

Inmaculada Spite Ri

Natalio Krasnogor

Jonathan M Garibaldi

Emad A Rakha

R Green

C Caldas

Ian O Ellis
email: ian.ellis@nottingham.ac.uk

Prof O Ian

Ellis

RERG (Ras-like, oestrogen-regulated, growth-inhibitor) expression in breast cancer: A marker of ER-positive luminal-like subtype

Keywords: Breast carcinoma, RERG, Gene expression, Luminal, Oestrogen receptor, Immunohistochemistry

Global gene expression profiling studies have classified breast cancer into a number of distinct biological and molecular classes with clinical relevance. The heterogeneous luminal group, which is largely characterised by oestrogen receptor (ER) expression, appears to contain distinct subgroups with differing behaviour.

In this study, we analysed 47,293 gene transcripts in 128 invasive breast carcinomas (BC) using Artificial Neural Networks and a cross-validation analysis used in combination with an ensemble sample classification to identify genes that can be used to subclassify ER+ luminal tumours. The results were validated using immunohistochemistry on TMAs containing 1,140 invasive breast cancers.

Our results showed that the RERG-gene is one of the highest ranked genes to differentiate between ER+ luminal tumours and ER-non-luminal cancers based on a 10-fold external cross-validation analysis with an average classification accuracy of 89%. This was confirmed in our protein expression studies that showed RERG positive associations with markers of luminal differentiation including ER, luminal cytokeratins (CK19, CK18, and CK7/8) and FOXA1 (p=0.004) and other markers of good prognosis in BC including small size, lower histologic grade and positive expression of androgen receptor, nuclear BRCA1, FHIT and cell cycle inhibitors p27 and p21. RERG expression was inversely associated with the proliferation marker MIB1 (p=0.005) and p53.

Strong RERG expression showed an association with longer breast cancer specific survival and distant metastasis free interval in the whole series as well as in the ER+ luminal group and these associations were independent of other prognostic variables

In conclusion, we used novel bioinformatics methods to identify candidate genes to characterise ER+ luminal-like breast cancer. RERG gene is a key marker of the luminal BC class and can be used to separate distinct prognostic subgroups.

Introduction

Gene expression profiling (GEP) studies have classified breast cancer (BC) into a number of distinct biological classes which show relationships with clinical outcome [START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sorlie | Gene expression patte rns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Sotiriou | Breast cancer classification and prognosis based on gene expression profiles from a population-based study[END_REF]. The luminal group is the largest class and is fundamentally characterised by oestrogen receptor (ER) expression, but it has increasingly been acknowledged that several subgroups exist within this class, each with different molecular features and clinical behaviour. The number and characterization of these subgroups however remains controversial. In one of the first GEP studies, Perou et al [START_REF] Perou | Molecular portraits of human breast tumours[END_REF] reported four molecular subtypes including the luminal subtype. In a subsequent study, three luminal subtypes were described (Luminal A, B and C) [START_REF] Sorlie | Gene expression patte rns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF]. Subsequent studies have settled for the existence of only two luminal classes (A and B) [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF]. Because luminal tumours constitute approximately 70% of all BC, there is a clear need to improve understanding of their heterogeneity through improved sub-classification and with emphasis on clinical behaviour.

Previously, we have reported on biomarkers selected for investigation on the basis of their biological function and their potential ability to distinguish different prognostic subclasses within the luminal group [START_REF] Habashy | The prognostic significance of PELP1 expression in invasive breast cancer with e mphasis on the ER-positive luminal-like subtype[END_REF][START_REF] Habashy | Transferrin receptor (CD71) is a marker of poor prognosis in breast cance r and can predict response to tamoxifen[END_REF][START_REF] Habashy | Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance[END_REF]. However, it is thought that novel approaches may be more appropriate for biomarker discovery in breast cancer.

Among the machine learning based methods, Artificial Neural Networks (ANNs) are emerging as valuable tools for this purpose. ANNs are a form of artificial intelligence inspired by learning in human neuronal systems and have been shown to be capable of modelling complex systems with high predictive accuracies on several large scale datasets [START_REF] Lancashire | Identification of gene transcript signatures predictive for estrogen receptor and lymph node status using a stepwise forward selection artificial neural network modelling approach[END_REF][START_REF] Rumelhart | LEARNING REPRESENTATIONS BY BACK-PROPAGATING ERRORS[END_REF][START_REF] Lancashire | A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks[END_REF]. We have used a combination of analytical computational approaches to identify genes associated with the luminal subtype of breast cancer and from the top ranked genes; RERG was validated as a prognostic luminal biomarker at the protein level in a well-characterised and annotated consecutive patient series of invasive breast cancer using immunohistochemistry and TMA analysis. This approach allowed us to assess the prognostic and biological importance of RERG protein in invasive BC and its role in subclassification of luminal-like cancer subtype.

Previous studies have shown that RERG mRNA expression was found to be induced by estradiol and repressed by ER-antagonist in ER-responsive MCF-7 cells [START_REF] Finlin | RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer[END_REF].

Material and Methods

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

Gene expression study

The study population used was derived from the Nottingham Tenovus Primary Breast Carcinoma Series of women aged 70 years or less, who presented with stage I and II primary operable invasive breast carcinomas.

Gene expression profiling has been previously described [START_REF] Chin | High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer[END_REF][START_REF] Blenkiron | MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype[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]. Briefly, total RNA was extracted from a series of frozen breast cancers retrieved from Nottingham Hospitals NHS Trust Tumour Bank between 1986 and 1992. RNA integrity and DNA contamination were analysed using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Total RNA was biotin-labelled using the Illumina TotalPrep RNA Amplification kit (Ambion, Austin, TX, USA) according to manufacturer's instructions. Biotin-labelled cRNA (1.5 µg) was used for each hybridisation on Sentrix Human-6 BeadChips (Illumina, San Diego, CA, USA) in accordance with the manufacturer's protocol. Illumina gene expression data containing 47,293 transcripts were analysed and summarised in the Illumina Bead Studio software. Analyses of the probe level data were done using the beadarray Bioconductor package. The expression data are available at the EBI website (http://www.ebi.ac.uk/miamexpress/) with the accession number E-TABM-576.

Bioinformatics analysis (1): Artificial neural network model

We identified the ER+ cases (n=84) identified by immunohistochemistry [START_REF] Habashy | Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance[END_REF][START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characte rised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF] in our patient cohort (n=128) and applied an artificial neural network (ANN) model for sample classification to the gene expression data comprising 47,293 inputs for each sample. The output node was coded as 0 if a case was low ER expression (the median H-score<140; n = 42), and 1 if high ER expression (H score> 140; n = 42). Each input (gene) was considered singly as an input to the model. More precisely, the data was analysed using multi-layer perception architecture with a sigmoidal transfer function, where weights were updated by a back propagation algorithm as previously described [START_REF] Lancashire | A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks[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]. Inputs were ranked in ascending order based on predictive error. The detailed methods are described previously [START_REF] Lancashire | A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks[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].

Bioinformatics analysis (2): Ensemble classification and c ross-validation analysis

In a second bioinformatics analysis step, we sought to obtain a robust ranking of genes that are differentially expressed between the ER+ (n=84) cases and the ER negative non-luminal cases (all other cases) and have high predictive power, by applying an ensemble sample classification method (see description below) within a leave-one-out cross-validation scheme. For this purpose, the 128 patient samples were first grouped into 128 different training/test set partitions, using 127 samples for the training sets and the remaining sample as the test set. For each of the 128 training sets differentially expressed genes were selected independently with the "Empirical Bayes moderated t-statistic" [START_REF] Smyth | Linear models and e mpirical bayes methods for assessing differential expression in microarray experiments[END_REF] [START_REF] Smyth | Linear models and e mpirical bayes methods for assessing differential expression in microarray experiments[END_REF] and used to train a machine learning model, which was evaluated based on the left-out sample (a procedure known as "external cross-validation") [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF]. To classify the left-out sample, the prediction results of four algorithms (Support Vector Machine, Random Forest, kNN and Prediction Analysis for Microarrays, with all parameters being optimised by using a grid search within a nested cross-validation) [START_REF] Tibshirani | Diagnosis of multiple cancer types by shrunken centroids of gene expression[END_REF] were combined to a majority-vote ensemble classifier as to compensate for the inevitable inherent biases and variances that exists amongst each of these machine learning algorithms. In order to rank the genes based on the cross-validation results, their frequency of occurrence in the list of significantly differentially expressed genes (p-value < 0.05) across different cross-validation cycles was recorded, and genes received higher scores the more often they had been selected. RERG belonged to the top-ranked genes which were selected in each of the 128 crossvalidation cycles. All steps of the analysis were conducted using an in-house webapplication for microarray analysis, available at www.arraymining.net [START_REF] Glaab | ArrayMining: a modular webapplication for microarray analysis combining ensemble and consensus methods with cross-study normalization[END_REF].

Patient selection and tissue microarray construction

To validate our main conclusion from the bioinformatics analysis of the gene expression data, tissue microarrays (TMAs) prepared from the whole series cases were used as described previously [START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characte rised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF]. Briefly, Formalin fixed paraffin embedded (FFPE) TMAs were prepared from a series cases of primary operable (stage I and II) breast carcinoma cases from patients age <70 presenting consecutively to the Nottingham Breast Unit with tumours of less than 5 cm diameter between 1988 and 1998. This is a well-characterized resource that contains patients' clinical and pathological data including patients' age, histologic tumour type [START_REF] Ellis | PATHOLOGICAL PROGNOSTIC FACTORS IN BREAST-CANCER .2. HISTOLOGICAL TYPE -RELATIONSHIP WITH SURVIVAL IN A LARGE STUDY WITH LONG-TERM FOLLOW-UP[END_REF], primary tumour size, lymph node status, mitotic count and histologic grade [START_REF] Elston | Pathological Prognostic Factors in Breast-Cancer .1. the Value of Histological Grade in Breast-Cancer -Experience from a Large Study with Long-Term Follow-Up[END_REF], Nottingham prognostic index (NPI) [START_REF] Galea | The Nottingham Prognostic Index in Primary Breast-Cancer[END_REF], vascular invasion (VI), development of recurrence, and distant metastases (DM). Patient management was based on Nottingham Prognostic Index (NPI) score and ER status as previously described [START_REF] Madjd | Loss of CD59 expression in breast tumours correlates with poor survival[END_REF]. The NPI, which is widely used in the UK, was developed for the prognostic management of breast cancer by using multivariate analysis to determine the most important prognostic factors. It includes 3 factors; tumour histologic grade (1-3 using the Nottingham Grading System [START_REF] Rakha | Prognostic significance of Nottingham histologic grade in invasive breast carcinoma[END_REF]), lymph node (LN) stage (1-3; 1=LN negative, 2=1-3 positive nodes and 3≥ 4 positive nodes) and primary tumour size (0.2x size in cm). NPI is then categorized into 3 groups: the good prognostic group (score ≤3.4), the moderate group (>3.4 -5.4) and the poor prognostic group (score > 5.4) [START_REF] Galea | The Nottingham Prognostic Index in primary breast cancer[END_REF].

Survival data including survival time and disease-free interval (DFI) were maintained on a prospective basis. 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 (or with active) breast cancer. DFI was defined as the interval (in months) from the date of the primary surgical treatment to the first locoregional or distant metastasis (DMFI).

Mean follow-up time of the studied cohort was 124 months. The data on other biomarkers with strong relevance to breast cancer including ER, progesterone receptor (PgR), androgen receptor (AR), BRCA1, p53, FHIT, HER2, E-cadherin, Pcadherin, basal and luminal cytokeratins (CKs) (CK5/6, CK14, CK18, CK19 and CK7/8), cell cycle inhibitors (p21 and p27), smooth muscle actin (SMA), MIB1, BCL2, and FOXA1 protein expression were available [START_REF] Habashy | Forkhead-box A1 (FOXA1) expression in breast cancer and its prognostic significance[END_REF][START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characte rised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Rakha | Triple-Negative Breast Cancer: Distinguis hing between Basal and Nonbasal Subtypes[END_REF] . Source, dilution, pretreatment and cut-offs of antibodies used are summarised in Table 1. MIB1 staining was done on full face tumour sections [START_REF] Aleskandarany | Growth fraction as a predictor of response to chemothe rapy in node-negative breast cancer[END_REF].

Immunohistochemistry

The RERG specific rabbit polyclonal antibody (Purified rabbit anti-human RERG polyclonal antibody, 10687-1-AP, Proteintech Group, Chicago, IL, USA) was optimized at a working dilution of 1:20 using randomly selected full-face sections of breast cancer tissue to assess the staining distribution in cancer and normal tissue.

After microwave antigen retrieval in citrate buffer pH 6 and protein blocking, the TMAs sections and control sections (normal breast tissue and a known positive carcinoma) were put in a humidity chamber followed by blocking of endogenous peroxidase by applying hydrogen peroxide in methanol for 10 min. The TMA slides were incubated in primary antibody at 4ºC overnight. The immunohistochemical detection of RERG was carried out using a labelled steptavidin biotin technique LSAB ® in accordance with the manufacturer's instructions (DakoCytomation, Cambridge, UK). The slides were incubated with the biotinylated secondary antibody for 30 min, followed by HRP-Streptavidin for another 30 min. Tris buffer saline (TBS) were used for washing between steps (3x2 with stirrer), for visualisation of the reaction, the slides were incubation in freshly prepared peroxidase substrate solution (DAB) dilated 1:50 for 10 min. After application of DAB, the slides were washed in running tap water, counterstained in haematoxylin, dehydrated in alcohols, cleared in xylene and coverslipped using DPX mounting medium. Negative controls were performed by omitting the primary antibody while positive control BC sections were used in each run.

Statistical analysis

Statistical analysis was performed using SPSS 15.0 statistical software (SPSS Inc., Chicago, IL, USA). Association between RERG immunoreactivity and different clinicopathological parameters was evaluated using Chi-square test. Standard cutoff values for the different biomarkers, needed to determine categorical scores before statistical analysis, were the same as those published in previous studies [START_REF] El-Rehim | High-throughput protein expression analysis using tissue microarray technology of a large well-characte rised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Rakha | Triple-Negative Breast Cancer: Distinguis hing between Basal and Nonbasal Subtypes[END_REF][START_REF] El-Rehim | Expression and co-expression of the me mbers of the epidermal growth factor receptor (EGFR) family in invasive breast carcinoma[END_REF] (Table 1). Survival curves were estimated by the Kaplan-Meier method with a log rank test to assess significance. Patients that died due to causes other than breast cancer were censored during survival analysis. We used REMARK guidelines [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF] for reporting on prognostic biomarkers and therefore studied the outcome in patients in the whole series and a subset of ER+ luminal like patients and according to the systemic therapy groups. Multivariate Cox proportional hazard regression models were used to evaluate any independent prognostic effect of the variables with 95% confidence interval. A p-value of <0.05 was considered to indicate statistical significance.

Results

Gene expression studies

Novel genes associated with ER+ status (high expression vs. low expression) using Artificial Neural Network

High ER expression is associated with better prognosis whe n compared to low ER expression. Our aim was to find; using a novel prediction method (ANN), a set of genes that can associate with high ER expression and to validated the genes using protein expression. To study this, ER+ cases (84 tumours) were categorized according to the level of ER expression into high and low expression using the median of the Hscore value (H-score 140).

The ranking order of ANN results was based on predictive error for the unseen cohort in the Monte Carlo Cross validation with the lowest being higher in the ranking order. Table 2 illustrates top transcripts according to ER status ranked by selection error.

RERG transcript was associated with high ER status that characterise luminal A molecular subtype.

In this study ESR1, the gene for ER, was ranked as the most important gene for ER membership and this was used a proof for the validation of the model.

Novel genes associated with ER+ luminal phenotype using the ensemble crossvalidation analysis

The RERG-gene was selected among the significantly differentially expressed genes in every cycle of a leave-one-out external cross-validation analysis. The prediction models obtained from this procedure distinguished the luminal from the non-luminal samples with an average accuracy of 88.3% (sensitivity: 95.2%, specificity: 75.0%).

Very similar results were obtained in a 10-fold cross-validation analysis, which was conducted for further verification (average accuracy: 89%, sensitivity: 95.2%, specificity: 77.3%). Table 3 lists the 10 top-ranked genes, which were identified as being differentially expressed in ER+ luminal and non-luminal samples. These were identified using both in a leave-one-out and a 10-fold cross-validation analysis, i.e. using different subsets of samples, and they were always selected as significantly differentially expressed in each cycle of the analysis (see details on the method in section "Bioinformatics analysis (2)"). RERG was therefore selected for further study using a protein expression assay to assess the biological and prognostic significance of its protein expression in large breast cancer patient cohort as well as in the ER+ subgroup.

Protein expression study Expression of RERG protein in breast tumours

Evaluation of RERG protein expression in breast cancer showed that the immunoreactivity was localised to the cytoplasm of invasive tumour cells with homogenous distribution (Fig 3a and3b) and was strongly expressed in the cytoplasm of luminal cells of normal acini.

Of the whole series, 1,140 informative cases for RERG expression were studied.

RERG cytoplasmic expression was scored as negative (no staining) in 28% of cases, low (weak staining hardly visible at low magnification) in 45.6% of cases or high (strong staining easily visible at low magnification) in 26.4%. When studying the expression in relation to BCSS we found no difference in outcome in patients between those with negative and low expression of RERG cases and therefore we combined them into one group of negative/low RERG expression.

Association of RERG with clinico-pathological parameters

Table 4 summarises the associations between RERG expression and clinicopathological variables in the whole series while Table 5 shows the associations in the ER+ luminal cohort. In the whole series, high RERG expression was positively associated with postmenopausal status (p=0.016), tumours of small size, (p=0.032), low grade (p=0.002), and low mitotic counts (p=0.006) with good NPI (p=0.006).

RERG expression also showed association with tumours that were less likely to develop DM (p=0.001) or tumour recurrence (p=0.003). No associations were found between RERG and other clinicopathological variables.

In the ER+ luminal cohort, RERG expression showed similar significant associations with regards to tumour size (p=0.022), NPI (p=0.026), DM (p=0.002) and recurrence.

(p=0.009).

Association of RERG with othe r biomarkers

In the whole patient series, RERG expression was positively associated with markers of luminal differentiation including ER (p=0.001), luminal CKs (CK19 (p=0.028), CK18 (p=0.001), CK7/8 (p=0.013)), E-cadherin (p=0.001), and the ER-related gene FOXA1(p=0.004), androgen receptor (AR) (p<0.001), nuclear BRCA1 (p=0.023), FHIT (p<0.001) and the cell cycle inhibitors p27 (p=0.005) and p21 (p=0.023). In contrast, RERG expression was inversely associated with the negative expression of the proliferation marker Ki67 (MIB1) (p=0.005) and p53 (p=0.050) (Table 6). In the ER+ luminal cohort RERG expression retained similar associations.

Patient Outcome

Univariate analysis

Breast cancer patients with strong RERG expression showed a significantly longer BCSS (LR=12.267, p<0.001;

According to systemic the rapy groups

The association between RERG protein expression and longer BCSS was also confirmed in the group of patients that did not receive systemic therapy (n=397) (LR=6.467, p=0.01) (Fig 4e). In ER+ tamoxifen-only treated patients, high RERG expression indicated better response to tamoxifen monotherapy (LR=4.871, p=0.027)

(Fig 4f).

Multivariate analysis

BCSS

Multivariate analyses including well-established prognostic variables showed that RERG expression was an independent prognostic marker for longer BCSS in the whole series (Hazard ratio (HR) =0.573, p =0.001, 95% CI =0.411-0.799) and in ER+ luminal-like cohort (HR =0.555, p =0.006, 95% CI =0.364-0.846) (Table 7).

DMFI

Multivariate analyses including the same well-established prognostic variables as above showed that RERG expression was not an independent prognostic marker for DMFI in the whole series (HR=0.752, p =0.053, 95% CI =0.563-1.003) and in ER+ luminal-like cohort (HR =0.748, p =112, 95% CI =0.522-1.070) (data not shown).

Discussion

Gene expression studies have classified breast cancer into a number of distinct biological classes which show an association with clinical outcome. The largest "luminal" group is mainly characterised by ER expression [START_REF] Sorlie | Gene expression patte rns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Sotiriou | Breast cancer classification and prognosis based on gene expression profiles from a population-based study[END_REF][START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF][START_REF] Sorlie | Gene expression profiles do not consistently predict the clinical treatment response in locally advanced breast cancer[END_REF]. However, this class is comprised of tumours showing wide variation at the morphological level, immunophenotypic features and a recognised heterogeneity in terms of response to therapy and clinical outcome indicating that the current molecular classification requires further refinement.

It is thought that novel approaches may be more appropriate for biomarkers discovery in breast cancer. Among the machine learning based methods, Artificial Neural Networks (ANNs) are emerging as valuable tools for this purpose. In the present study, we used a transcript expression profiling of 128 frozen breast cancer cases and analysed the normalised expression values using ANNs. Additionally, a crossvalidation analysis in combination with a majority-vote ensemble sample classification was applied in order to obtain a more robust selection and ranking of genes to characterise luminal breast cancer samples. The ensemble classifier combines the prediction results of four algorithms (Support Vector Machine, Random Forest, k-Nearest Neighbour, and Prediction Analysis for Microarrays ) [START_REF] Tibshirani | Diagnosis of multiple cancer types by shrunken centroids of gene expression[END_REF]. We identified Ras-related, oestrogen-regulated growth inhibitor (RERG) as a candidate marker for differentiating between luminal and non-luminal BC classes among other genes including GATA3, CA12 and ESR1 which have been reported previously for characterising luminal class membership.

RERG is a GTP-binding protein with intrinsic GTPase activity [START_REF] Finlin | RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer[END_REF] and was initially identified as one of the genes that characterise luminal A tumours using gene expression arrays. The expression of RERG has been reported to be decreased in the aggressive ER negative subtypes [START_REF] Sorlie | Gene expression patte rns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF].

In our analysis of BC gene expression data, RERG shown to correlate with high ER expression status using an ANN model and clearly belonged to the best-ranked genes for differentiating between luminal and non-luminal BC, being selected in each cycle a leave-one-out external cross-validation analysis that provided an average classification accuracy of 88.3%. Although RERG gene expression has been reported to be associated with ER+ breast cancer in our analysis and previous studies [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF], its protein expression has not been studied in BC. To validate the gene expression findings we studied RERG protein expression in invasive BC using TMAs and immunohistochemistry. We found good agreement between protein and gene expression results, highlighting the importance of RERG as a candidate luminal marker. RERG protein expression showed a highly significant association with luminal CKs, FOXA1, E-cadherin, steroid receptors, BRCA1, cell cycle inhib itors (p21 and p27) and FHIT which are all markers associated with good prognosis and luminal phenotype. In vitro studies also lend support to its association with goodprognostic phenotypes because RERG mRNA expression is induced rapidly in ERresponsive MCF-7 cells stimulated by estradiol and repressed by ER-antagonist tamoxifen treatment [START_REF] Finlin | RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer[END_REF].

In contrast, we found an inverse relation between RERG protein expression and indicators of cell proliferation such as tumour grade, mitosis and MIB1 expression and this observation of altered cellular proliferation has been proposed as an explanation for the difference in prognosis seen within luminal tumours. The association of RERG with high tumour grade was also seen on mRNA level as shown in Fig 1a . In agreement with this observation, our protein expression results confirmed that RERG expression is associated with tumours displaying low MIB1 expressio n supporting the growth inhibitory function of RERG. For these reasons, we propose that within the luminal classes, expression of ER, RERG and MIB1 could be used to define biological subgroups with different prognoses. ER+ RERG+ MIB low could represent a luminal subgroup with good prognosis while those with an ER+ RERG-MIB1 high phenotype could define a luminal subclass with poor prognosis. Further work is currently underway to better characterise the luminal subclasses with emphasis on predicting prognostic signatures.

A key aim of this study was to assess the prognostic ability of RERG protein expression in ER-positive luminal-like BC patients. In this important group of patients, we found that RERG expression was significantly associated with longer BCSS and longer DMFI which implies its role in subclassification of ER-positive groups into prognostic subgroups. To overcome the confounding effects of systemic therapy, these findings were also confirmed in a cohort of patients that did not receive adjuvant treatment.

In conclusion, a novel bioinformatics approach was used to analyse high dimensional datasets and we were able to identify candidate genes to characterise the ER+ luminal like BC. Subsequently, these genes were translated into protein biomarkers that were shown to subclassify BC in terms of biology and prognosis. In particular, we propose that RERG can differentiate between patients with luminal-like tumours with good and poor clinical outcome and as such it could be a useful marker for a more precise definition of the luminal phenotype.

Gene identifier Gene

GI_4503602-S ESR1

GI_14249703-S RERG

GI_9951924-S CA12

GI_37551139-S C6orf115

GI_34452698-S ACTR3

GI_22779933-S WDR19

GI_38455428-S AGR3

GI_38146007-A TTC8

GI_40788002-S PSME4

GI_4503928-S GATA3

Fig

  Fig 1a shows a box plot of RERG gene (mRNA) expression (normalised expression

  Fig 4a) and longer DMFI (LR=7.472, p=0.006) (Fig 4b). Similar associations were found in the ER+ luminal group in terms of BCSS (LR=9.887, p=0.002; Fig 4c) and DMFI (LR=7.205, p=0.007; Fig 4d).

Figures legends Figure 1 :

 1 Figures legends

Figure 2 :

 2 Figure 2:

Figure 3 :Figure 4 :

 34 Figure 3:

Figure 4b :

 4b Figure 4b: Kaplan Meier plot of RERG expression with respect of DMFI shows a

Figure 4d :

 4d Figure 4d: Kaplan Meier plot of RERG expression with respect of DMFI in ER+

Figure 4e :

 4e Figure 4e: Kaplan Meier plot of RERG expression patterns with respect to BCSS in

Table 1 :

 1 Source, dilution and pretreatment of antibodies used

	# =scored according to Hercept test guidelines
	*=H-score
	^=Scored as (0, absent; 1, weak; 2, moderate; 3, strong cytoplasmic expression)

Table 2 :

 2 A gene signature of ER expression status (summary of step 1 of the ANN approach-10 genes shown)

	Gene	Selection Error
	ESR1	0.403422327
	RERG	0.438626499
	AMN1	0.441492448
	ZNF271	0.445580899
	PCDHA5	0.446326207
	PRKAR2B	0.447933195
	TCEAL1	0.448787999
	CTBP2	0.449052205
	LDB3	0.449750785
	DDIT4	0.451080472

Table 3 :

 3 A gene signature to differentiate between luminal (ER+) and non-luminal cases (ER-) using cross validation analysis

Table 4 :

 4 Relation of RERG immunostaining with various clinicopathological parameters in whole series of breast cancer patients

	Variable	Negative	Positive	χ 2	P value
	Age			4.870	0.182
	<40	70(79.5)	18(20.5)
	40-50	251(76.1)	79(23.9)
	51-60	277(73.3) 101(26.7)
	>60	241(70.1) 103(29.9)
	Size			4.604	0.032
	≤2 cm	406(70.9) 167(29.1)
	>2 cm	432(76.5) 133(23.5)
	Lymph Node (LN) Stage			1.334	0.513
	1(Negative)	497(72.6) 188(27.4)
	2(1-3 LN)	261(74.6)	89(25.4)
	3(>3 LN)	79(77.5)	23(22.5)
	Grade			12.419	0.002
	1	127(66.8)	63(33.2)
	2	260(70.3) 110(29.7)
	3	451(78)	127(22)
	NPI(Nottingham			10.330	0.006
	Prognostic Index
	Good	212(67.3) 103(32.7)
	Moderate	470(75	157(25)
	Poor	157(79.3)	41(20.7)
	DM (Distant Metastasis)			11.948	0.001
	No	565(70.9) 232(29.1)
	Positive	266(80.9)	63(19.1)
	Recurrence			8.642	0.003
	No	474(70.6) 197(29.4)
	Positive	348(78.6)	95(21.4)
	Tumour type			4.783	0.443
	Ductal/NST	498(75)	166(25)
	Lobular	94(72.9)	35(27.1)
	Tubular and Tubular mixed	163(70.6)	68(29.4)
	Medullary	25(83.3)	5(16.7)
	Other special types*	12(75)	4(25)
	Mixed**	47(67.1)	23(32.9)
	Mitotic Frequency			10.274	0.006
	1	268(68.7) 122(31.3)
	2	144(73.5)	52(26.5)
	3	407(78.1) 114(21.9)
	Menopause			6.116	0.016
	Premenopausal	341(77.7)	98(22.3)
	Postmenopausal	498(71)	203(29)
	*Includes Mucoid, invasive cribriform and invasive papillary carcinoma, ** Include
	ductal/NST mixed with lobular or special types

Table 5 :

 5 Relation of RERG immunostaining with various clinicopathological parameters in ER+ cohort of breast cancer patients

	Variable	Negative	Positive	χ 2	P value
	Age			1.875	0.599
	<40	31(70.5)	13(29.5)
	40-50	160(73.4)	58(26.6)
	51-60	187(72.2)	72(27.8)
	>60	177(68.1)	83(31.9)
	Size			5.335	0.021
	≤2 cm	288(67.6) 138(32.4)
	>2 cm	266(75.1)	88(24.9)
	LN Stage			2.044	0.360
	1(Negative)	325(69.1) 145(30.5)
	2(1-3 LN)	182(73.4)	66(26.6)
	3(>3 LN)	46(75.4)	15(24.6)
	Grade			5.379	0.068
	1	111(65.7)	58(34.3)
	2	227(69.8)	98(30.2)
	3	216(75.5)	70(24.5)
	NPI			7.313	0.026
	Good	185(65.8)	96(34.2)
	Moderate	285(72.7) 107(27.3)
	Poor	85(78.7)	23(21.3)
	DM			9.537	0.002
	No	382(68.2) 178(31.8)
	Positive	170(79.4)	44(20.6)
	Recurrence			7.189	0.007
	No	321(67.9) 152(32.1)
	Positive	226(76.9)	68(23.1)
	Tumour type			3.301	0.654
	Ductal/NST	272(70.6) 113(29.4)
	Lobular	90(76.9)	27(23.1)
	Tubular and Tubular mixed	142(70)	61(30)
	Medullary	3(75)	1(25)
	Other special types*	9(75)	3(25)
	Mixed**	39(65)	21(35)
	Mitosis			3.223	0.200
	1	238(68.6) 109(31.4)
	2	116(72)	45(28)
	3	186(75.3)	61(24.7)
	Menopause			2.984	0.084
	Premenopausal	203(74.9)	68(25.1)
	Postmenopausal	352(69)	158(31)
	*Includes Mucoid, invasive cribriform and invasive papillary carcinoma, ** Include
	ductal/NST mixed with lobular or special types

Table 6 :

 6 Relation of RERG immunostaining with various biomarkers parameters in whole cohort of breast cancer patients

	Variable	Negative	Positive	χ 2	p-value
	CK 5/6
	Negative	688(73.7)	245(26.3)	0.003	0.954
	Positive	125(73.5)	45(26.5)
	CK 14
	Negative	711(73.8)	253(26.2)	0.290	0.590
	Positive	92(76)	29(24)
	CK18
	Negative	118(84.9)	21(15.1)	10.823	0.001
	Positive	647(71.7)	256(28.3)
	CK19
	Negative	90(82.6)	19(17.4)	4.829	0.028
	Positive	726(72.8)	271(27.2)
	CK7/8
	Negative	17(100)	0(0)	6.128	0.013
	Positive	800(73.4)	290(26.6)
	ER
	Negative	249(80.8)	59(19.2)	10.938	0.001
	Positive	555(71.1)	226(28.9)
	PgR
	Negative	384(74)	122(26)	0.026	0.872
	Positive	449(73.6)	161(26.4)
	AR
	Negative	300(82.9)	62(17.1)	23.614	<0.001
	Positive	469(69)	211(31)
	P53
	Negative	571(71.7)	225(28.3)	3.839	0.050
	Positive	223(77.7)	64(22.3)
	FHIT
	Negative	158(84.9)	28(15.1)	14.235	<0.001
	Positive	574(71.5)	229(28.5)
	BRCA1
	Negative	114(82)	25(18)	5.163	0.023
	Positive	586(72.9)	218(27.1)
	Bcl2
	Negative	268(77.5)	78(22.5)	2.280	0.131
	Weak	385(72.9)	143(27.1)
	MIB1
	low	172(67.7)	82(32.3)	7.915	0.005
	High	488(76.9)	147(23.1)
	P-Cad
	Negative	321(71.7)	127(28.3)	2.601	0.107
	Positive	382(76.2)	119(23.8)
	E-Cad
	Negative	332(79.6)	85(20.4)	11.370	0.001
	Positive	473(70.4)	199(29.6)
	FOXA1
	Negative	344(78.9)	92(21.1)	8.082	0.004
	Positive	272(70.3)	115(29.7)
	HER2
	Negative	711(73.8)	253(26.2)	0.243	0.622
	Positive	103(75.7)	33(24.3)
	Actin
	Negative	678(73)	251(27)	2.631	0.105
	Positive	125(79.1)	33(29.1)
	p21
	Negative	366(77.9)	104(22.1)	5.162	0.023
	Positive	281(71.1)	114(28.9)
	p27
	Negative	317(79.1)	84(20.9)	7.711	0.005
	Positive	264(70.4)	111(29.6)

Table 7 :

 7 COX model for predictors of BCSS in the whole patient series and in the ER+ luminal subgroup

			Whole series		ER-positive cohort
	Variable	p value	HR	95 % CI	p value	HR	95 % CI
				Lower	Upper			Lower	Upper
	RERG expression	0.001	0.573	0.411	0.799	0.006	0.555	0.364	0.846
	Endocrine therapy	0.020	0.670	0.478	0.938	0.126	0.713	0.463	1.100
	Chemotherapy	0.001	0.508	0.338	0.762	0.028	0.521	0.292	0.931
	Tumour size (>2cm)	<0.001	1.951	1.472	2.585	<0.001	2.083	1.464	2.963
	Tumour stage 1*	<0.001	1			<0.001	1
	Tumour stage 2 vs. 1	<0.001	1.814	1.341	2.454	0.004	1.769	1.201	2.606
	Tumour stage 3 vs. 1	<0.001	4.604	3.204	6.617	<0.001	3.948	2.388	6.526
	Tumour grade 1*	<0.001	1			<0.001	1
	Tumour grade 2 vs.1	0.011	1.982	1.174	3.348	0.021	1.955	1.107	3.454
	Tumour grade 3 vs. 1	<0.001	4.175	2.495	6.985	<0.001	4.487	2.483	8.107
	*fitted as categorical variable

Table 1 :

 1 Source, dilution and pretreatment of antibodies us

Table 2 :

 2 A gene signature of ER expression status (summary of step 1 of the ANN approach)

Table 3 :

 3 A gene signature to differentiate between luminal (ER+) and non-luminal cases (ER-) using cross validation analysis

Table 4 :

 4 Relation of RERG immunostaining with various clinicopathological parameters in whole series of breast cancer patients

Table 5 :

 5 Relation of RERG immunostaining with various clinicopathological parameters in ER+ cohort of breast cancer patients

Table 6 :

 6 Relation of RERG immunostaining with various biomarkers parameters in whole cohort of breast cancer patients

Table 7 :

 7 COX model for predictors of BCSS

Acknowledge ment

We thank the ministry of higher education (Egypt) for funding HO Habashy and E Rakha. Funding (salaries and infrastructure) was provided by the University of Nottingham and Nottingham University Hospitals Trust. N Krasnogor and E Glaab would like to acknowledge funding by Marie Curie Early Stage Training Programme (MEST-CT-2004-007597).

All authors declare no conflict of interest.


Gabriel Farante
email: gabriel.farante@ieo.it

Stefano Zurrida

Viviana Galimberti

Paolo Veronesi

Giuseppe Curigliano

Alberto Luini

Aron Goldhirsch

Umberto Veronesi

The management of Ductal Intraepithelial Neoplasia (DIN): Open controversies and

Keywords:

come

Introduction

DIN, is the new acronym (corresponding to ductal intraepithelial neoplasia) that replaces the traditional definition of ductal carcinoma in situ (DCIS) of the breast. This is because some authors [START_REF] Tavassoli | Ductal carcinoma in situ: introduction of the concept of ductal intraepithelial neoplasia[END_REF] found it difficult to accept the intraductal proliferation of tumor cells being defined as a malignant tumor and others [START_REF] Veronesi | Breast cancer[END_REF][START_REF] Veronesi | Rethinking TNM: Breast cancer TNM classification for treatment decisionmaking and research[END_REF] think that in these cases, the N and M categories should not be applied and that there is therefore no reason to keep them within the TNM classification, as "intraductal carcinoma".

The incidence of breast DIN has been progressively increasing; it currently represents 15-25% of all breast carcinomas and approximately 35% of those diagnosed only by mammography. For example, in the USA, DIN increased from 1.87/100.000 in 1973-1975 to 32.5/100.000 in 2004 [START_REF] Virnig | Ductal carcinoma in situ of the Breast: a systematic review of incidence, treatment and outcomes[END_REF]. Being overall 10-year survival close to 100% of affected patients, the surgery and radiotherapy interventions employed are sometimes more aggressive for this condition than those used for invasive cancer [START_REF] Veronesi | Breast cancer[END_REF]. Some aspects of DIN management are still controversial, as reported in studies across the United States and Europe [START_REF] Baxter | Trends in the treatment of ductal carcinoma in situ of the breast[END_REF][START_REF] Ernster | Incidence of and treatment for ductal carcinoma in situ of the breast[END_REF][START_REF] Ceilley | The Management of Ductal Carcinoma In Situ in North America and Europe Results of a Survey[END_REF] and in different areas within the United States [START_REF] Katz | Patterns and Correlates of Local Therapy for Women With Ductal Carcinoma-In-Situ[END_REF]. These controversies are mainly due to the heterogeneity of its clinical presentation and of its biological and pathological characteristics.

BIOLOGICAL FEATURES OF DIN

DIN is characterised by multiple clinical-pathological and biological features which differentiate it from both normal breast tissue and other benign proliferative breast lesions. As hyperplastic lesions progress through ductal carcinoma in situ to invasive breast cancer, chromosomal alterations usually occur with gain or loss at multiple foci [START_REF] Aubele | Accumulation of chromosomal imbalances from intraductal proliferative lesions to adjacent in situ and invasive ductal breast cancer[END_REF]. Estrogen receptors (ER) are usually expressed in more than 70% of DIN lesions.

The HER2/neu proto-oncogene is over-expressed in roughly half of all DIN lesions but not in atypical hyperplasia [10]. The p53 tumor-suppressor gene is mutated in approximately 25% of all DIN lesions, but is rarely mutated in normal proliferative breast tissue [START_REF] Rudas | p53 Protein expression, cell proliferation and steroid hormone receptors in ductal and lobular in situ carcinomas of the breast[END_REF]. The most dramatic changes in patterns of gene expression during breast tumorigenesis appear during the transition from normal tissue to DIN [START_REF] Luzzi | Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays[END_REF], which is biologically heterogeneous, with variable molecular features. The gene-expression profile of grade 3 (G3) DIN differs from that of grade 1 (G1) lesions and exhibits a greater overall genetic change from normal breast tissue [START_REF] Seth | Gene expression profiling of ductal carcinomas in situ and invasive breast tumors[END_REF][START_REF] Allred | The progression of DCIS to IBC: a cDNA expression microarray study[END_REF]. A great majority of G1 DIN lesions are positive for estrogen receptors, and less than 20% exhibit overexpression of HER2/neu or p53 mutations. In contrast, overexpression of HER2/neu or p53 mutations [START_REF] Allred | Biologic characteristics of ductal carcinoma in situ[END_REF] arise in more than 75% of G3 DIN lesions, whereas only one quarter of them express ER [START_REF] Lampejo | Evaluation of infiltrating ductal carcinomas with a DCIS component: correlation of the histologic type of the in situ component with grade of the infiltrating component[END_REF].

DIN can be considered a step in the development of breast cancer in which most of the molecular changes that characterize invasive breast cancer are already present [START_REF] Buerger | Different genetic pathways in the evolution of invasive breast cancer are associated with distinct morphological subtypes[END_REF].

MANAGEMENT OF DIN PATIENTS

Surgical Treatment

Mastectomy is considered today an overtreatment in most DINs due to the biological and pathological features and also its high survival rate and low absolute risk of local recurrences (LR). However, mastectomy is still indicated in DIN patients with multicentricity, diffuse microcalcification or large palpable masses, as well as when there is an inability to obtain negative margins.

The main goal of surgical treatment for women with DIN is breast conserving surgery (BCS) [START_REF] Fisher | Conservative management of intraductal carcinoma (DCIS) of the breast[END_REF][START_REF] Schwartz | Consensus Conference on the Treatment Classification of Ductal Carcinoma in Situ of the Breast[END_REF][START_REF] Silverstein | Ductal carcinoma in situ: 228 cases without microinvasion[END_REF] particularly for those patients with small solid masses, mammographically-detected lesions, or limited microcalcification areas, and because it is a disease with an extremely high survival rate and low absolute risk of LR.

At present, mastectomy is performed in about 30%, BCS without radiotherapy in about 30% and BCS followed by radiotherapy in about 40% in DIN patients [START_REF] Guerreri-Gonzaga | Ductal Intraepithelial Neoplasia: Postsurgical out come for 1.267 woman cared for in one single Institution over 10 years[END_REF][START_REF] Schmidt | Rationalization and regionalization of treatments for DCIS of the breast[END_REF][START_REF] Kuerer | DCIS: State of the science and roadmap to advance the field[END_REF].

One important issue in breast cancer surgery is that of surgical margins, mainly in DIN patients, since these lesions are typically vague masses, which often cannot be adequately seen or felt and, thus, the pathological sampling of margins is fairly random [START_REF] Fisher | Pathologic findings from the NSABP eight years update of Protocol B-17: Intraductal Carcinoma[END_REF][START_REF] Allred | Ductal Carcinoma In Situ of the Breast: Pathologic and Biologic Perspectives[END_REF]. There are two currently-held concepts that meet with general agreement on this topic: the first is the evidence that wide surgical excision and negative margins of resection decrease the risk of LR in patients with mammographically-detected DIN treated with BCS and radiation [START_REF] Mirza | Ductal carcinoma-in situ: Long-term results of breast conserving therapy[END_REF]. The second concept is a general consensus that patients with positive margins show a significantly higher rate of LR than do patients with negative margins, regardless of irradiation or tamoxifen administration [START_REF] Fisher | Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomized controlled trial[END_REF][START_REF] Bijker | Risk factors for recurrence and metastasis after breast-conserving therapy for ductal carcinoma in-situ: Analysis of European Organization for Research and Treatment of Cancer Trial 10853[END_REF].

In practice, there are two opposite approaches to surgical margins in a DIN patient: the anatomical approach, for which a clear margin width (of at least 10 mm) is more important than any other prognostic factor or subsequent adjuvant therapy [START_REF] Kerlikowske | Characteristics associated with recurrence among women with ductal carcinoma in situ treated by lumpectomy[END_REF][START_REF] Silverstein | The influence of margin width on local control of ductal carcinoma in situ of the breast[END_REF]; and the biological approach, for which the pathological characteristics of the lesion (G, comedo-necrosis, etc) are decisive in decision-making related to surgery and radiotherapy.

The current treatment of "positive" or focally involved margins in DIN patients is re-excision.

Results of the UK Sloane Project showed that in 30% of patients undergoing BCS for DIN, preoperative imaging underestimates the extent of disease resulting in a requirement for further surgery [START_REF] Thomas | Radiological and pathological side estimations of pure ductal carcinoma in situ of the breast: a review of 2564 cases from the Sloane Project[END_REF]. On the other hand, some authors suggest that re-excision may be avoided and satisfactory local control achieved by increasing the radiation dose to the tumour bed to at least 66 Gy [START_REF] Monteau | Ductal carcinoma in situ of the breast with close or focally involved margins following breast-conserving surgery: treatment with reexcision or radiotherapy with increased dosage[END_REF].

Regarding the width of surgical margins, about ten years ago, the "Consensus on DCIS of Philadelphia" [START_REF] Schwartz | Consensus Conference on the Treatment Classification of Ductal Carcinoma in Situ of the Breast[END_REF], and Silverstein et al [START_REF] Silverstein | The influence of margin width on local control of ductal carcinoma in situ of the breast[END_REF] proposed 10 mm as a limit of oncological safety. Since then other authors proposed progressively smaller measures, i.e., 3mm [START_REF]tamoxifen for good risk ductal carcinoma in situ (DCIS) of the female breast[END_REF][34][START_REF] Hughes | Local excition alone without irradiation for ductal carcinoma in situ of the breast: a trial of the Eastern Cooperative Oncology Group[END_REF], 2-3 mm[136-37], 2 mm [START_REF] Mokbel | Heterogeneity the ductal carcinoma in situ and its effects of managements[END_REF][START_REF] Morrow | Breast conservation and negative margins: hoow much is enough?[END_REF][START_REF] Dunne | Effect of the margin status on local recurrence after breast conservation and radiation therapy for ductal carcinoma in situ[END_REF] and 1mm [START_REF] Mansell | Ductal Carcinoma in Situ: Surgery and Radiotherapy[END_REF]. At the 2009 Saint Gallen International Experts Consensus, there was consensus in avoiding the need to insist on a large (e.g. 1 cm) free margins [START_REF] Goldhirsch | Threeholds for the therapies: highlights of the Saint Gallen International Expert Consensus, on the primary therapy of early breast cancer[END_REF].

The issue of surgical margins is closely related to another important issue in DIN patients: LR. The risk of LR for DIN after BCS is the main concern, because approximately 50% of the recurrences are invasive [START_REF] Fisher | Lumpectomy compared with lumpectomy and radiation therapy for the treatment of intraductal breast cancer[END_REF]. LR is rare after total mastectomy, while it is observed at a higher rate in patients treated with breast conservation, even if the impact of these LR on overall survival is small. LR after 10 years of followup in DIN patiens is about 1% when mastectomy is performed, 30% in case of BCS alone and about 10-15% when BCS is followed by radiotherapy [START_REF] Boughey | Current treatment and clinical trial developments for ductal carcinoma in situ of the breast[END_REF].

An interesting study (with a four-decade-long follow up) of women with G1 DIN treated by biopsy alone from 1950 to 1968 reveals the natural history of this poorly understood breast cancer [START_REF] Sanders | The natural history of low-grade DCIS of the breast in women treated by biopsy only revealed over 30 years long-terms follow-up[END_REF]. This study shows that even G1 DIN left without further treatment have a risk of LR and will eventually develop invasive carcinoma in the same site in 30% of cases within 15 years.

Most LR in patients undergoing BCS and radiation for DIN occur in the proximity of the primary tumor. Virtually all patients who develop a non invasive LR, and approximately 75% of those with an invasive LR, are long-term survivors after mastectomy [START_REF] Silverstein | Outcome after invasive LR in patients with ductal carcinoma in situ of the breast[END_REF]. The risk of LR for clinically-evident DIN is approximately twice that of mammographically-detected DIN [START_REF] Solin | Mammographically detected clinically occult ductal carcinoma in situ (intraductal carcinoma) treated with breast conserving surgery and definitive breast irradiation[END_REF]. However, the recurrences after BCS are today diagnosed at a very early stage and almost always treated successfully; for that reason, breast cancer mortality in these patients is very low, about 1% [START_REF] Ernster | Mortality among women with ductal carcinoma in situ of the breast in the population-based Surveillance, Epidemiology and End Results program[END_REF][START_REF] Lee | Breast cancer specific mortality after invasive local recurrence in patients with duct carcinoma in site of the breast[END_REF] and probably due to an underestimated histological evaluation.

Several prognostic factors (PF) influencing LR in DIN patients have been identified [START_REF] Guerreri-Gonzaga | Ductal Intraepithelial Neoplasia: Postsurgical out come for 1.267 woman cared for in one single Institution over 10 years[END_REF][START_REF] Bijker | Risk factors for recurrence and metastasis after breast-conserving therapy for ductal carcinoma in-situ: Analysis of European Organization for Research and Treatment of Cancer Trial 10853[END_REF][START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast Project (NSABP) protocol B-17. Intraductal carcinoma (ductal carcinoma in situ)[END_REF][START_REF] Fisher | Pathologic variables predictive of Breast events in patients with DCIS[END_REF][START_REF] Mamounas | Effects of breast irradiation following lumpectomy in intraductal carcinoma (DCIS): Updated results from NSABP B-17[END_REF][START_REF] Vicini | Age at diagnosis and outcome for women with ductal carcinoma in situ of the breast. A critical review of the literature[END_REF][START_REF] Collins | Relationship between clinical and pathologic features of ductal carcinoma in situ and patient age: an analysis of 657 patients[END_REF][START_REF] Turaka | Young age is not associated with increased local recurrence for DCIS treated by breast-conservation surgery and radiation[END_REF][START_REF] Silverstein | Ductal carcinoma in situ: USC/Van Nuys Prognostic Index and the impac of margin status[END_REF].

The most important PFs are the following: a) High nuclear grade and/or necrosis (particularly extensive comedo-necrosis); b) Tumor size; c) Margin status; and d) young age. In this respect, a recent study observed that DIN in younger women is more often symptomatic, is more extensive and more often shows cancerization of lobules than DIN in older women, although it requires further studies to conclude if these features contribute to the higher local recurrence risk in young patients [START_REF] Collins | Relationship between clinical and pathologic features of ductal carcinoma in situ and patient age: an analysis of 657 patients[END_REF]. In fact, another analysis concludes that young age may be a smaller contributor to LR risk in DIN than previously suggested [START_REF] Turaka | Young age is not associated with increased local recurrence for DCIS treated by breast-conservation surgery and radiation[END_REF]. In order to organize the information about PF, Silverstein [START_REF] Silverstein | Ductal carcinoma in situ: USC/Van Nuys Prognostic Index and the impac of margin status[END_REF] developed the Van Nuys Prognostic Index (VNPI), (including tumor size, margin width, histological classification and age), to define three risk groups of patients. But this Index was criticized by most authors [START_REF] Fisher | Pathologic findings from the NSABP eight years update of Protocol B-17: Intraductal Carcinoma[END_REF][START_REF] Harris | Radiotherapeutic Considerations for DCIS[END_REF].

One important controversial topic in DIN patients is the management of the axilla. We have recently examined this point [START_REF] Farante | No more axillary dissection in patients with ductal intraepithelial neoplasia (DIN)[END_REF] observing that, before the sentinel lymph node biopsy (SLNB) era, axillary dissection (AD) was a part of the standard surgical treatment for these patients. Thanks to this procedure, the status of the axillary lymph nodes was reported as metastatic between 1-2%[34, [START_REF] Winchester | Treatment trends for ductal carcinoma in situ of the breast[END_REF][START_REF] Porembka | Factors associated with lymph node assessment in DCIS: analysis of 1988-2002 Seer Data[END_REF]. A logical and consequent conclusion from these low percentages, would be that AD should never be indicated as first treatment in DIN patients. However, both in theory and in practice it happens the contrary[34, [START_REF] Porembka | Factors associated with lymph node assessment in DCIS: analysis of 1988-2002 Seer Data[END_REF][START_REF] Bishop | Significant Variation in the management of non invasive breast carcinoma in the UK: Initial findings from the Sloane Project, a UK National Prospective Audit[END_REF][START_REF] Cutuli | Breast conserving surgery with or without radiotherapy vs mastectomy for ductal carcinoma in situ: French survey experience[END_REF]. In the light of these analyses, we can conclude that AD should no longer be performed as a first indication of treatment in patients with pure DIN and that AD should be considered in these cases an unnecessary overtreatment without any rational justification.

But not only is AD not necessary, neither should SLNB always be required. However, this is still controversial in DIN patients. In fact, for some authors SLNB should not, in general, be indicated [START_REF] Edge | Counterpoint: Sentinel lymph node biopsy is not indicated for ductal carcinoma in situ[END_REF]; for others, SLNB should always be carried out [START_REF] Pendas | Sentinel node biopsy in ductal carcinoma in situ patients[END_REF][START_REF] Ansari | Meta-analysis of sentinel node biopsy in ductal carcinoma in situ of the breast[END_REF], and for others still, it should be performed only in certain specific and particular cases [START_REF] Intra | Axillary sentinel lymph node biopsy in patients with pure ductal carcinoma in situ of the breast[END_REF][START_REF] Cody | Sentinel Lymph node biopsy for breast cancer: Indications, contraindications, and new directions[END_REF][START_REF] Julian | Is sentinel node biopsy necessary in conservately treated DCIS?[END_REF][START_REF] Yi | Role of primary tumor characteristics in predicting positive sentinel lymph nodes in patiens with ductal carcinoma in situ or microinvasive breast cancer[END_REF]. Major cancer centers agree that SLNB should be conducted a) always when mastectomy is performed [START_REF] Cody | Sentinel Lymph node biopsy for breast cancer: Indications, contraindications, and new directions[END_REF][START_REF] Yi | Role of primary tumor characteristics in predicting positive sentinel lymph nodes in patiens with ductal carcinoma in situ or microinvasive breast cancer[END_REF][START_REF] Dominguez | SLN biopsy is important in mastectomy for DCIS[END_REF][START_REF] Intra | Unnecessary axillary node dissections in the sentinel lymph node era[END_REF]; b) with large lesions (masses or micro-calcifications) and G3 tumors [START_REF] Cody | Sentinel Lymph node biopsy for breast cancer: Indications, contraindications, and new directions[END_REF][START_REF] Julian | Is sentinel node biopsy necessary in conservately treated DCIS?[END_REF]; c) after performing core or mammotome biopsies. In these cases, because an invasive ductal carcinoma can be found at the final pathological diagnosis after surgery: generally, this upstaging represents about 16-20% of these cases, with variations from 11-13% (in cases of either T1 or G1 lesions) to 36-45%(in cases of extended or G3 lesions) [START_REF] Mittendorf | Core biopsy diagnosis of ductal carcinoma in situ: an indication for sentinel lymph node biopsy[END_REF][START_REF] Sayed | Predictive value of needle core biopsy diagnoses of lesions of uncertain malignant potential (B3) in abnormalities detected by mammographic screening[END_REF][START_REF] Sakr | The sentinel lymph node procedure for patients with preoperative diagnosis of ductal carcinoma in situ: risk factors for unsuspected invasive disease and for metastatic sentinel lymph nodes[END_REF][START_REF] Yen | Predictors of invasive breast cancer of an initial diagnosis of DCIS. A guide in the selective use of sentinel lymph node biopsy in management of DCIS[END_REF][START_REF] Fadare | High and intermediate grade ductal carcinoma in situ of the breast: a comparison of pathologic features in core biopsies and excisions and an evaluation of core biopsy features that may predict a close of positive margin in the excision[END_REF][START_REF] O'flynn | Prediction of the presence of invasive disease from the measurement of extent of malignant micro-calcifications on mammography and ductal carcinoma in situ grade at core biopsy[END_REF][START_REF] Doyle | Sentinel Lymph node biopsy in patients with a needle core biopsy diagnosis of ductal carcinoma in situ: is it justified?[END_REF][START_REF] Murphy | Do sentinel node micrometastases predict recurrence risk in ductal carcinoma in situ and ductal carcinoma in situ with microinvasion?[END_REF].

The last question related to the management of the axilla in DIN patients is: should we perform AD in cases of positive SLNB in DIN patients, as we do in cases of infiltrating breast carcinoma? In the few cases published in the literature [START_REF] Pendas | Sentinel node biopsy in ductal carcinoma in situ patients[END_REF][START_REF] Yi | Role of primary tumor characteristics in predicting positive sentinel lymph nodes in patiens with ductal carcinoma in situ or microinvasive breast cancer[END_REF][START_REF] Dominguez | SLN biopsy is important in mastectomy for DCIS[END_REF][START_REF] Intra | Unnecessary axillary node dissections in the sentinel lymph node era[END_REF][START_REF] Murphy | Do sentinel node micrometastases predict recurrence risk in ductal carcinoma in situ and ductal carcinoma in situ with microinvasion?[END_REF][START_REF] Moore | Outcomes for women with DCIS and positive sentinel Node: a multi-institutional audit[END_REF][START_REF] Tunon-De-Lara | Sentinel node procedure is warranted in ductal carcinoma in situ with high risk of occult invasive carcinoma treated by mastectomy[END_REF][START_REF] Leidenius | Tumour-positive sentinel node findings in patients with ductal carcinoma in situ[END_REF], in which AD was performed after an positive SLNB, in all cases but one (67/68, 98,5%) all the non-SLNs were negative and the SLN already biopsied was the only positive LN. That is why we conclude that in cases of DIN patients with a positive SLNB, AD should not be immediately performed except for only those cases that present mammary invasion on final pathologic evaluation.

Open questions remain regarding the significance of isolated tumor cells or micrometastases in the lymph nodes as well as the role SLNB plays in DIN with microinvasion [34,[START_REF]Diagnosis and management of DCIS[END_REF].

Radiation Therapy

External RT

The role of radiotherapy in DIN patients with conservative treatment has been mainly defined by four randomized trials: the NSABP B-17 [START_REF] Bryan | DCIS: evidence from randomized trials[END_REF], the EORTC 10853 [START_REF] Bijker | Breast-conserving treatment with or without radiotherapy in ductal carcinoma in situ: ten-year results of European Organisation for Research and Treatment of Cancer Randomized Phase III Trial 10853 -A Study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotheraphy Group[END_REF], the United Kingdom-Australia-New Zealand (UK-A-NZ) trials [START_REF] Houghton | UK Coordinating Committee on Cancer Research: Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial[END_REF] and the Swedish study on women screened with mammography [START_REF] Emdin | SwDCIS: Radiotherapy after sector resection for ductal carcinoma in situ of the breast. Results of a randomized trial in a population offered mammography screening[END_REF]. A recent review of these trials confirm the following results [START_REF] Goodwin | Post-operative radiotherapy for ductal carcinoma in situ of the breast[END_REF]: all four studies demonstrated that additional radiotherapy reduced the LR rate by about 50%, with no effect on survival. Comparable reductions were seen for the risk of invasive and non invasive LR. Non-significant long term toxicity from radiotherapy was found. These trials were organized to discover whether radiation therapy decreases LR and this was something they clearly demonstrated.

The controversy is, instead, related if all DIN patients have to undergo RT. According to the 2009 St Gallen Consensus [START_REF] Goldhirsch | Threeholds for the therapies: highlights of the Saint Gallen International Expert Consensus, on the primary therapy of early breast cancer[END_REF], RT could be avoided in elderly patients and in those with G1 DIN and clearly negative margins. There are two aspects that are to be taken into consideration at the moment of performing RT in DIN: the margins of resection and the tumor grading. Regarding margins, the recent ECOG report [START_REF] Hughes | Local excition alone without irradiation for ductal carcinoma in situ of the breast: a trial of the Eastern Cooperative Oncology Group[END_REF] showed that a margin of 3 mm or wider could be an adequate width for only excision without irradiation (in G1-G2 DIN patients). Regarding tumor grading, clinicians agree that patients with high-grade DIN and with close margins benefit from adjuvant radiotherapy. However, there are controversies regarding the need for radiotherapy in patients with low-grade DIN which has been resected with wide (>10 mm) negative margins.

Two prospective studies showed contradictory results: in the trial from the Dana-Farber Cancer Institute [START_REF] Wong | Prospective study of wide excision alone for ductal carcinoma in situ of the breast[END_REF][START_REF] Solin | Is excision alone adequate treatment for low-risk ductal carcinoma in situ of the breast?[END_REF], the omission of radiation therapy after wide excision alone resulted in a high cumulative LR rate of 12% at 5 years of follow-up in patients with G1 DIN. On the contrary, the EORTC 10850 trial showed that within the group of DIN, patients treated with BCS alone (with 26% of 10-year LR-free rate) in the subgroup G1 DIN after surgery alone, presented a risk of LR <10% at 10 years' follow up [START_REF] Bijker | Breast-conserving treatment with or without radiotherapy in ductal carcinoma in situ: ten-year results of European Organisation for Research and Treatment of Cancer Randomized Phase III Trial 10853 -A Study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotheraphy Group[END_REF].

In fact this is one of the more important controversies regarding the management of DIN patients: are all these patients candidates to receive RT after BCS? At the IEO in Milan, the answer to this question has been no, for more than a decade. In fact, RT is not administered to DIN patients with G1 or G2 without comedonecrosis. On the other hand, only last year, at least four significant papers (from the Saint Gallen Consensus [START_REF] Goldhirsch | Threeholds for the therapies: highlights of the Saint Gallen International Expert Consensus, on the primary therapy of early breast cancer[END_REF], the ECOG trial [START_REF] Hughes | Local excition alone without irradiation for ductal carcinoma in situ of the breast: a trial of the Eastern Cooperative Oncology Group[END_REF], the Newport Consensus Conference III [START_REF] Silverstein | Image-detected breast cancer: State-of-the-art diagnosis and treatment[END_REF], and from the National Consensus Cancer Network [91]) suggested that some DIN patient sub-groups (i.e., G1 or G2 tumors without comedonecrosis, and other low-risk sub-grups) could not be candidates to receive RT after BCS.

Another open question is related to the use of a boost after RT in DIN patients. In a multicentric, retrospective study, Omlin et al [START_REF] Omlin | Boost radiotherapy in young women with DCIS: a multicentre, retrospective study of the Rare Cancer Network[END_REF] demonstrated that an RT boost decreases LR in patients <45 years of age. The ten-year relapse-free survival was 46% for patients not receiving any RT, 72% for patients with RT without boost and 86% for patients receiving RT including a boost, thus suggesting that an RT boost should be considered in addition to surgery for BCS in patients with DIN. A French multicentric randomized trial, the first study evaluating in young DIN patients the impact of a 16 Gy boost after 50 Gy delivered to the whole breast in 25 fractions and 33 days, has started inclusions recently [START_REF] Azria | Ductal carcinoma in Situ: role of the boost[END_REF].

Intraoperative Radiotherapy (IORT).

The standard course of current external RT after BCS for DIN, delivers a total dose of 50 Gy in daily fractions of 1.8/2 Gy without boost. Intraoperative Radiotherapy (IORT), instead, is an attractive new treatment, an accelerated partial breast irradiation (APBI), and offers a new approach to irradiate the breast directly during the surgery itself when BCS is performed. The recent Consensus Statement from the American Society for Radiation Oncology agreed that IORT can be used in some particular group of breast cancer patients [START_REF] Smith | Accelerated partial breast irradiation consensus statement from the American society for radiation oncology (ASTRO)[END_REF], but there is no significant data of the use of APBI in DIN patients. The American Society of Breast Surgeons Registry Trial includes the largest published collection of DIN treated with APBI. In this series, four-year follow-up shows result similar to those with invasive cancer treated with APBI, as well as DIN treated with whole breast irradiation [START_REF] Keisch | American Society of Breast Surgeons Mammosite Radiation Therapy System registry trial: Ductal carcinoma in situ subset analysis-4 year data in 194 treated lesions[END_REF] . In some cases with DIN, IORT is also used on the nipple-areola-complex (NAC) when a NAC-sparing mastectomy in performed. This is a new surgical procedure whose risks and complications are acceptable when compared to the traditional surgical treatment of breast cancer [START_REF] Petit | Nipple sparing mastectomy with nipple areola intraoperative radiotherapy: 1001 cases of a five years experience at the European Institute of Oncology in Milan (EIO)[END_REF]. In some institutions, such as the IEO in Milan, the NAC-sparing mastectomy is associated with IORT (so-called E [START_REF] Tavassoli | Ductal carcinoma in situ: introduction of the concept of ductal intraepithelial neoplasia[END_REF]LIOT, one shot of 16 Gy) to the NAC [START_REF] Petit | Nipple sparing mastectomy with nipple areola intraoperative radiotherapy: 1001 cases of a five years experience at the European Institute of Oncology in Milan (EIO)[END_REF]. In other centres, it is performed without IORT [START_REF] Garcia-Etienne | Nipple-sparing mastectomy: initial experience at the Memorial Sloan-Kettering cancer Center and a comprehensive review of literature[END_REF][START_REF] Brachtel | Occult nipple involvement in breast cancer: clinicopathologic findings in 319 consecutive mastectomy specimens[END_REF].

Medical treatment

Two known international trials studied tamoxifen effects after BCS and RT. One of them (NASBP B-24) [START_REF] Fisher | Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomized controlled trial[END_REF], showed that tamoxifen reduces LR of DIN patients, whereas the other trial [START_REF] Houghton | UK Coordinating Committee on Cancer Research: Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial[END_REF] did not demonstrate that advantage.

Currently, systemic therapy is related to ER status and is only considered for ER-positive patients.

Barnes et al [START_REF] Barnes | A prospective study of estrogen and progesterone receptor status in ductal carcinoma-in-situ[END_REF] found that most DIN lesions are ER-positive, especially in well and moderately differentiated G1 and G2 tumors, while a large fraction of G3 and comedonecrosis DIN lack or have less marked ER expression.

Patients with ER-positive tumors

The role of tamoxifen was addressed in the above-mentioned NSABP B-24 trial [START_REF] Fisher | Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomized controlled trial[END_REF], reducing the number of overall breast cancer events by 37% (43% in invasive and 31% in noninvasive breast cancer events). This overall reduction was secondary to a 31% decrease in the incidence rate of ipsilateral breast tumors (p = 0.02) and a 47% reduction in contralateral breast cancers (p = 0.01). With regard to ER, there were 77% ERpositive patients and 23% ER-negative patients. The reduction of all breast cancer events was 58% for ERpositive patients (p. 0.0001) and 23% for ER-negative patients (p. 0.45) [START_REF] Bryan | DCIS: evidence from randomized trials[END_REF]. In women under 50 years of age, tamoxifen reduced the risk of ipsilateral breast tumors by 38% whereas in patients aged 50 and older the reduction was 22%. On the other hand, tamoxifen was found to reduce the risk of recurrence in patients with both positive and negative margins. However, even with tamoxifen there were fewer local failures in the group with negative margins (22%) than in the group with positive margins (44%) [START_REF] Fisher | Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomized controlled trial[END_REF]. The risk/benefit ratio should be considered in each patient: women undergoing BCS, premenopausal women, and postmenopausal histerectomized women are likely to achieve the greatest benefit from tamoxifen.

A possible alternative for low-risk patients could be the use of low-dose tamoxifen, as shown by three IEO papers: firstly, Decensi et al. [100], demonstrated that the effects on Ki-67 expression of lower doses of tamoxifen (1mg, 5mg/day) were proven to be comparable to those achieved with the standard dose (20mg/day), although the effects on blood biomarkers were variable. Secondly, the cases of low-dose tamoxifen were confirmed in a randomized dose-ranging trial in hormone replacement therapy (HRT) users.

A dose of 5 mg/day was the most effective and was selected for a phase III trial in HRT users; this dose modulates favourably biomakers of breast carcinogenesis and cardiovascular risk, with no increase of endometrial proliferation and menopausal symptoms [101]. Thirdly, low doses of tamoxifen were associated to a lower incidence of recurrence in patients with ER-positive and HER-2-negative disease [START_REF] Guerreri-Gonzaga | Ductal Intraepithelial Neoplasia: Postsurgical out come for 1.267 woman cared for in one single Institution over 10 years[END_REF].

At present two selective estrogen receptor modulators, tamoxifen and raloxifene, are approved in the US for reduction of breast cancer risk in high-risk women. Tamoxifen, but not raloxifene, is also approved for adjuvant therapy in patients treated with breast-conserving excision and radiation for DIN. In the NSABP-P2 prevention trial, raloxifene was as effective as tamoxifen in reducing the risk of Infiltrating Breast Cancer (IBC), but was 30% less effective than tamoxifen in reducing the risk of DIN [102].

Interestingly, the UK-A-NZ trial on adjuvant tamoxifen and radiation therapy in DIN patients found that tamoxifen did not significantly reduce the overall event rate or the rate of IBC events, but did reduce the overall rate of DIN (hazard ratio, 0.58; 95% CI, 0.49 to 0.96; P =.03) [START_REF] Houghton | UK Coordinating Committee on Cancer Research: Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial[END_REF]. The different results may be a consequence of a different patient population: the NSABP B-24 study had a higher proportion of patients younger than 50 (34% vs 10%), and tamoxifen was associated with a greater risk reduction in this age group [START_REF] Fisher | Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomized controlled trial[END_REF].

On the other hand, the impact of the NSABP B-24 results on tamoxifen use in patients with DIN was evaluated and it was reported that the overall acceptance was 41%[103]. That's why, considering a) this low acceptance, b) the controversy regarding tamoxifen benefit in ER-negative DIN and c) the side effects related to therapy with tamoxifen (that might not be acceptable to an otherwise healthy women), other agents should be evaluated for adjuvant therapy in patients with DIN.

The aromatase inhibitors (AI) group[104] reduces the risk of contralateral breast cancer by 50%

when compared with tamoxifen in adjuvant breast cancer trials. Two trials are currently evaluating the role of the aromatase inhibitor anastrozole as adjuvant therapy in patients with DIN: the NSABP B-35 and the International Breast Cancer Intervention Study-II. Furthermore, non-endocrine agents should also be considered, especially for high-grade DIN, including COX-2 inhibitors, retinoids and tyrosine kinase inhibitors such as trastuzumab [START_REF] Kuerer | DCIS: State of the science and roadmap to advance the field[END_REF].

Patients with ER-negative tumors

Patients with ER-negative DIN are not candidates for tamoxifen. However, a preventive strategy is needed to reduce the incidence of ER-negative tumors, which contribute to breast cancer-related mortality. Some studies suggest that the administration of COX-2 inhibitors to patients with ER-negative DIN may represent such a strategy[105-106]. The association of COX-2 with HER-2 expression, is a surrogate marker of an aggressive DIN phenotype and links non-estrogen growth factor-signaling pathways with COX-2 overexpression. There is no evidence that herceptin, the antibody approved for the treatment of HER-2positive invasive breast cancer, is beneficial for patients with DIN (even if the lesion is HER-2 positive). At present, Lapatinib, an approved oral agent for HER-2-positive breast cancer, is under investigation for DIN [107]. There is an ongoing non-randomized clinical trial in the USA to find out whether a single dose of the drug Herceptin (trastuzumab) administered before surgery kills cancer cells or slows the growth of cancer cells in women who have DIN, and to evaluate the effect of a single dose of Herceptin on the apoptotic index of Her-2/neu-overexpressing DIN [108]. So, COX-2 inhibitors and trastuzumab could play a role in preventing invasive breast cancer in patients with ER-negative DIN [109] .

New agents and new strategies for medical treatment?

The biological alterations controlling DIN proliferation are not clear. The fact that antiestrogens are not effective in ER-negative breast cancers, suggests that other factors are promoting proliferation in ERnegative DIN. Mutated or over expressed tyrosine kinases are frequently associated with tumor development.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TKI) that is involved with mitogenesis and is expressed in ER-negative DIN. It has been hypothesized that EGFR is central in driving proliferation in ER-negative/EGFR-positive DIN [110]. Preclinical studies have demonstrated that EGFR-TKI therapy reduces proliferation and increases apoptosis in the epithelium, and, either alone or combined with antiestrogen therapy, EGFR-TKI may represent a novel form of chemoprevention. Clinical trials should investigate evidence that breast cancers that have become tamoxifen-resistant use the EGFR signalling pathway which would suggest that the combination of tamoxifen and Iressa would be potent at chemopreventing cancer [111].

Preclinical studies have involved the development of genetically engineered mice (GEMs) to investigate cancer prevention. GEMs harbour activated oncogenes and/or inactivated tumour-suppressor genes that predispose them to develop cancer [112]. These studies provide evidence of the great potential of vaccines to induce prevention of cancer and demonstrate that a vaccine-alerted immune system effectively blocks the carcinogenesis process driven by the overexpression of specific oncogenes [112][113][114]. In GEMs that are transgenic for the rat HER2 oncogene, mammary and salivary carcinogenesis is driven by HER2receptor expression, whereas down-modulation or loss of the receptor blocks tumor progression [115][116][117].

As preventive vaccines operate during the early phases of carcinogenesis, effective inhibition of the HER2 receptor arrests the whole process and renders the selection of HER2-receptor-loss variants unlikely.

Preclinical data available in GEMs models cannot be directly translated to humans for preventive treatments; these require more attention since the plan would be to vaccine a healthy individual.

Vaccination against selected oncoantigens of healthy people who have a specific genetic risk of cancer, who have been exposed to an exogenous carcinogen, or who bear multifocal pre-neoplastic lesions would provide the most appropriate scenario. New clinical trial targeting HER-2/neu using a dendritic cell vaccine in a neoadjuvant trial for treatment of DIN patients are ongoing [118] . . Starting from the hypothesis that trastuzumab will have substantial activity against DIN, some trials are studying the use of trastuzumab a) after breast-conserving surgery with negative margins, for patients with HER-2/neu-overexpressing DIN (NSABP phase III randomized trial) [START_REF] Kuerer | DCIS: State of the science and roadmap to advance the field[END_REF]; b) before surgery as neoadjuvant treatment in DIN patients (at the MDAnderson) [START_REF] Kuerer | DCIS: State of the science and roadmap to advance the field[END_REF] and c) some studies used trastuzumab during breast irradiation [119] .

MANAGEMENT OF DIN PATIENTS AT THE Istituto Europeo di Oncologia (IEO)

Between June 1994 and December 2009, a total of 24,668 breast cancer patients were treated at the IEO, in Milan, Italy; 4,350 of these (17,6%) were DIN patients. About 80% of these patients received BCS and 20% mastectomy, in accordance with the following IEO guidelines for the management of DIN patients.

Guidelines for surgery

The surgical decisions depend on the amount of breast tissue with DIN, the size of the breast, other health and physical factors and the patient's preferences.

a. Indications for breast conservative surgery (BCS):

BCS is performed in cases of detected mammographically DIN lesions or palpable masses, without evidence of multicentricity or diffuse malignant calcifications. The extent of DIN for BCS should be near 3-4 cm but this depends mainly on an appropriate cosmetic final result (the rate tumor/breast side should be considered).

In cases of non-palpable lesions the procedure is as follows: a) for small or non-palpable masses or for clusters of microcalcifications, the surgical excision is guided by the ROLL (Radioguided Occult Lesions Localization), a technique developed at the IEO[120]; b) for extended or diffuse microcalcifications, the skin over the area of microcalcifications is mapped by the radiologist, and the surgeon takes into consideration the extension of the mapped area to decide if performing either BCS or mastectomy.

b. Indications for mastectomy

Mastectomy is performed in cases of a) large and extended DIN areas, in which BCS would create a bad final cosmetic result; b) multiple areas of DIN in the same breast, i.e., multicentricity or extended multifocality. The NAC-sparing mastectomy, with IORT (ELIOT) applied to the nipple areola complex is more often proposed. c. Breast reconstruction after mastectomy: When mastectomy is performed, immediate breast reconstruction is always proposed to the patient, via different techniques (expander, definitive prostheses or flap) depending on the size of the breast, the local situation, the possible contraindications and the wishes of the patient.

d. Surgical treatment of recurrences:

The decision to perform a new BCS or a mastectomy after a LR, depends on the cosmetic final results and the patient's opinion: both are important elements to be considered when making the decision. Regarding the width of surgical margins, the IEO considers 1 mm as a limit of oncological safety.

e. Axillary management:

AD is never indicated as a first surgical treatment in DIN patients. SLNB is only considered: a) in all cases of mastectomy; b) when there exists a strong doubt of invasion or microinvasion (i.e., in cases of large solid lesions or diffuse microcalcifications) and c) after a DIN diagnosis by core or mammotome biopsy. If the SLNB is positive, AD should be performed only in cases with mammary invasion on final pathologic evaluation.

Guidelines for radiotherapy

DIN patients are divided into two groups based on the pathological features as follows: on the one hand, radiotherapy (50 Gy to the whole breast, without boost) is given to all patients with Grade 3 and with Grade 2 and comedonecrosis, considering that radiotherapy is more effective over cells in phase G2 of the cellular cycle. On the other hand, no radiotherapy after BCS is indicated in Grade 1 and Grade 2 in patients without comedonecrosis; in these cases, RT is considered an overtreatment.

Guidelines for systemic treatment

DIN patients are divided according to their ER status: a. ER-positive patients: premenopausal and postmenopausal patients receive low-dose tamoxifen (20mg/week) or participate in the IBIS-2 trial, which has 2 arms: after quadrantectomy, Tamoxifen 20mg/day) vs Anastrazole (100mg/day); after mastectomy, Anastrazole (100mg/day) vs placebo.

b. ER-negative patients:

The standard for ER-negative both pre and postmenopausal patients is no treatment.

As an alternative a randomized pilot study of pharmacoprevention is proposed to high risk patients. This study includes 150 patients divided into three groups, receiving during one year: a) Nimesulide (100mg/day), b) Sinvastatina (20mg/day) and c) placebo

FINAL CONSIDERATIONS

Data for irradiation in DIN patients are strong and consistent, after the results of the known randomized trials [START_REF] Bryan | DCIS: evidence from randomized trials[END_REF][START_REF] Bijker | Breast-conserving treatment with or without radiotherapy in ductal carcinoma in situ: ten-year results of European Organisation for Research and Treatment of Cancer Randomized Phase III Trial 10853 -A Study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotheraphy Group[END_REF][START_REF] Houghton | UK Coordinating Committee on Cancer Research: Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial[END_REF][START_REF] Emdin | SwDCIS: Radiotherapy after sector resection for ductal carcinoma in situ of the breast. Results of a randomized trial in a population offered mammography screening[END_REF], while the recommendations for adjuvant tamoxifen must be reviewed in light of the differences in reported outcomes from two mentioned randomized trials [START_REF] Fisher | Tamoxifen in treatment of intraductal breast cancer: National Surgical Adjuvant Breast and Bowel Project B-24 randomized controlled trial[END_REF][START_REF] Houghton | UK Coordinating Committee on Cancer Research: Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial[END_REF]. Treatment with radiation and tamoxifen can reduce the risk of recurrence, including invasive recurrence, but for the overall population of patients with DIN, such risk reduction comes with the knowledge that most patients would be overtreated.

Moreover, some questions remain open and call for new international consensus and agreements, The variations on DIN management in practice, demonstrated, for example, by the ASTRO/ESTRO investigation [START_REF] Katz | Patterns and Correlates of Local Therapy for Women With Ductal Carcinoma-In-Situ[END_REF], the UK Sloane Project [START_REF] Bishop | Significant Variation in the management of non invasive breast carcinoma in the UK: Initial findings from the Sloane Project, a UK National Prospective Audit[END_REF]and the French experience [START_REF] Cutuli | Breast conserving surgery with or without radiotherapy vs mastectomy for ductal carcinoma in situ: French survey experience[END_REF] show that breast cancer professionals are not convinced about the optimal method to treat DIN patients. New large trials are needed in order to evaluate the best management of DIN which is in most respects still complex and controversial.

  i.e.: a) Regarding surgery: Which margin width can be considered oncologically safe(1 mm? 3 mm? 10 mm?). Are micrometastasis in a SLNB of any clinical significance in DIN patients? Are macrometastasis in DIN patients responsible for long term axillary recurrences? Could macrometastasis be the only situation that justifies a complete axillary dissection? Is it necessary to perform a complete axillary dissection in cases of SLNB positivity in DIN patients? If not, why perform SLNB in this group of patients? b) Regarding radiotherapy: Which group of DIN patients does not need RT after BCS? Do young DIN patients benefit from the application of a boost after 50-Gy radiotherapy? Is RT really needed when there are safe margins of 10 mm or more, regardless of tumor size, nuclear grade and the presence or absence of comedo-necrosis? c) Regarding systemic therapy: Are collateral effects of tamoxifen justified in low-risk ER-positive DIN patients? How should ER-negative DIN patients be treated? What is the potential role of vaccines and Trastuzumab in cancer prevention (including DIN)?.

AUTHORS' DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST:

The authors indicate no potential conflicts of interest.

ACKNOWLEDGEMEHNTS

The authors would like to thank Marina Nordiana Baruzzi and Maria Grazia Villardita for the preparation of the manuscript and William Russell-Edu for the MEDLINE research and editing.