Mohamed Kamal

Abeer M Shaaban

Liqun Zhang

Clare Walker

Sally Gray

Nalin Thakker

Carmel Toomes

Valerie Speirs

Sandra M Bell

A E Abeer

M Shaaban

A E Liqun

Zhang Ae

Clare Walker Ae

A E Nalin

Thakker Ae

A E Valerie

Speirs Ae

Á C Walker

Á C Toomes

Á S M Bell

Á S Gray

Á N Thakker

Loss of CSMD1 expression is associated with high tumour grade and poor survival in invasive ductal breast carcinoma

Keywords: CSMD1, Breast cancer, Prognosis, Differentiation, Immunohistochemistry

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Introduction

Breast cancer is one of the leading causes of death in women [START_REF] Bray | Estimates of cancer incidence, mortality in Europe in 1995[END_REF][START_REF] Parkin | Estimating the world cancer burden: globocan 2000[END_REF]. It is a complex and heterogeneous disease at the molecular level. Abnormal expression of growth factor receptors, cell-cycle regulatory molecules and cell-adhesion proteins frequently occurs [START_REF] Fabre-Lafay | Nectin-4 is a new histological and serological tumor associated marker for breast cancer[END_REF]. Investigating the expression and functions of these molecules, and their association with patient outcome, is extremely important to aid the understanding of the disease process and assist in clinical decision-making [START_REF] Alford | Cell adhesion molecules in the normal and cancerous mammary gland[END_REF].

CUB and SUSHI multiple domains-1 (CSMD1) encodes multiple mRNA transcripts with the largest being 14.3 Kb long. The gene spans over 2 Mb of genomic DNA and contains 71 exons, which encode a 3,565 amino acid protein consisting of 14 CUB domains and 28 SUSHI domains. CSMD1's function is unknown; however, CUB and SUSHI domains are thought to be involved in receptorligand interactions. The full-length CSMD1 protein is predicted to be a membrane protein with an extracellular region containing the CUB and SUSHI domains, a single transmembrane domain and a short cytoplasmic domain that contains a tyrosine phosphorylation site. From these features, one could predict that CSMD1 is a receptor or coreceptor for unknown ligand(s) and is involved in signal transduction [START_REF] Sun | Transcript map of the 8p23 putative tumor suppressor region[END_REF][START_REF] Marinad | Structure and flexibility of the multiple domain proteins that regulate complement activation[END_REF].

Many lines of evidence indicate CSMD1 could be a tumour suppressor gene. Firstly, CSMD1 spans chromosomal band 8p23, which is known to exhibit frequent deletions in many cancers, including breast cancer [START_REF] Sigbjornsdottir | Chromosome 8p alterations in sporadic and BRCA2 999del5 linked breast cancer[END_REF][START_REF] Yokota | Localization of a tumor suppressor gene associated with the progression of human breast carcinoma within a 1-cm interval of 8p22-p23.1[END_REF][START_REF] Emi | Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer[END_REF][START_REF] Wu | Deletion mapping defines three discrete areas of allelic imbalance on chromosome arm 8p in oral and oropharyngeal squamous cell carcinomas[END_REF][START_REF] Toomes | The presence of multiple regions of homozygous deletion at the CSMD1 locus in oral squamous cell carcinoma question the role of CSMD1 in head and neck carcinogenesis[END_REF][START_REF] Sunwoo | Evidence for multiple tumor suppressor genes on chromosome arm 8p in supraglottic laryngeal cancer[END_REF][START_REF] Scholnick | Chromosome 8 allelic loss and the outcome of patients with squamous cell carcinoma of the supraglottic larynx[END_REF][START_REF] Fujiwara | Evidence for the presence of two tumor Suppressor genes on chromosome 8p for colorectal carcinoma[END_REF][START_REF] Ishwad | Frequent allelic loss and homozygous deletion in chromosome band 8p23 in oral cancer[END_REF]. Secondly, loss of CSMD1 has been reported in many cancers. At the DNA level, an array-based comparative genomic hybridization (CGH) study detected the loss of CSMD1 in a large proportion of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (SCC) and breast cancers [START_REF] Ma | Characterization CSMD1 in a large set of primary lung, head and neck, breast and skin cancer tissues[END_REF]. At the RNA level, quantitative real-time PCR (qPCR) detected the loss of CSMD1 expression in HNSCCs and lung SCCs [START_REF] Ma | Characterization CSMD1 in a large set of primary lung, head and neck, breast and skin cancer tissues[END_REF], liver [START_REF] Midorikawa | Allelic imbalances and homozygous deletion on 8p23.2 for stepwise progression of hepatocarcinogenesis[END_REF], colorectal [START_REF] Farrell | Somatic mutations to CSMD1 in colorectal adenocarcinomas[END_REF] and prostate cancer [START_REF] Henshall | Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse[END_REF][START_REF] Paris | Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors[END_REF]. Thirdly, CSMD1 shares homologies with many other proteins playing roles in cancer progression by the virtue of its CUB and SUSHI domains such as SEZ6L and DMBT1. SEZ6L is homozygously deleted in small cell lung cancer cell lines [START_REF] Sun | Transcript map of the 8p23 putative tumor suppressor region[END_REF][START_REF] Nishioka | Identification of a 428-kb homozygously deleted region disrupting the SEZ6L gene at 22q12.1 in a lung cancer cell line[END_REF][START_REF] Kraus | CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues[END_REF], and DMBT1 expression is reduced in malignant brain tumours [START_REF] Mollenhauer | DMBT1, a new member of the SRCR superfamily, on chromosome 10q25. 3-26.1 is deleted in malignant brain tumours[END_REF], lung cancer [START_REF] Wu | Expression of DMBT1, a candidate tumor suppressor gene, is frequently lost in lung cancer[END_REF] and breast cancer [START_REF] Mollenhauer | Carcinogen inducibility in vivo and down-regulation of DMBT1 during breast carcinogenesis[END_REF]. A variety of developmental proteins have been implicated in carcinogenesis, and CUB and SUSHI domains are also found in many of these, including bone morphogenetic proteins, tolloid, neuropilin and spermadhesins [START_REF] Bork | The CUB domain: a widespread module in developmentally regulated proteins[END_REF][START_REF] Topfer-Petersen | Spermadhesins: a new protein family. Facts, hypotheses and perspectives[END_REF][START_REF] Lu | HNPCC associated with germline mutation in the TGF-[beta] type II receptor gene[END_REF][START_REF] Su | ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma[END_REF].

The genomic localisation, expression pattern and protein homology of CSMD1 suggest that it is a tumour suppressor gene. In this study, we investigate its role in breast cancer progression. We have generated an anti-CSMD1 antibody and identified the expression pattern of CSMD1 in 275 IDC patients with a follow-up period of 152 months. Univariate and multivariate Cox regression analyses have been performed to correlate CSMD1 expression with OS and DFS. Loss of CSMD1 expression was associated with high tumour grade and poor survival outcome.

Materials and methods

Patients

All patients gave informed consent to participate in this study, and ethical approval was obtained from the Local Research Ethics Committee of the Leeds Teaching Hospitals (LTH), NHS Trust, Leeds, UK. Paraffin blocks from two independent cohorts of breast cancer cases were retrieved from the archives of the pathology departments of the LTH, NHS Trust. The first cohort containing 50 cases was designated as the training set, and the second cohort containing 446 cases was used as the validation set. These cases were selected from patients presenting between 1987 and 2005. Only patients with IDC of no special type, NST (40 in the training set and 331 in the validation set) were included in the study due to the small numbers of patients with other tumour subtypes. Immunohistochemical data were available on all the 40 patients of the training set. While in the validation set, from 331 cases, immunohistochemical data were available on a maximum of 275 patients due to loss of some tissue microarray (TMA) cores during antigen retrieval and staining, a well-recognised phenomenon associated with TMA immunohistochemistry. Clinical history and tumour characteristics (age, tumour type, size, histological grade, lymph node status and Nottingham prognostic index (NPI)) were available. DFS was defined as the interval (months) from primary surgical treatment to the first loco regional or distant recurrence. OS was taken as time (months) from primary surgical treatment to time of death from breast cancer.

Generation of anti-CSMD1 antibody

Two peptide sequences HFTSDSNHRRKGFN and QRVTE TLAAWSDHR from the N-terminus of human CSMD1 were selected as targets for generating a polyclonal antibody. In vitro synthesis of the peptides and immunisation of chickens with keyhole limpet hemocyanine-conjugated CSMD1 peptides, followed by Ig purification from the egg yolks, were performed by Abcam (Cambridge, UK).

Anti-CSMD1 antibody validation

Initially, the binding specificity of the anti-CSMD1 antibody was tested by an enzyme-linked immunosorbent assay (ELISA) against the BSA-conjugated peptides stated earlier [START_REF] Williams | Characterization of an antibody to the human melatonin mt1 receptor[END_REF]. Antibody specificity was also confirmed on histological tissue sections by pre-absorption with the peptides. The antibody was diluted to its working concentration and incubated with a tenfold excess of relevant peptide overnight at 4°C before application to the tissue.

For further validation of the specificity and sensitivity of the antibody and to control the effects of formalin fixation and antigen retrieval, CSMD1 was silenced in the MCF10A cell line (ATCC, Manassas, VA, USA) using short hairpin RNA (shRNA). Cells were plated at 1 9 10 6 cells/ml in 6well plates. Next day, they were transfected with pRS vector (HuSH TM , ORIGENE, Rockville, MD, USA) containing shRNA sequences against CSMD1 mRNA using Lipofectamine TM 2000 (Invitrogen, Paisley, UK). Transfected cells were selected by incubation in DMEM-F12 media (Invitrogen) that contains 0.5 lg puromycin (SIGMA-ALDRICH, Dorset, UK) for 2 weeks. To obtain a homogenous cell population and to avoid copy number variation, single cells were plated into the wells of a 96-well plate and grown up to give single-cell colonies. All colonies were then screened by qRT-PCR. Colonies that showed the highest level of knockdown were formalin fixed and paraffin embedded and used as a control for antibody optimisation. To optimise the CSMD1 staining protocol, full sections of paraffin-embedded tissue from both normal and tumour samples were stained using a range of primary antibody dilutions, incubation times and antigen retrieval methods.

Tissue microarrays construction and immunohistochemistry

CUB and SUSHI multiple domains-1 (CSMD1) was stained on full sections of patient's samples in the training set. Whereas in the validation set, tissue microarrays (TMAs) were constructed from the breast cancer patients using 0.6 mm cores selected from the most representative tumour area. Immunohistochemical analysis of CSMD1 expression was performed with the chicken anti-CSMD1 antibody at a 1:3,000 dilution. TMA sections measuring 4 lm were deparaffinised in graded alcohols. Endogenous peroxide activity was inhibited by immersing the sections in 0.5% H 2 O 2 . Antigen retrieval was carried out by pressure-cooking slides for 90 s in 1% Antigen Unmasking Solution (Vector Laboratories Ltd, Peterborough, UK). A casein incubation (1/2 dilution) was also carried out to reduce non-specific staining (Vector Laboratories). Sections were incubated with the antibody for 2 h at room temperature. Bound antibody was detected with EnVision TM polymer (DAKO, Ely, Cambridgeshire, UK), using diaminobenzine as the substrate (Sigma, Poole, Dorset, UK). Sections were counterstained with Mayer's haematoxylin (VWR International Ltd, Poole, UK). Negative controls, in which the pre-immune serum was applied, and positive controls of normal breast tissue were included in each batch of immunohistochemistry.

Immunohistochemical evaluation

Cytoplasmic and membranous staining was scored as a percentage of the positive cells in relation to the total number of tumour cells present. Expression status was dichotomised and the cut-off point that showed the biggest difference between patients groups with regard to survival was chosen. Cases were scored blindly and independently by two authors (M. K. and A. M. S., a specialist consultant breast histopathologist). Discordant results were re-evaluated jointly to reach consensus.

Statistical analysis

Using SPSS version 15.0, Pearson correlation was used to correlate CSMD1 staining as a percentage of the positive cells versus tumour size, age of patient, number of positive nodes, OS time and DFS time as continuous variables. The chi-square test/Fisher exact test or Mann-Whitney U-test (non-parametric) was used for comparisons between groups/categorical data as needed. Survival curves were calculated using the Kaplan-Meier method, and differences between groups were analysed by the log-rank test and confirmed by applying Cox regression model. The optimal cut-off for immunohistochemical expression was evaluated by testing different cut-off values versus OS using Kaplan-Meier curves. For systematic modelling, a stepwise multivariable Cox regression model was used, testing the independent prognostic relevance of CSMD1 immunoreactivity. All statistical tests were two-sided, and a P B 0.05 value was considered statistically significant.

Results

Anti-CSMD1 antibody characterisation

Anti-CSMD1 antibody specificity was confirmed by ELISA (supplementary Fig. 1a). The staining pattern of CSMD1 was abolished when the antibody was incubated in the presence of the peptides it was raised against (supplementary Fig. 1b). To test the validity of the antibody to stain paraffin-embedded tissues, the antibody was tested on paraffin-embedded CSMD1 shRNA stable MCF10A cells that showed very weak staining relative to the control shRNA (supplementary Fig. 1c). Anti-CSMD1 antibody was then optimised to stain paraffin-embedded tissues on full sections. Since the CSMD1 staining was homogenous on full sections, we were able to use it to stain TMAs. Optimal results were identified at a 1:3,000 antibody dilution and pressure cooking for 90 s in unmasking solution for antigen retrieval. The vast majority of normal tissues showed CSMD1 staining providing positive controls.

Patient's characteristics

This study included two groups of patients after excluding non-IDC tumour subtypes, core loss and unscorable cores.

The first group had 40 patients and is considered as a training set, while the second group had 275 patients and is considered as a validation set. Detailed patient characteristics of the validation set are summarised in Table 1.

We dichotomised the percentage of CSMD1 staining. A cut-off point of 30% showed the most significant difference between the two groups of patients (low and high CSMD1) in overall survival in the validation study and 10% in the training study. There was not enough follow-up information to meet the statistical analysis requirements in the training set. However, in the validation set, during a median followup period of 107 months (range, 1-152 months), death occurred in 99/275 patients (36%) and metastasis or/and recurrence occurred in 59/275 patients (21.5%).

CSMD1 expression in normal and tumour breast tissues

CUB and SUSHI multiple domains-1 (CSMD1) expression pattern was determined in breast epithelium by immunohistochemistry on 10 paraffin embedded normal breast samples. The staining pattern of CSMD1 was luminal, membranous and cytoplasmic. Luminal staining is shown in Fig. 1a. In the tumour samples, CSMD1 expression exhibited both cytoplasmic and cell membrane staining (Fig. 1b,c).

In the training set, low CSMD1 expression was identified in 22/40 (55%). Low CSMD1 expression is shown in an IDC sample (Fig. 1e). While in the validation set, 79/ 275 (28.7%) of cases showed low CSMD1 expression. Low and high CSMD1 expression in TMA samples with IDC are shown in Fig. 1f-i. The adjacent normal epithelium and blood vessels, which have been used as internal controls, showed high CSMD1 staining in the majority of cases. Interestingly, high CSMD1 expression is observed in the normal tissues, and the intact ducts in the tumour samples exhibit low CSMD1 expression (Fig. 1d).

As chromosome 8p, and in particular 8p23, is frequently deleted in tumours, we wanted to check whether the low CSMD1 expression we were observing in our breast cancer samples was caused by the specific loss of CSMD1 and not simply a consequence of larger non-specific deletions in this region. We did this by retesting a subset of our samples with an antibody raised to MCPH1 (encoded by a neighbouring 8p23 gene (http://genome.ucsc.edu/index.html)). In total, 28 samples were stained with both antibodies. Low expression of CSMD1 was detected in 64% of cases (18/ 28), while low expression of MCPH1 was detected in 25% of cases (7/28). Low expression of both proteins in the same sample was shown in 18% of cases (5/28).

Relation of CSMD1 expression to histological parameters

In each patient sample, the level of CSMD1 expression was correlated with the severity of the disease. Low expression of CSMD1 was significantly associated with higher tumour grade (P \ 0.001 in the training set and P = 0.003 in the validation set). In the training set, low expression of CSMD1 was identified in 4.45% (1/22) in grade 1, 40.9% (9/22) in grade 2 and 54.4% (12/22) in grade 3. In the validation set, low expression of CSMD1 was identified in 21.5% (17/79), 25.58% (21/79) and 51.89% (41/79) in grade 1, 2, and 3, respectively. Low or high CSMD1 expressing tumours did not differ significantly for the distribution of other clinicopathological variables (Table 2).

Univariate and multivariate analyses

In the validation set, low expression of CSMD1 was significantly associated with shorter OS, with an estimated median OS of 65 months (95%CI: 38.8-91.1) compared to 135 months in patients with high CSMD1 expression (P = 0.01; Fig. 2). There was no significant association between CSMD1 expression and DFS (P = 0.48). However, the mean DFS in patients with low CSMD1 was 108.7 months (95%CI: 93.4-123.9) compared to 120 months (95%CI: 110.9-128.1) of those who highly express CSMD1 (Fig. 3). Univariate analysis showed significant association between CSMD1 expression and OS (HR = 0.607, 95%CI: 0.4-0.91, P = 0.018; Table 3) but not with DFS (HR = 0.81, 95%CI: 0.46-1.43, P = 0.48; Table 4).

A multivariate Cox regression model was carried out to examine the prognostic impact of CSMD1 expression on OS and DFS in relation to other established clinicopathological and tumour-biological factors such as tumour grade, lymph node status and NPI. CSMD1, together with NPI, appeared to be independent predictors of OS (HR = 0.6, 95%CI: 0.3-0.9, P = 0.03; Table 3). However, unlike tumour grade, CSMD1 was not shown to be an independent predictor of DFS (HR = 0.84, 95%CI: 0.46-1.5, P = 0.573; Table 4).

Discussion

The short arm of chromosome 8p is frequently deleted in a wide range of cancers including breast cancer [START_REF] Sigbjornsdottir | Chromosome 8p alterations in sporadic and BRCA2 999del5 linked breast cancer[END_REF][START_REF] Yokota | Localization of a tumor suppressor gene associated with the progression of human breast carcinoma within a 1-cm interval of 8p22-p23.1[END_REF][START_REF] Emi | Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer[END_REF][START_REF] Wu | Deletion mapping defines three discrete areas of allelic imbalance on chromosome arm 8p in oral and oropharyngeal squamous cell carcinomas[END_REF][START_REF] Toomes | The presence of multiple regions of homozygous deletion at the CSMD1 locus in oral squamous cell carcinoma question the role of CSMD1 in head and neck carcinogenesis[END_REF][START_REF] Sunwoo | Evidence for multiple tumor suppressor genes on chromosome arm 8p in supraglottic laryngeal cancer[END_REF][START_REF] Scholnick | Chromosome 8 allelic loss and the outcome of patients with squamous cell carcinoma of the supraglottic larynx[END_REF][START_REF] Fujiwara | Evidence for the presence of two tumor Suppressor genes on chromosome 8p for colorectal carcinoma[END_REF][START_REF] Ishwad | Frequent allelic loss and homozygous deletion in chromosome band 8p23 in oral cancer[END_REF]. Interestingly, 8p23 deletions are associated with poor survival in patients with various types of cancers including breast cancer [START_REF] Wu | Deletion mapping defines three discrete areas of allelic imbalance on chromosome arm 8p in oral and oropharyngeal squamous cell carcinomas[END_REF][START_REF] Scholnick | Chromosome 8 allelic loss and the outcome of patients with squamous cell carcinoma of the supraglottic larynx[END_REF][START_REF] Fujiwara | Evidence for the presence of two tumor Suppressor genes on chromosome 8p for colorectal carcinoma[END_REF][START_REF] Paris | Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors[END_REF][START_REF] Muscheck | High density deletion mapping of bladder cancer localizes the putative tumor suppressor gene between loci D8S504 and D8S264 at chromosome 8p23.3[END_REF][START_REF] Washburn | 8pter-p23 deletion is associated with racial differences in prostate cancer outcome[END_REF][START_REF] Bockmu ¨hl | Association of 8p23 deletions with poor survival in head and neck cancer[END_REF][START_REF] Weber-Mangal | Breast cancer in young women (\ or =35 years): genomic aberrations detected by comparative genomic hybridization[END_REF][START_REF] Wright | Frequent loss of heterozygosity and three critical regions on the short arm of chromosome 8 in ovarian adenocarcinomas[END_REF]. The properties of CSMD1 make it one candidate for the putative 8p23 tumour suppressor genes [START_REF] Sun | Transcript map of the 8p23 putative tumor suppressor region[END_REF]. In this study, we have undertaken the first investigation of the role of CSMD1 in breast cancer. We have developed an anti-CSMD1 antibody and investigated its protein distribution in normal and malignant breast tissues.

The specificity of our anti-CSMD1 antibody was confirmed by ELISA and incubation in the presence/absence of the peptides it was raised against. Unfortunately, our antibody is not suitable for western blot analysis. However, we have silenced CSMD1 in a breast cell line (MCF10A) using shRNA and shown that these cells show a much weaker staining pattern with the CSMD1 antibody in comparison with control cells.

In normal breast duct epithelial cells, luminal, membranous and cytoplasmic CSMD1 staining was observed. In the tumour cells, membranous and/or cytoplasmic CSMD1 staining was identified. The luminal and cytoplasmic expression may represent expression and/or secretion of protein(s) encoded by alternatively spliced CSMD1 transcripts. The cytoplasmic CSMD1 expression may alternatively represent internalisation of the full-length CSMD1 protein. Indeed, the cytoplasmic tail of CSMD1 contains a receptor internalisation motif (FENPMYDT/RN motif) that could potentially mediate clathrin-dependent endocytosis [START_REF] Paccaud | Clathrin-coated pit-mediated receptor internalization. Role of internalization signals and receptor mobility[END_REF]. The CSMD1 membrane-staining pattern is consistent with the presence of a transmembrane domain and is in agreement with its putative role as a receptor or coreceptor playing a role in signal transduction.

In samples that contained both normal and tumour tissues, we observed higher CSMD1 expression in the normal tissues and intact ducts in comparison with the surrounding tumour cells. This raises the question of whether reduction of CSMD1 expression is a consequence or a cause of the disrupted tissue morphology. Studies on the rat ortholog of CSMD1 have shown that CSMD1 expression is low in brain regions exhibiting high levels of cell proliferation and very high in regions of neuronal differentiation, suggesting that CSMD1, like neuropilin and other neuronal growth cone proteins, may be involved in mechanisms of signal transduction, substrate adhesion and/or motility that help guide axons towards their synaptic targets during development [START_REF] Kraus | CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues[END_REF].

Initially, we recorded low CSMD1 expression in 55% (22/40) of the training set. Our larger validation set also showed a reduction in CSMD1 expression, however: the percentage loss dropped to 28.7% (79/275). Our results agree with other studies that report reduced CSMD1 mRNA expression in prostate cancer, hepatocellular carcinoma, HNSCCs and lung SCC [START_REF] Midorikawa | Allelic imbalances and homozygous deletion on 8p23.2 for stepwise progression of hepatocarcinogenesis[END_REF][START_REF] Henshall | Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse[END_REF][START_REF] Paris | Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors[END_REF].

In this study, we evaluated the protein expression of CSMD1 in breast cancer but have not experimentally determined the way in which the CSMD1 gene may be lost or inactivated. Epigenetic modification of the genomic DNA preceding the first exon of CSMD1 has been shown to be associated with its silencing in squamous cancers [START_REF] Richter | Epigenetic inactivation and aberrant transcription of CSMD1 in squamous cell carcinoma cell lines[END_REF], and aberrant splicing and point mutations have also been shown to lead to loss of CSMD1 in a variety of cancers [START_REF] Ma | Characterization CSMD1 in a large set of primary lung, head and neck, breast and skin cancer tissues[END_REF][START_REF] Farrell | Somatic mutations to CSMD1 in colorectal adenocarcinomas[END_REF]. However, deletion of the genomic region encompassing CSMD1 is probably the most common mechanism. Chromosome 8p12-ptel deletions are commonly found in multiple cancers and intragenic homozygous deletions in CSMD1 have been reported in oral SCC [START_REF] Toomes | The presence of multiple regions of homozygous deletion at the CSMD1 locus in oral squamous cell carcinoma question the role of CSMD1 in head and neck carcinogenesis[END_REF]. Similarly, array-based CGH studies have detected loss of CSMD1 in HNSCCs, lung SCC and breast cancers, cutaneous SCC and basal cell carcinomas. While we believe deletion of CSMD1 is the most common mechanism of inactivation, we wanted to rule out that we were detecting random deletion of 8p23. To prove this, we stained a cohort of our IDCs with an antibody for a neighbouring protein, MCPH1. A far higher proportion of samples showed low CSMD1 expression compared to MCPH1 and only 18% of these cases showed reduced expression of both proteins, indicating that reduction of CSMD1 expression is specific.

Our results revealed that low CSMD1 expression was significantly associated with high tumour grade in both our sample sets. In the validation set, reduction of CSMD1 expression was significantly associated with shorter OS. This agrees with other studies that have reported an association between loss of the 8pter-p23 region or CSMD1 and poor survival and high stage disease in prostate, bladder and head and neck cancer patients. This suggests that CSMD1 may be a marker of advanced stage disease and disease recurrence [START_REF] Paris | Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors[END_REF][START_REF] Washburn | 8pter-p23 deletion is associated with racial differences in prostate cancer outcome[END_REF][START_REF] Bockmu ¨hl | Association of 8p23 deletions with poor survival in head and neck cancer[END_REF][START_REF] Wright | Frequent loss of heterozygosity and three critical regions on the short arm of chromosome 8 in ovarian adenocarcinomas[END_REF].

Multivariate Cox regression model showed that CSMD1 expression and NPI both appeared to be independent predictors of OS. This result agrees with a study in supraglottic SCC where allelic loss at 8p23 appears to be a statistically significant independent predictor of poor prognosis [START_REF] Scholnick | Chromosome 8 allelic loss and the outcome of patients with squamous cell carcinoma of the supraglottic larynx[END_REF]. However, unlike tumour grade, CSMD1 expression was not shown to be an independent predictor of DFS. This result is in contrast to an earlier study of prostate cancer that showed that loss at 8p23.2 is a predictive indicator of postoperative recurrence independent of stage and grade [START_REF] Henshall | Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse[END_REF]. Further large confirmatory studies are warranted to determine the potential use of CSMD1 expression assessment of clinical practice in breast and other cancer types.

In summary, the predominant staining pattern of CSMD1 was identified on the luminal epithelial cells, which supports the idea that CSMD1 may be a receptor or co-receptor involved in the process of signal transduction. Moreover, the high CSMD1 expression levels observed in the well-differentiated tumours compared to the poorly Together with the multivariate analysis, which identified CSMD1 as an independent prognostic factor for OS, the present results provide clinical evidence to support the idea that CSMD1 is a tumour suppressor gene, which may prove to be a new prognostic biomarker.

Fig. 1

 1 Fig. 1 Immunohistochemical analysis of CSMD1 expression in normal and malignant breast samples. a Normal mammary duct showing luminal cytoplasmic expression of CSMD1 (arrow) (940). b Invasive ductal carcinoma showing cytoplasmic staining (arrow) (940). c Invasive ductal carcinoma showing strong membranous staining (940). d Invasive ductal carcinoma, the malignant cells shows no expression, but the normal duct shows cytoplasmic staining (arrow) (940). e Invasive ductal carcinoma with no CSMD1 expression. Blood vessels (internal controls) positive expression

Fig. 2 Fig. 3

 23 Fig. 2 Comparison of overall survival based on CSMD1 status in the validation set

Table 1

 1 Tumour characteristics

	Variables	Validation set n = 275 (%)
	Age
	Median	57
	Range	27-92
	Age distribution
	\50	91 (33.1)
	[50	184 (66.9)
	Tumour size (mm)
	Median	20
	Range	1-100
	Size distribution (mm)
	\20	160 (58.2)
	[20	108 (39.3)
	Missing	7 (2.5)
	Tumour grade
	G1	38 (13.8)
	G2	114 (41.5)
	G3	123 (44.7)
	Lymph node status
	Positive	144 (52.4)
	Negative	121 (44)
	Missing	10 (3.6)
	Nottingham prognostic index
	Good	60 (21.8)
	Moderate	139 (50.5)
	Poor	64 (23.3)
	Missing	12 (4.4)

Table 2

 2 Clinicopathological parameters in relation to CSMD1

	Variables		CSMD1 expression a
			Low n (%)	High n (%)	P value
	Age distribution
	\50		24 (30.37)		67 (34.18)	0.54
	[50		55 (69.62)	129 (65.81)
	Size distribution (mm)
	\20 mm	50 (65.78)	110 (57.29)	0.20
	[20 mm	26 (34.21)		82 (42.70)
	Tumour grade
	G1		17 (21.51)		21 (10.7)	0.003
	G2		21 (26.58)		93 (47.4)
	G3		41 (51.89)		82 (41.83)
	Lymph node status
	Positive	34 (45.94)		87 (45.54)	0.95
	Negative	40 (54.05)	104 (54.45)
	Nottingham prognostic index
	Good		21 (28.76)		39 (20.52)	0.31
	Moderate	34 (46.57)	105 (55.26)
	Poor		18 (24.65)		46 (24.21)
	a 30% cut-off was used to dichotomise CSMD1 expression into low
	and high groups
					High CSMD1 (n=147)
		1.0
	Over all Survival probability	0.8 0.6 0.4 0.2	P= 0.016	Low CSMD1 (n=58)
		0.0
		0.00	50.00	100.00	150.00	200.00
					Month

Table 3

 3 Univariate and multivariate analyses of overall survival in the validation set Multivariate analysis: n = 195; deceased = 90, censored = 105, missing = 80 a 30% cut-off was used to dichotomise CSMD1 expression into low and high groups

	Variable	Univariate (P value)	HR (95%CI)	Multivariate (P value)	HR (95%CI)
	Grade
	G1 vs. G2 vs. G3	0.000	1.78 (1.28-2.45)	0.34	1.2 (0.79-1.97)
	Lymph node status
	Negative vs. positive	0.009	1.79 (1.16-2.76)	0.947	0.98 (0.53-1.8)
	NPI
	Good vs. moderate vs. poor	0.000	2.09 (1.53-2.87)	0.015	1.96 (1.14-3.36)
	CSMD1
	High vs. low a	0.018	0.607 (0.4-0.91)	0.03	0.6 (0.3-0.9)

Table 4

 4 Univariate and multivariate analyses of DFS in the validation setMultivariate analysis: total n = 200; deceased = 57, censored = 143, missing = 75 a 30% cut-off was used to dichotomise CSMD1 expression into low and high groups differentiated areas, suggests that CSMD1 may play a role in cell morphogenesis and/or cell differentiation and duct formation. Reduction of CSMD1 expression was recorded in almost a third of breast carcinoma cases and significantly associated with high tumour grade and decreased OS.

	Variable	Univariate (P value)	HR (95%CI)	Multivariate (P value)	HR (95%CI)
	Grade
	G1 vs. G2 vs. G3	0.000	3.9 (2.27-6.7)	0.003	2.72 (1.4-5.26)
	Lymph node status
	Negative vs. positive	0.000	3.8 (1.99-7.46)	0.057	2.26 (0.97-5.25)
	NPI
	Good vs. moderate vs. poor	0.000	3.41 (2.21-5.26)	0.167	1.6 (0.82-3.11)
	CSMD1
	High vs. low a	0.48	0.81(0.46-1.43)	0.573	0.84(0.46-1.5)

Breast Cancer Res Treat (2010) 121:555-563

Acknowledgments This study was supported by grants from Yorkshire Cancer Research and Breast Cancer Campaign (SMB & VS), the Association for International Cancer Research (LZ & NT) and the Egyptian government (MK). CT is a Royal Society University Research Fellow.


Mats Lambe
email: mats.lambe@ki.se

Annette Wigertz

Marit Holmqvist

Jan Adolfsson

Carola Bardage

Tommy Fornander

Per Karlsson

Viveca Odlind

Ingemar Persson

Johan Ahlgren

Leif Bergkvist

Á A Wigertz

Á M Holmqvist

Á J Adolfsson

Á V Odlind

Á I Persson

Reductions in use of hormone replacement therapy: effects on Swedish breast cancer incidence trends only seen after several years

Keywords: Breast cancer, Incidence, Trends, Hormone replacement therapy, Sweden

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Following a continuous increase in breast cancer incidence rates since the early 1980s, studies from several Western countries have found evidence of recent sharp decreases in postmenopausal women. In an analysis based on data from nine US cancer registries, Ravdin et al. reported a 7% overall decrease in incidence between 2002 and 2003 in women older than 50 years of age and particularly for estrogen receptor positive tumors [START_REF] Ravdin | The decrease in breastcancer incidence in 2003 in the United States[END_REF]. Subsequent studies from the US, Canada, Australia, France, Switzerland, Germany, Great Britain, Belgium, and the Nordic countries have also reported reductions in breast cancer incidence [START_REF] Clarke | Recent declines in hormone therapy utilization and breast cancer incidence: clinical and population-based evidence[END_REF][START_REF] Glass | Breast cancer incidence, 1980-2006: combined roles of menopausal hormone therapy, screening mammography, and estrogen receptor status[END_REF][START_REF] Jemal | Recent trends in breast cancer incidence rates by age and tumor characteristics among U.S. women[END_REF][START_REF] Keegan | Recent changes in breast cancer incidence and risk factor prevalence in San Francisco Bay area and California women: 1988 to 2004[END_REF][START_REF] Kerlikowske | Declines in invasive breast cancer and use of postmenopausal hormone therapy in a screening mammography population[END_REF][START_REF] Robbins | Regional changes in hormone therapy use and breast cancer incidence in California from 2001 to 2004[END_REF][START_REF] Canfell | Decrease in breast cancer incidence following a rapid fall in use of hormone replacement therapy in Australia[END_REF][START_REF] Hemminki | Postmenopausal hormone drugs and breast and colon cancer: nordic countries 1995-2005[END_REF][START_REF] Katalinic | Decline in breast cancer incidence after decrease in utilisation of hormone replacement therapy[END_REF][START_REF] Bouchardy | Changing pattern of age-specific breast cancer incidence in the Swiss canton of Geneva[END_REF][START_REF] Parkin | Is the recent fall in incidence of post-menopausal breast cancer in UK related to changes in use of hormone replacement therapy?[END_REF][START_REF] Seradour | Changes by age in breast cancer incidence, mammography screening and hormone therapy use in France from 2000 to 2006[END_REF][START_REF] Vankrunkelsven | Reduction in hormone replacement therapy use and declining breast cancer incidence in the Belgian province of Limburg[END_REF]. These changes have been hypothesized to be a result of a dramatic decline in the use of hormone replacement therapy (HRT) following the 2002 release of the report from the Women's Health Initiative (WHI) hormone trial [START_REF] Rossouw | Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial[END_REF]. However, it remains unclear whether a reduced use of HRT represents the main and only cause behind the reversal of breast cancer incidence trends. In the US, earlier increases in breast cancer incidence have primarily been discussed in terms of higher rates of detection because of rising mammography rates [START_REF] Krieger | Hormone therapy and the rise and perhaps fall of US breast cancer incidence rates: critical reflections[END_REF]. Also, the recent downward trend have by some authors been suggested to reflect saturation in mammography screening [START_REF] Li | Changes in breast cancer incidence rates in the United States by histologic subtype and race/ethnicity, 1995 to 2004[END_REF]. However, based on results from a mammography screening population other investigators have argued that stable rates of ductal carcinoma in situ disease support the notion of an important role of changes in HRT use [START_REF] Kerlikowske | Declines in invasive breast cancer and use of postmenopausal hormone therapy in a screening mammography population[END_REF].

Sweden has a high incidence of breast cancer, and a historically relatively high prevalence of HRT use. Using data from three population-based Regional Clinical Quality Registers on breast cancer, we examined in detail incidence trends in Sweden between 1997 and 2007, a period characterized by a drop in the use of HRT.

Materials and methods

Use of hormone replacement therapy

Aggregated information on annual sales of HRT to women aged 50-69 years was obtained from the drug sales database of The Swedish National Corporation of Pharmacies in which all drugs are classified by the Anatomical Therapeutic Chemical (ATC) classification system. Sales figures over time for the group G03C (estrogens) and G03D (progestogens) and G03F (preparations with fixed combinations of estrogen and progestogens) were assessed. In the estrogen group, only medium-potency estrogens were included. Data were collected in the form of defined daily dosages (DDDs) per 1,000 women in age groups, i.e., the number of average recommended maintenance doses per day for a drug for its main indication in adults relative to the number of women in the respective age group.

Breast cancer incidence

Incidence trends between 1997 and 2007 were estimated for women in three administrative health care regions in Sweden, covering more than 5 million inhabitants corresponding to 62% of the Swedish population (Uppsala-O ¨rebro, Va ¨stra Go ¨taland, and Stockholm-Gotland). Data on incident cases of breast cancer (invasive and in situ) were retrieved from Regional Clinical Quality Registries on breast cancer that include information on date of diagnosis, tumor stage, tumor characteristics, and primary treatment on all newly diagnosed breast cancer patients. The Regional Quality Registers are validated against the national Swedish Cancer Register and all have a completeness exceeding 96%.

Statistical analysis

Breast cancer incidence rates were calculated based on the number of cases for each 5 year age group, divided by the corresponding mean population per 100,000. Age specific incidence rates were adjusted to the age distribution of the Swedish population on January 1, 2000.

Breast cancer incidence data are presented with estimates per 100,000 women by year of diagnosis (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007). In order to test incidence trends, a regression model was used where the breakpoint was set to 2003. Temporal patterns of HRT use from pharmacy sales data are presented as DDD/1,000 women by calendar year.

Results

Following a sharp increase in sales beginning in the late 1980s, HRT use peaked in 1999 (Fig. 1) with an estimated prevalence in age group 50-69 years of 27% compared to 23% in 2002 and 9% in 2007. The corresponding estimates in women aged 50-59 were 36, 27, and 9% and in women aged 60-69 were 19, 19, and 9%. Thus, in the age group 50-59 where HRT use was most common, the prevalence declined by 25% between 1999 and 2002, and by more than 70% between 1999 and 2007.

Among women 50-59 years of age and prescribed HRT in 1999, 59% used fixed combinations of estrogen and progestogens, either combined continuously or sequentially, and 41% were given estrogen, either unopposed or together with a separate prescription of a progestogen. The estrogen component in the majority of HRT prescriptions in Sweden is estradiol, whereas a small proportion used to be conjugated estrogens. Since 2007 conjugated estrogens are no longer marketed in Sweden.

Among women using fixed combinations the use of combined continuous preparations increased between 2000 and 2005. The progestogen component used in fixed combinations, both in continuous and sequential preparations was most often norethisterone acetate (NETA) during the same time period, but medroxyprogesterone acetate preparations (MPA) were also used (Table 1).

The incidence of invasive breast cancer increased in all age groups in women 50 years and older between 1997 and 2003. A statistically significant decrease in incidence between 2003 and 2007 was confined to women 50-59 years of age (P \ 0.05) and corresponded to a reduction of 4.5% per year (Figs. 2,3). Among women 50-59 years the incidence pattern for ER ? (estrogen receptor positive) tumors was similar to that of all invasive tumors. No significant changes in incidence rates of lobular cancers or of in situ cancers were observed during the period under study (Fig. 3).

Among all invasive tumors in women aged 50-59 years, 72% were ER ? (estrogen receptor positive), 17% were ER -(estrogen receptor negative), and 11% had an unknown estrogen receptor status. Ductal and lobular type cancers represented 69 and 15%, respectively, of all invasive cases. breast cancer disappear within a few years after discontinuation of use [START_REF]Breast cancer and hormone replacement therapy: collaborative reanalysis of data from 51 epidemiological studies of 52, 705 women with breast cancer and 108, 411 women without breast cancer[END_REF][START_REF] Chlebowski | Breast cancer after use of estrogen plus progestin in postmenopausal women[END_REF]. In some, but not all, earlier reports the decrease in incidence was most pronounced for ER ? -tumors [START_REF] Ravdin | The decrease in breastcancer incidence in 2003 in the United States[END_REF][START_REF] Jemal | Recent trends in breast cancer incidence rates by age and tumor characteristics among U.S. women[END_REF][START_REF] Kerlikowske | Declines in invasive breast cancer and use of postmenopausal hormone therapy in a screening mammography population[END_REF]. In the present study, ER ? -tumors represented 3/4 of all tumors in women 50-59 years of age with a decrease in incidence closely resembling temporal patterns for all cancers in this age group. Lobular cancers have also been postulated to be particularly sensitive to hormonal stimulation. While a few investigators have reported larger recent decreases in incidence for lobular cancers than for ductal cancers [START_REF] Vankrunkelsven | Reduction in hormone replacement therapy use and declining breast cancer incidence in the Belgian province of Limburg[END_REF][START_REF] Eheman | The changing incidence of in situ and invasive ductal and lobular breast carcinomas: United States, 1999-2004[END_REF], we found no evidence of a different incidence pattern for the small subgroup of lobular carcinomas available for analyses in the present study. Saturation of mammography screening has been suggested to explain a slight decline in US breast cancer incidence observed in the years preceding the WHI report [START_REF] Jemal | Recent trends in breast cancer incidence rates by age and tumor characteristics among U.S. women[END_REF], and also that it played a role for the continued decrease up until 2004 [START_REF] Li | Changes in breast cancer incidence rates in the United States by histologic subtype and race/ethnicity, 1995 to 2004[END_REF]. Organized, invitational mammography service screening was introduced gradually in Sweden beginning in 1974, covered 85% of Swedish counties in 1992, and achieved full national coverage in 1997 [START_REF] Olsson | Implementation of service screening with mammography in Sweden: from pilot study to nationwide programme[END_REF]. While the age groups invited have varied between geographical regions and over time, the age group of particular interest in the present study (50-59 years) was included from the start in all programs with at least biennial invitations, and a steady state can be expected to have been reached well before year 2000. The incidence of in situ cancers can be seen as a measure of mammography saturation and utilization since this subtype most often is detected by mammography. During the period under study, the incidence rates of breast cancer in situ remained stable indicating no major changes in the use of mammography in Sweden. Thus, it appears unlikely that patterns of mammography use could explain our findings.

Discussion

In Sweden, the most commonly used HRT therapies have been continuous or sequential combinations of estradiol and NETA or MPA (Fig. 1, Table 1). In the US conjugated estrogens have predominated. The WHI randomized placebo-controlled trial tested conjugated equine estrogens (CEE) in continuous combination with MPA [START_REF] Rossouw | Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial[END_REF]. Types of estrogen and progestogen and doses of the hormones, as well as routes of administration may be associated with different risks for the development of breast cancer. Combinations of estrogen and progestogen have been shown to confer a higher risk than estrogen-only treatment [START_REF] Beral | Breast cancer and hormone-replacement therapy in the Million Women Study[END_REF], and continuous combined HRTs has been suggested to confer a higher risk than sequentially administrated HRTs [START_REF] Olsson | Hormone replacement therapy containing progestins and given continuously increases breast carcinoma risk in Sweden[END_REF]. However, in a review of the risks and benefits of different HRT regimens it was concluded that the WHI findings based on CEE also are consistent with the risk profile for estradiol preparations [START_REF] Warren | A comparative review of the risks and benefits of hormone replacement therapy regimens[END_REF].

Compared to the US and other countries, the decrease in breast cancer incidence in Sweden appears to have been more gradual and less rapid. This may reflect the differences in types of HRT used and that the prevalence of HRT use started to decline in 1999, several years before the release of the results of the WHI study. However, it should be noted that a slight decline in HRT use was detectable prior to 2002 also in the US [START_REF] Kerlikowske | Declines in invasive breast cancer and use of postmenopausal hormone therapy in a screening mammography population[END_REF]. The observation that breast cancer risk increases after only a few years of HRT exposure and that the risk declines within years of its discontinuation is compatible with HRT acting through growth stimulation of preexisting premalignant breast epithelial cells. Somewhat later decline in breast cancer incidence in Sweden compared with the US may reflect not only differential growth promoting effects of different HRT regimens, but possibly also differences in the proportions of short-versus long-term users discontinuing their treatment.

Strengths of the present study included the availability of high quality detailed population-based clinical data to assess incidence trends of breast cancer by subtypes. Like most earlier post-WHI reports on breast cancer incidence trends, individual level data on mammography use, or hormonal therapy were unavailable. Our sales-based estimates of time trends of prevalence of HRT use were in line with results from earlier Swedish studies that have assessed use from questionnaire data [START_REF] Hoffmann | Changes in women's attitudes towards and use of hormone therapy after HERS and WHI[END_REF][START_REF] Thunell | Changes in attitudes, knowledge and hormone replacement therapy use: a comparative study in two random samples with 6-year interval[END_REF].

In conclusion, our finding of a decreasing breast cancer incidence trend in the age group previously most exposed to HRT is unlikely to be explained by changes in mammography utilization, registration routines in the Clinical Quality Registries on breast cancer, or shifts in established risk factors. If anything, an increase in the prevalence of postmenopausal obesity and changes in reproductive risk factors (e.g., earlier menarche and postponement of child bearing) would drive incidence trends in the opposite direction. Taken together, our results indicate that HRT use has been a driver of population rates of breast cancer also in Sweden with changes in incidence patterns reflecting country specific particulars of use.

Fig. 1 Fig. 2

 12 Fig.1HRT pharmacy sales data for women 50-59 years of age in Sweden

Fig. 3

 3 Fig. 3 Breast cancer incidence in women 50-59 years of age for invasive cancer, ER-positive and ER-negative cancer, lobular and in situ cancer per year in Stockholm-Gotland, Va ¨stra Go ¨taland, and Uppsala-O ¨rebro region in Sweden

Breast Cancer Res Treat (2010) 121:679-683

Acknowledgments

The study was supported by grants from the Swedish Council for Working life and Social research (#2007-214), Regional Research Council Uppsala-O ¨rebro (#10316), and ALF Uppsala University Hospital (#43159).


Simona Borstnar
email: sborstnar@onko-i.si

Aleksander Sadikov

Barbara Mozina

Tanja Cufer

Á B Mozina

High levels of uPA and PAI-1 predict a good response to anthracyclines

Keywords: uPA, PAI-1, Predictive value, Breast cancer, Anthracycline-based chemotherapy, Balancing patients' and tumor characteristics

Introduction

The overview of the Early Breast Cancer Trialists' Collaborative Group demonstrates that adjuvant hormonal therapy and polychemotherapy substantially reduce the risk of both recurrence and death from breast cancer and that anthracycline-based chemotherapy is more effective than non-anthracycline, taking into account that the most frequently used non-anthracycline-based chemotherapy in the overview trials was CMF [START_REF]Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials[END_REF]. However, since the adjuvant systemic therapy does have associated risks, it would be useful to optimally select the patients who are more likely to respond to and benefit from a certain kind of systemic therapy. Therefore, in the clinical management of breast cancer, the identification of predictors of response to therapy may have an important impact on treatment decision, thereby improving clinical outcome.

A predictive marker can be defined as a factor which predicts response or resistance to a particular therapy. Standard predictive markers include hormone receptor status for response to endocrine therapy and HER-2 amplification and/or overexpression for response to anti-HER-2 therapy in both metastatic and adjuvant setting. Recently, HER-2 status was also recognized as a predictive marker for response to anthracycline-containing chemotherapy. In a meta-analysis with 8 large prospective trials included, the significant benefit of anthracycline-containing regimens over non-anthracycline ones was observed in women with HER-2 positive early breast cancer [START_REF] Gennari | HER2 status and efficacy of adjuvant anthracyclines in early breast cancer: a pooled analysis of randomized trials[END_REF]. Preclinical as well as clinical data indicate that the increased anthracycline sensitivity might actually not be due to HER-2 amplification/overexpression, but rather to topoisomerase IIa gene (TOP2A) amplification or protein overexpression [START_REF] Pritchard | HER-2 and topoisomerase II as predictors of response to chemotherapy[END_REF][START_REF] O'malley | Prognostic and predictive value of topoisomerase II alpha in a randomized trial comparing CMF to CEF in premenopausal women with node positive breast cancer (NCIC CTG MA. 5)[END_REF], the major molecular target of anthracyclines.

Urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-1) were shown with level 1 evidence to be prognostic factors for primary breast cancer. In numerous individual trials (reviewed [START_REF] Andreasen | The urokinase-type plasminogen activator system in cancer metastasis: a review[END_REF][START_REF] Schmitt | Clinical impact of the plasminogen activation system in tumor invasion and metastases: prognostic relevance and target for therapy[END_REF][START_REF] Duffy | Urokinase plasminogen activator: a prognostic marker in multiple types of cancer[END_REF][START_REF] Look | Clinical relevance of the urokinase plasminogen activator system in breast cancer[END_REF]), in our own preliminary work [START_REF] Borstnar | Prognostic value of plasminogen activator inhibitors in breast cancer[END_REF][START_REF] Borstnar | Prognostic value of the urokinase-type plasminogen activator, and its inhibitors and receptor in breast cancer patients[END_REF][START_REF] Cufer | Prognostic and predictive value of the urokinase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer[END_REF], as well as in a pooled analysis of 8,377 breast cancer patients from 18 different data sets [START_REF] Look | Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients[END_REF] uPA and PAI-1 uniformly showed independent prognostic value. Most of the patients included in these early studies did not receive any chemotherapy, while endocrine therapy was performed in a majority of hormonal receptor positive patients. A prospective randomized clinical study Chemo N0 [START_REF] Ja ¨nicke | 0 Study Group (2001) Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1[END_REF][START_REF] Harbeck | Chemo N0 Study Group (2009) Final 10-year analysis of prospective multicenter Chemo N0 trial for validation of ASCO-recommended biomarkers uPA/PAI-1 for therapy decision making in node-negative breast cancer[END_REF] randomizing patients with high levels of uPA or PAI-1 between chemotherapy and no chemotherapy while leaving patients with low levels of uPA and PAI-1 out of any adjuvant systemic therapy established a level 1 prognostic value of these two markers in early breast cancer. This was actually the first prospective randomized trial demonstrating that patients with good prognosis, according to low uPA and PAI-1 levels, could be spared toxic chemotherapy.

The observation that the prognostic value of both uPA and PAI-1 was lost in patients who received the adjuvant therapy observed in Chemo N0 trial [START_REF] Ja ¨nicke | 0 Study Group (2001) Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1[END_REF][START_REF] Harbeck | Chemo N0 Study Group (2009) Final 10-year analysis of prospective multicenter Chemo N0 trial for validation of ASCO-recommended biomarkers uPA/PAI-1 for therapy decision making in node-negative breast cancer[END_REF] as well as in other trials [START_REF] Harbeck | Prognostic impact of proteolytic factors (uPA, PAI-1, cathepsins B, D, L) in primary breast cancer reflects effects of adjuvant systemic therapy[END_REF][START_REF] Harbeck | Clinical relevance of invasion factors uPA and PAI-1 for individualized therapy decisions in primary breast cancer is greatest when used in combination[END_REF][START_REF] Ferno | Urokinase plasminogen activator in breast cancer, analysed in steroid receptors cytosols with a luminometric immunoassay[END_REF][START_REF] Manders | Predictive impact of urokinase-type plasminogen activator: plasminogen activator inhibitor type-1 complex on the efficacy of adjuvant systemic therapy in primary breast cancer[END_REF] resulted in the hypothesis that these two markers might have predictive value for response to systemic therapy as well. The correlation of inherited worse prognosis associated with a particular prognostic factor diminishing with treatment efficacy points out to a possible predictive value of this particular factor. The difference between the prognostic and predictive value of a marker was nicely explained and graphically displayed by Hamilton and Piccart [START_REF] Hamilton | The contribution of molecular markers to the prediction of response in the treatment of breast cancer: a review of the literature on HER-2, p53 and BCL-2[END_REF].

The findings of different clinical trials which investigated this issue regarding uPA and PAI-1 are not uniform. We reported on a possible predictive value of uPA/PAI-1 for response to hormonal therapy in a group of 460 breast cancer patients [START_REF] Cufer | Prognostic and predictive value of the urokinase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer[END_REF]. In our collective of patients treated with tamoxifen, the bad prognosis associated with high levels of uPA/PAI-1 was lost. Similar observations were made by the Munich [START_REF] Harbeck | Prognostic impact of proteolytic factors (uPA, PAI-1, cathepsins B, D, L) in primary breast cancer reflects effects of adjuvant systemic therapy[END_REF][START_REF] Harbeck | Clinical relevance of invasion factors uPA and PAI-1 for individualized therapy decisions in primary breast cancer is greatest when used in combination[END_REF], Swedish [START_REF] Ferno | Urokinase plasminogen activator in breast cancer, analysed in steroid receptors cytosols with a luminometric immunoassay[END_REF] and Dutch [START_REF] Manders | Predictive impact of urokinase-type plasminogen activator: plasminogen activator inhibitor type-1 complex on the efficacy of adjuvant systemic therapy in primary breast cancer[END_REF] groups, indicating that high levels of uPA and/or PAI-1 in the primary tumor could predict for a better response to the adjuvant tamoxifen treatment. On the contrary, in a larger data set obtained from the German and Dutch groups, containing 3,424 patients, no significant interactions between hormonal therapy and uPA/PAI-1 levels were found. However, in the frame of this pooled analysis, the observation that the patients with high uPA/PAI-1 levels might have benefited more from the adjuvant chemotherapy than those with low levels of uPA/PAI-1 was made for the first time [START_REF] Harbeck | Enhanced benefit from adjuvant systemic chemotherapy in breast cancer patients classified high-risk according to urokinasetype plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1)[END_REF]. In another Dutch study with 1,119 patients included, it was also found that the expression of the uPA:PAI-1 complex independently predicted the efficacy of adjuvant chemotherapy [START_REF] Manders | Predictive impact of urokinase-type plasminogen activator: plasminogen activator inhibitor type-1 complex on the efficacy of adjuvant systemic therapy in primary breast cancer[END_REF]. In the already mentioned prospective randomized Chemo N0 trial, high uPA and PAI-1 levels were associated with a greater benefit (i.e. 52%) from chemotherapy, namely CMF, than expected according to the overview data [START_REF] Ja ¨nicke | 0 Study Group (2001) Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1[END_REF][START_REF] Harbeck | Chemo N0 Study Group (2009) Final 10-year analysis of prospective multicenter Chemo N0 trial for validation of ASCO-recommended biomarkers uPA/PAI-1 for therapy decision making in node-negative breast cancer[END_REF].

Anthracycline-based adjuvant chemotherapy results in significantly (10%) higher benefit compared to CMF chemotherapy in unselected breast cancer populations according to the overview data. However, serious longterm toxicities such as cardiotoxicity and secondary cancers have been observed in a small proportion of patients treated with anthracyclines. The selection of patients that might benefit from anthracyclines is therefore of utmost importance.

In our collective of early breast cancer patients treated in clinical practice, we have already reported that patients with high uPA and PAI-1 levels in primary tumor might benefit more from the anthracycline-based regimens than from CMF. Our preliminary retrospective observation was based on a cohort of 1,220 consecutive patients, the majority of whom were treated with some kind of adjuvant systemic therapy. Already in this unselected population of patients we observed a significant difference in disease-free survival (DFS) with respect to uPA and PAI-1 levels in the whole cohort of patients and in patients treated by CMF, while the significance was lost in the subgroup of patients treated by anthracycline-containing regimens [START_REF] Borstnar | High levels of uPA and PAI-1 predicts a good response to antracyclines (abstract)[END_REF]. The retrospective study, however, suffered from severely uneven patient and tumor characteristics as the patients were treated per institutional guidelines valid at the time and were not randomized between the anthracycline and CMF arms. In the present paper, we attempted to remedy this shortcoming and recheck our previous observations on more balanced data. To this end we employed a custommade computer algorithm to select well balanced anthracycline and CMF arms according to patient and tumor characteristics. This retrospective study adheres to REMARK criteria [START_REF] Mcshane | Statistics Subcommittee of the NCI-EORTC Working Group on Cancer Diagnostics (2005) Reporting recommendations for tumor marker prognostic studies[END_REF].

Patients and methods

Patients

From 1997 until 2001, uPA and PAI-1 levels were prospectively determined in tumor extract of 1,220 patients with early breast cancer treated at the Institute of Oncology, Ljubljana. All of the patients were female and had a histologically confirmed invasive breast cancer. The established clinical and histomorphological factors, such as menopausal status, tumor size, nodal status, pathological grade, hormonal receptors and tumor vessel invasion, were determined. Tumor grade was scored according to the Scarff-Bloom-Richardson classification modified by Elston [START_REF] Elston | Grading of invasive carcinoma of the breast[END_REF]. Steroid hormone receptors were determined either in cytosol fractions using a biochemical method or by immunohistochemical staging on paraffin-embedded tissue sections. The values equal to or greater than 10 fmol/mg protein as measured by biochemical method or positive staining in more than 10% of tumor cells by immunohistochemical method for one or both steroid hormone receptors were considered as positive hormone receptor status.

Treatment decisions regarding primary surgery and adjuvant systemic therapy were based primarily on the guidelines valid at the time and did not take into account either uPA or PAI-1 levels. All patients underwent radical local treatment. Most of the patients were treated with some kind of adjuvant systemic therapy: chemotherapy (ChT, n = 331), hormonal therapy (HT, n = 379), or a combination of both (n = 365). Of 696 patients treated with ChT, either alone or in combination with HT, 368 (53%) received CMF and 328 (47%) were treated with anthracycline-containing regimens (28 patients in combination with taxanes). The values of either uPA or PAI-1 were not determined for 41 of these 696 patients, therefore these patients were excluded from the analysis. After the completion of primary treatment, the patients underwent regular follow-up examinations at our institute. All the procedures were in accordance with the ethical standards of the Medical Ethics Committee at our Institute and with the Helsinki declaration.

Statistical methods

The endpoint in this study was disease-free survival (DFS). It was calculated from the date of the start of primary therapy to the date of breast cancer recurrence (locoregional or distant), the date of death from any cause, or the date of the last follow-up; censored observations correspond to patients alive and without evidence of disease recurrence at the time of the last follow-up. DFS as a function of the markers studied was estimated by the Kaplan-Meier method and the log-rank test was used to test for differences. The Cox multivariate hazards models were used to calculate the hazard ratios (HR) and their 95% confidence intervals (95% CI) in the analysis of DFS. Computations were performed with the use of the SPSS 12 statistical package. All reported P values are two-tailed.

Laboratory assays of uPA and PAI-1

Tumor specimens were obtained from primary tumors and stored in liquid nitrogen until extraction. The frozen tumor tissue samples were pulverized using a micro-dismembrator. The tumor powder was suspended in a buffer (pH 8.5) containing 0.02 mM Tris-HCl, 0.125 mM NaCl and 2% Triton X-100, and shaken for 3 h at 4°C. The obtained suspension was then centrifuged for 30 min at 100,009g. Protein content was determined by using the Pierce assay. Both biological markers were determined in tumor detergent extracts by commercially available enzyme-linked immunosorbent assays (American Diagnostica Inc., Greenwich, CT). International quality assurance of the kits is guaranteed, and the kits have been assessed by the EO-RTC RBG [START_REF] Sweep | External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunoabsorbent assay) of urokinase-type plasminogen activator (Upa) and its type 1 inhibitor (PAI-1) in human breast cancer tissue extracts[END_REF]. Validated cut-off values were assigned for uPA (3 ng/mg protein) and PAI-1 (14 ng/mg protein). According to the combination of uPA and PAI-1 values, patients were divided into two categories: both factors low (low group) versus one or both factors high (high group).

Balancing the treatment groups

In the retrospective evaluation of a prognostic and/or predictive factor, the research and the comparator groups are usually unbalanced with respect to other patient and tumor characteristics. The purpose of a matching algorithm is to find such subgroups of the given initial groups as to minimize the differences in the distributions of selected characteristics. The matching algorithms in the literature, surveyed by Gu and Rosenbaum [START_REF] Gu | Comparison of multivariate matching methods: structures, distances, and algorithms[END_REF], usually pick tuples of patients in each step-one (or more) belonging to each group that differ less than some prescribed threshold between one another. In their most basic and rigorous form, such algorithms pick only the tuples of patients that are identical with respect to all characteristics of interest. The algorithms repeat this selection step for as long as possible.

The main goal of our research was to investigate the potential predictive value of uPA and PAI-1 in treatment groups as well-balanced as possible with respect to standard prognostic factors. Our problem was as follows: we had to match the patients in four observation groups as given in Fig. 1 (CMF low, CMF high, ANT low, and ANT high) with respect to seven characteristics: menopausal status, adjuvant hormonal treatment, tumor grade, tumor size, hormonal receptor status, vascular invasion, and lymph node status. In our case, matching algorithms failed to yield subgroups of sufficiently large sizes. Our problem was specific in the sense that we had to match four groups with severely different distributions of characteristics due to inherent characteristic distributions related to low versus high uPA/PAI-1 levels, as well as due to the bias in chemotherapy selection. One look at Table 1 shows that, in our setting, there simply were not enough tuples of four patients (one from each group) to match completely in every characteristic-there are extreme differences in distributions, especially in lymph node status, vascular invasion, tumor size, and tumor grade.

Since the matching algorithms failed, we decided to match the groups as a whole and not on a patient-by-patient level. However, since matching on the group level is weaker in terms of matching quality, we actually combined the two approaches to get a reasonable compromise between the quality of matching and subgroup sizes. The algorithm was custom-built specifically for our problem and its main ideas were as follows (these ideas, however, are general and can be applied to other difficult cases where matching algorithms fail).

First, we took the two smallest (and thus most problematic) groups, namely CMF low and ANT low, and matched them in two steps. In the first step, we selected the patients with identical characteristics from the two groups (perfect matches). In the second step, we additionally selected patients that differed in at most two characteristics, but not in the lymph node status. Furthermore, in the second step, we selected the new patients only if they did not violate the predescribed maximum difference in the distributions between groups (the difference was measured in terms of the Pearson chi-square statistic). Such a procedure is greedy and myopic in the sense that a subsequently selected patient could repair the imbalance created by the current patient; however, taking that into account would be computationally too ineffective due to combinatorial explosion. This procedure yielded two smallest subgroups (CMF low and ANT low). From the two remaining groups we then selected the patients that matched as closely as possible with the patients in the two already set-up subgroups.

The selection of patients from the two larger groups was slightly different. For each patient from the two smaller subgroups, we selected up to three (if possible) patients from these two groups that best matched it-but never allowed a mismatch in more than two characteristics and never a mismatch in lymph node status. Violation in distribution was not checked in this step as it was found to be too restrictive. Since this procedure yielded the subgroups that violated the maximum predescribed difference in the distributions between subgroups, they had to be trimmed down. We only trimmed the two larger subgroups and did this as follows. If a violation in any of the characteristics of interest remained, we excluded the patient that most contributed to the violation in general (not just in one characteristic), while not making another characteristic violated. The patient that most skewed the distributions was selected by a greedy search with a specially designed heuristic using as its basis the Pearson chi-square statistic. Finally, the patients that remained were grouped in the two remaining subgroups (CMF high and ANT high). The quality of the final match and the subgroup sizes are two contradicting criteria. To get larger subgroup sizes we sacrificed menopause as the least important characteristic.

The success of our balancing algorithm (the term balancing is used here to separate this algorithm from the matching algorithms and because it better describes the idea of the algorithm) can be seen in Table 2. It shows the distributions of the aforementioned characteristics after applying the balancing algorithm. We can see that the groups are now well balanced with respect to the selected characteristics. The characteristic, whose distribution most varies between any two subgroups, is the hormonal receptor status between CMF high and ANT high subgroups-yet, this difference is not so large. Heuristic (Pearson) thresholds were arbitrarily selected and experimented with to give us the best possible compromise between the number of included cases (arm strength) and similarity of distributions. This experimentation, however, was performed in advance and independently of the data analysis. Only after being satisfied with both the number of included patients and the similarity of distributions within the observed groups did we perform the analysis, not alternating between the two steps to give us the sought after results.

The balancing algorithm is deterministic; the same input data consisting of thresholds and patients with their characteristics always give the same result. The algorithm with final threshold settings was run on lexicographically ordered patients. This enables us to reproduce and verify its results and at the same time helps prevent any possible biases since the lexicographic order of patients does not correlate with any selected or observed characteristic.

Results

The experimental design is as given in Fig. 1. The median follow-up in the cohort of patients under observation was 37 months (range 1-84 months). Either local (just in one single case) or distant disease recurrence was observed in 49 out of 180 patients; 31 in the CMF arm and 18 in the anthracycline arm.

Survival data

The 3-year DFS was 86.0% in the group of 54 patients with both factors low, and 78.7% in the group of 126 patients with one or both factors high (Fig. 2); the difference did not reach the level of significance (P = 0.157). With respect to treatment groups, the 3-year DFS rates in the CMF group of 100 patients and in anthracycline group of 80 patients were similar, 79.7 and 82.6%, respectively (P = 0.390) (Fig. 3).

CMF arm

In the group of 100 patients treated with CMF, a significant difference in DFS with respect to uPA and PAI-1 status was found (Fig. 4). The 3-year DFS was 87.1% in the low group (n = 27) and 77.0% in the high group (n = 73) (P = 0.044; HR = 2.81; 95% CI = 0.98-8.04).

Anthracycline arm

In the group of 80 patients treated with anthracycline-based chemotherapy, the difference in DFS with respect to uPA and PAI-1 disappeared (Fig. 5). The 3-year DFS was 85.2% in the low group (n = 27) and 81.8% in the high group (n = 53) (P = 0.807; HR = 0.88; 95% CI = 0.33-2.40). Low versus high levels of uPA and PAI-1

Patients with low levels of uPA and PAI-1 exhibited similar 3-year DFS rates regardless of treatment: 87.1% in the CMF group of 27 patients and 85.1% in the anthracycline group of 27 patients (P = 0.334). On the other hand, patients with high uPA and/or PAI-1 levels revealed a trend towards better survival when treated with anthracyclines instead of CMF; the curves split after one and a half years of follow-up. The 3-year DFS was 77.0% in the CMF group of 73 patients and 81.1% in the anthracycline group of 53 patients (P = 0.147) .

Discussion

In breast cancer, markers that predict sensitivity of tumors to a particular systemic therapy are necessary to support the selection of the most optimal adjuvant treatment for each individual breast cancer patient since more precise selection of systemic therapy can improve outcomes and reduce adverse effects. Currently, hormone receptor status and HER-2 status are the only established factors predicting responsiveness to hormonal and HER-2 targeted therapy. The components of the plasminogen activator system uPA and PAI-1 are the only novel tumor biological factors that have reached level-1 evidence with regard to their prognostic impact in primary breast cancer. There is no doubt that at least the patients with high protease levels need some form of systemic therapy. However, there still exists a lack of data on whether uPA and/or PAI-1 levels can also serve as predictive markers for response to a particular systemic therapy, thus supporting the selection of the most appropriate systemic therapy for each individual patient.

The positive predictive value of the plasminogen activator system for response to adjuvant chemotherapy, mostly CMF, was already found in two prior retrospective observations [START_REF] Manders | Predictive impact of urokinase-type plasminogen activator: plasminogen activator inhibitor type-1 complex on the efficacy of adjuvant systemic therapy in primary breast cancer[END_REF][START_REF] Harbeck | Enhanced benefit from adjuvant systemic chemotherapy in breast cancer patients classified high-risk according to urokinasetype plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1)[END_REF]. Additionally, the Chemo N0 trial showed that nodenegative breast cancer patients with high uPA/PAI-1 levels benefit significantly from adjuvant chemotherapy with CMF [START_REF] Ja ¨nicke | 0 Study Group (2001) Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1[END_REF][START_REF] Harbeck | Chemo N0 Study Group (2009) Final 10-year analysis of prospective multicenter Chemo N0 trial for validation of ASCO-recommended biomarkers uPA/PAI-1 for therapy decision making in node-negative breast cancer[END_REF]. In the ongoing NNBC-3 trial, optimal chemotherapy (anthracycline-taxane versus anthracycline-based) for patients with high uPA/PAI-1 levels is being evaluated and the results are eagerly awaited [START_REF] Annecke | uPA and PAI-1 in breast cancer: review of their clinical utility and current validation in the prospective NNBC-3 trial[END_REF].

This paper is the first to show a difference in response to the anthracycline-containing regimen compared to response to CMF with respect to uPA and PAI-1 status in primary tumors. We found that patients with high uPA and/ or PAI-1 levels in primary tumor may benefit more from adjuvant anthracycline-containing regimens than from CMF. Our preliminary data based on the complete data set containing 1,220 patients of which the majority had been treated with some kind of adjuvant systemic therapy already pointed to the possible predictive value of uPA/ PAI-1 levels for response to anthracycline-based chemotherapy [START_REF] Borstnar | High levels of uPA and PAI-1 predicts a good response to antracyclines (abstract)[END_REF]. Since the selection of the type of adjuvant chemotherapy was based on the institutional guidelines valid at the time, the standard prognostic factors in two treatment groups were not balanced; the patients treated with anthracyclines tended to have anatomically more widespread disease, higher tumor grades and higher rates of vascular invasion that might have influenced our preliminary results. To avoid the unevenness in tumor characteristics, a further step with balancing the treatment groups was called for. With the balancing algorithm described in this paper we reached a well-balanced distribution of 180 patients in four groups determined by CMF and anthracycline-based chemotherapy and uPA/PAI-1 status. The algorithm in a way created a similar setting to that of a randomized study at the expense of greatly reducing the number of patients included into the study itself. In this setting, we observed that the 3-year DFS in the CMF arm differs significantly between patients with low and patients with high levels of markers (87.1% vs. 77.0%, P = 0.044), while, on the other hand, the 3-year DFS in the anthracycline arm did not differ much between the two marker level subgroups (85.2% vs. 81.8%, P = 0.807). Our observations show that worse prognosis correlated to high uPA and PAI-1 levels can be reversed by treatment efficacy achieved through anthracycline-based chemotherapy. This points to a possible predictive value of uPA/PAI-1 for sensitivity to anthracyclines. The situation is graphically represented in Fig. 6 and corresponds to ''type 4 marker'' as defined by Hamilton and Piccart [START_REF] Hamilton | The contribution of molecular markers to the prediction of response in the treatment of breast cancer: a review of the literature on HER-2, p53 and BCL-2[END_REF]. Parallels can be drawn to a similar situation with HER-2 and trastuzumab, where same as in our case worse prognosis correlates with treatment efficacy.

It must be noted that the distributions of different characteristics after the balancing are not like those usually encountered in clinical settings with, for example, approximately half of the patients being node-negative; most of the patients were lymph node-positive in our selected subgroups. This was due to the fact that only very few lymph node-negative patients were treated with anthracycline-containing therapy at the time. However, there is no reason to believe that our observations made in the cohort of mostly node-positive early breast cancer patients cannot be extended to node negative disease. To confirm our observations, however, a prospective randomized trial comparing the efficacy of anthracyline and non-anthracycline-based chemotherapy in different uPA/ Marker value (uPA/PAI-1) 3-year disease-free survival (%) CMF ANT Fig. 6 Three-year DFS according to uPA/PAI-1 categories. This graphical representation shows parallels to the one introduced by Hamilton and Piccart [START_REF] Hamilton | The contribution of molecular markers to the prediction of response in the treatment of breast cancer: a review of the literature on HER-2, p53 and BCL-2[END_REF] for ''type 4 marker'' PAI-1 subgroups of patients is needed. On the other hand, there is the question of whether our results can be applicable to different molecular subgroups of patients, as classified by the HR and HER-2 status. Unfortunately, due to the small groups of patients and a lack of routine HER-2 determination at the time of our study, no data on the influence of uPA/PAI-1 levels on response to different chemotherapeutic schedules in various molecular subtypes of early breast cancer can be obtained from our work.

HER-2 status has already been confirmed as a reliable predictive marker for response to anthracyclines in early breast cancer [START_REF] Gennari | HER2 status and efficacy of adjuvant anthracyclines in early breast cancer: a pooled analysis of randomized trials[END_REF], and topoisomerase IIa gene/protein co-expression, which is frequently found in HER-2 positive tumors and represents the major target for anthracyclines, was found to add some additional information [START_REF] O'malley | Prognostic and predictive value of topoisomerase II alpha in a randomized trial comparing CMF to CEF in premenopausal women with node positive breast cancer (NCIC CTG MA. 5)[END_REF][START_REF] Knoop | Retrospective analysis of topoisomerase IIa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide, epirubicin, and fluorouracil: Danish Breast Cancer Cooperative Group[END_REF]. There is the question of whether uPA/PAI-1 levels, influencing stroma and cancer cells microinvasion, can add any additional independent information on anthracycline-based chemotherapy sensitivity in molecular subtypes defined by HER-2 status. Only limited data are available on the correlation between HER-2 status and uPA and/or PAI-1 levels in primary breast tumors. In our observation, performed on a cohort of 308 early breast cancer patients treated between the years 2004 and 2006 at the Maribor Teaching Hospital, Slovenia, a weak correlation between HER-2 status and uPA levels, but not PAI-1 levels, was found [START_REF] Ravnik | Correlation between invasive markers (uPA/PAI-1) and HER-2 status in early breast cancer[END_REF]. A French group also found a weak positive correlation between uPA/PAI-1 and HER-2 [START_REF] Bouchet | Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma[END_REF]. The study by Urban et al. indicates that overexpression of uPA mRNA levels in HER-2 positive breast cancer determines a worse outcome of the disease [START_REF] Urban | Increased expression of urokinase-type plasminogen activator mRNA determines adverse prognosis in ErbB2-positive primary breast cancer[END_REF]. On the other hand, Harbeck et al. demonstrated that uPA/PAI-1 and HER-2 gave independent information on disease-free survival in lymph node-negative breast cancer patients [START_REF] Harbeck | HER-2/neu gene amplification determined by in-situ hybridization (FISH) allows risk group assessment in node-negative breast cancer[END_REF] with a similar finding described by Konecny et al. [START_REF] Konecny | HER-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer[END_REF] and by Zemzuoum et al. [START_REF] Zemzoum | Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patients outcome in node-negative breast cancer[END_REF]. Therefore, uPA and PAI-1 levels might, independently of HER-2 status, help us to better select patients that will benefit from anthracyclines. According to a meta-analysis, there is the question of whether HER-2 negative patients do benefit from anthracyclines at all; however, a precise look at the pooled data, analyzed by Gennari et al., tells us that in the trials not so extensively contaminated by endocrine-responsive, luminal-type cancers, namely the Danish and NSABP B 11 trials, the benefit of anthracyclines over non-anthracycline schemes was observed also in HER-2 negative disease as well. This gives us confidence that even among HER-2 negative early breast cancer patients, there is a subset of patients that might benefit from anthracyclines. There is therefore an urgent need to identify the predictive markers that will help us to select such patients. Given that the methodology for uPA/PAI-1 determination is simple, validated and quality assured, measuring the uPA/PAI-1 levels might be another valuable marker for determining anthracycline sensitivity in the future.

In conclusion, the present paper confirms our previous observation that worse prognosis correlated with high uPA and PAI-1 levels can be reversed with improved treatment efficacy achieved by anthracycline-based chemotherapy, and not by CMF, which points to a possible predictive value of uPA/PAI-1 for sensitivity to anthracyclines. Patients with high uPA and/or PAI-1 levels in the primary tumor may benefit more from the adjuvant anthracyclinecontaining regimens than from CMF. The clinical application of the present work is to offer additional information that might facilitate the selection of the most appropriate candidates for anthracycline-based chemotherapy. However, for any firm conclusions on the predictive value of uPA/PAI-1 levels for the response to different systemic therapies in various molecular subtypes of early breast cancers, large prospective translational research trials are required.

Fig. 1

 1 Fig.1 Experimental design

Fig. 4 Fig. 5 Fig. 3 Fig. 2

 4532 Fig. 4 DFS in the CMF-treated patients according to uPA and PAI-1 levels (N = 100)

Table 1

 1 Patient and tumor characteristics before balancing

					Table 1 continued
	Characteristic	CMF	ANT	P (treatment)	Characteristic	CMF	ANT	P (treatment)
	uPA/PAI-1 status = both low			p(uPA/PAI1)*	0.089	0.759
	Menopausal status				HR status
	Pre/peri	43	36	0.340	Negative	97	71	0.924
	Post	36	42		Positive	191	137
	Adjuvant treatment				Unknown	1	0
	ChT only	25	25	1.000	p(uPA/PAI1)*	\0.001	0.014
	ChT & HT	55	53		Vascular invasion
	Tumor grade				No	184	96	\0.001
	Grade I	11	3	0.045	Yes	79	103
	Grade II	43	37		Unknown	26	9
	Grade III	26	35		p(uPA/PAI1)*	0.307	0.786
	Unknown	0	3		Lymph node status
	Tumor size				Negative	153	23	\0.001
	B20 mm	36	18	0.007	1-3 positive	103	60
	[20 mm	44	58		[3 positive	32	125
	Unknown	0	2		Unknown	1	0
	HR status				p(uPA/PAI1)*	0.658	0.072
	Negative	11	15	0.396	Note: Groups were compared using Fisher's exact test where possible,
	Positive	69	63		otherwise the Kruskall-Wallis test was used
	Vascular invasion
	No	55	34	\0.001
	Yes	17	40
	Unknown	8	4
	Lymph node status
	Negative	39	2	\0.001
	1-3 positive	32	23
	[3 positive	7	53
	Unknown	2	0
	uPA/PAI-1 status = one or both high
	Menopausal status
	Pre/peri	111	105	0.013
	Post	169	100
	Unknown	9	3
	p(uPA/PAI1)*	0.021	0.506
	Adjuvant treatment
	ChT only	168	95	0.006
	ChT & HT	121	113
	p(uPA/PAI1)*	\0.001	0.044
	Tumor grade
	Grade I	12	10	0.058
	Grade II	87	43
	Grade III	189	155
	Unknown	1	0
	p(uPA/PAI1)*	\0.001	\0.001
	Tumor size
	B20 mm	98	55	0.076
	[20 mm	187	152
	Unknown	4	1

* These P-values (uPA/PAI1) are for the characteristics grouped by treatment, e.g. comparing lymph node status for ANT-treated patients separately from CMF-treated patients with respect to uPA/PAI-1 status

Table 2

 2 Patient and tumor characteristics after balancing

					Table 2 continued
	Characteristic	CMF	ANT	P (treatment)	Characteristic	CMF	ANT	P (treatment)
	uPA/PAI-1 status = both low			No	45	29	0.573
	Menopausal status				Yes	26	22
	Pre/peri	12	11	1.000	Unknown	2	2
	Post	15	16		p(uPA/PAI1)*	0.471	0.808
	Adjuvant treatment				Lymph node status
	ChT only	7	6	1.000	Negative	6	4	0.656
	ChT & HT	20	21		1-3 positive	49	32
	Tumor grade				[3 positive	18	17
	Grade I	2	2	0.958	p(uPA/PAI1)*	0.880	0.973
	Grade II	16	15		* These P-values (uPA/PAI1) are for the characteristics grouped by
	Grade III	9	10		treatment, e.g. comparing lymph node status for ANT-treated patients
	Tumor size				separately from CMF-treated patients with respect to uPA/PAI-1
	B20 mm	8	6	0.757	status
	[20 mm	19	21
	HR status
	Negative	3	4	1.000
	Positive	24	23
	Vascular invasion
	No	19	16	0.555
	Yes	7	10
	Unknown	1	1
	Lymph node status
	Negative	2	2	1.000
	1-3 positive	17	17
	[3 positive	8	8
	uPA/PAI-1 status = one or both high
	Menopausal status
	Pre/peri	27	33	0.007
	Post	46	20
	p(uPA/PAI1)*	0.500	0.096
	Adjuvant treatment
	ChT only	22	12	0.418
	ChT & HT	51	41
	p(uPA/PAI1)*	0.806	1.000
	Tumor grade
	Grade I	4	6	0.434
	Grade II	34	21
	Grade III	35	26
	p(uPA/PAI1)*	0.425	0.395
	Tumor size
	B20 mm	22	14	0.693
	[20 mm	51	39
	p(uPA/PAI1)*	1.000	0.789
	HR status
	Negative	6	8	0.260
	Positive	67	45
	p(uPA/PAI1)*	0.699	1.000
	Vascular invasion

Breast Cancer Res Treat (2010) 121:615-624