Laura La Paglia

Anthony Laugé

Jérémie Weber

Jérôme Champ

Eve Cavaciuti

Antonio Russo

Jean-Louis Viovy

Dominique Stoppa-Lyonnet
email: dominique.stoppa-lyonnet@curie.net

A E Anthony

Lauge ´ae

Je ´re ´mie

Weber Ae

Je ´ro ˆme

Champ Ae

A E Antonio

Russo Ae

Jean-Louis Viovy

Á A Lauge

´á J Champ

Á D Stoppa-Lyonnet

Á A Russo

J.-L Viovy

ATM germline mutations in women with familial breast cancer and a relative with haematological malignancy

Keywords: Ataxia-telangiectasia, Breast cancer, BRCA1, BRCA2, ATM

come

germline mutations in women with familial breast cancer and a relative with haematological malignancy

Introduction

Breast cancer (BC) is the most common cancer in western countries. Most cases of BC are sporadic, but almost 13% of women have at least one first-degree relative with BC [START_REF]Collaborative group on hormonal factors in breast cancer (2001) Familial breast cancer: collaborative reanalysis of individual data from 52 epidemiological studies including 58, 209 women with breast cancer and 101, 986 women without the disease[END_REF]. The BRCA1 and BRCA2 genes are the two genes whose germline monoallelic inactivating mutations are identified in 15-20% of women with BC and a strong family history of BC [START_REF] Easton | How many more breast cancer predisposition genes are there?[END_REF], suggesting the presence of other BC predisposing genes explaining at least a portion of the remaining non-mutated familial cases. Three other genes are associated with an increased BC risk: TP53, STK11, and PTEN. As mutations of these genes are associated with rare autosomal dominant syndromes, these genes must be considered in a specific familial context. They also have a very small contribution to familial aggregations (\1% of familial cases) [START_REF] Easton | How many more breast cancer predisposition genes are there?[END_REF]. The ATM gene (ataxia telangiectasia mutated) encodes a lipid kinase phosphatidylinositol 3-kinase (PI3K) expressed in a wide range of tissues. It is the apex of a signalling cascade that detects and repairs DNA double-strand breaks, a cascade in which the BRCA1 and BRCA2 proteins are involved [START_REF] Shiloh | ATM and related protein kinases: safeguarding genome integrity[END_REF][START_REF] Lavin | Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer[END_REF]. Biallelic inactivation of the ATM gene causes ataxia-telangiectasia (A-T), a neurodegenerative disorder that develops in early childhood. Ataxia-telangiectasia children present a severe clinical phenotype characterised by progressive cerebellar ataxia and dysarthria, associated with ocular telangiectasia, immunodeficiency, sensitivity to ionising radiation, and an increased risk of cancers, especially leukaemias and lymphomas [START_REF] Chun | Ataxia-telangiectasia, an evolving phenotype[END_REF]. A large number of ATM mutations have been identified, extending over a very long 10 kb coding sequence scattered in 62 exons. Point or small size mutations and large gene rearrangements have been identified and some deleterious missense mutations located in the PI3K domain have been reported (ATM mutation database: http://chromium.liacs.nl/LOVD2/ home.php) [START_REF] Concannon | Diversity of ATM gene mutations detected in patients with ataxia-telangiectasia[END_REF][START_REF] Taylor | Molecular pathology of ataxia telangiectasia[END_REF].

The mothers of A-T children, obligate ATM heterozygote mutation carriers, have been shown to present an increased BC risk [START_REF] Swift | Incidence of cancer in 161 families affected by ataxia-telangiectasia[END_REF]. Additional studies conducted in families of A-T children have shown a relative risk (RR) of about three attributable to ATM heterozygosity in women [START_REF] Easton | Cancer risks in A-T heterozygotes[END_REF][START_REF] Janin | Breast cancer risk in ataxia telangiectasia (AT) heterozygotes: haplotype study in French AT families[END_REF][START_REF] Olsen | Cancer in patients with ataxia-telangiectasia and in their relatives in the nordic countries[END_REF][START_REF] Thompson | Cancer risks and mortality in heterozygous ATM mutation carriers[END_REF]. Various BC population studies have tried to estimate the risk of developing BC amongst ATM mutation carriers, sometimes with controversial results, as reviewed in Ahmed and Rahman [START_REF] Ahmed | ATM and breast cancer susceptibility[END_REF]. In a more recent study, Renwick et al. analysed 443 cases of familial BC and 521 controls, and found 12 women carrying an ATM mutation in the case group (2.70%) and two in the control group (0.38%) [START_REF] Renwick | ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles[END_REF]. They estimated that ATM mutation heterozygosity was associated with a BC RR of 2.37 (95% CI: 1.51-3.78) [START_REF] Renwick | ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles[END_REF]. As the rate of BRCA1/2 mutation detection in BC women with a family history of BC is 15-20%, the rate of ATM mutations is five to seven times lower than the rate of BRCA1/2 mutations.

ATM mutations are involved in the development of lymphoid malignancies as A-T patients are at high risk of T-cell acute lymphocytic leukaemia (T-ALL) and especially prolymphocytic leukaemia (T-PLL) [START_REF] Taylor | Leukemia and lymphoma in ataxia telangiectasia[END_REF]. Somatic inactivation of the ATM gene has been reported in T-PLL and B-cell chronic lymphocytic leukaemia (B-CLL) occurring in patients outside an A-T context [START_REF] Vorechovsky | Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia[END_REF][START_REF] Stilgenbauer | Biallelic mutations in the ATM gene in T-prolymphocytic leukemia[END_REF][START_REF] Stoppa-Lyonnet | Inactivation of the ATM gene in T-cell prolymphocytic leukemias[END_REF]. Mutations observed in these reports are essentially missense mutations and mostly occur in the PI3 K domain. Monoallelic ATM germline mutations have also been reported in some patients with B-CLL, mantle cell lymphoma (MCL), and T-cell ALL [START_REF] Stankovic | Inactivation of ataxia telangiectasia mutated gene in B-cell chronic lymphocytic leukaemia[END_REF][START_REF] Bullrich | ATM mutations in B-cell chronic lymphocytic leukemia[END_REF][START_REF] Camacho | ATM gene inactivation in mantle cell lymphoma mainly occurs by truncating mutations and missense mutations involving the phosphatidylinositol-3 kinase domain and is associated with increasing numbers of chromosomal imbalances[END_REF][START_REF] Gumy-Pause | ATM alterations in childhood non-Hodgkin lymphoma[END_REF]. In Stankovic's series, 18% of patients were carriers of a monoallelic ATM germline mutation [START_REF] Stankovic | Inactivation of ataxia telangiectasia mutated gene in B-cell chronic lymphocytic leukaemia[END_REF]. In a short series of T-ALL affected children, a monoallelic ATM germline mutation was identified in 20% of cases [START_REF] Liberzon | Germ-line ATM gene alterations are associated with susceptibility to sporadic T-cell acute lymphoblastic leukemia in children[END_REF]. In a Nordic study of relatives of A-T children, an increased standardised incidence ratio (SIR) of 37 (95% CI: 13-80) was observed for leukaemias and non-Hodgkin lymphomas [START_REF] Olsen | Cancer in patients with ataxia-telangiectasia and in their relatives in the nordic countries[END_REF]. A trend towards an increased RR of leukaemias and lymphomas was also observed in a French study of the relatives of A-T children [START_REF] Geoffroy-Perez | Cancer risk in heterozygotes for ataxia-telangiectasia[END_REF].

To further our clinical understanding of familial BC without BRCA1/2 mutation and to limit the volume of ATM gene screening, we estimated the frequency of ATM mutation carriers in BC women with a family history of both BC and haematological malignancy. In other words, this study was designed to examine whether the presence of leukaemia and/or lymphoma amongst relatives in BC families was a predictive factor for ATM mutation. A second objective was to report a new high throughput method, called enhanced mismatch mutation analysis (EMMA), that we have adapted for ATM gene mutation screening. We report the results of EMMA ATM gene analysis in a series of 122 unrelated BRCA1 and BRCA2 mutation negative women with at least one-first or second degree relative with leukaemia or lymphoma.

Patients and methods

Patients

Between January 1995 and December 2005, 2,009 unrelated patients with BC (index case) were tested for BRCA1 and BRCA2 mutations at the Institut Curie. They had a family history of breast and/or ovarian cancer and/or developed BC before the age of 36. A family history was defined as either two first-degree relatives with cancer: (1) at least one with invasive BC before the age of 41, or (2) one with ovarian cancer or breast cancer at any age, or three first-degree or second degree relatives from the same lineage with invasive BC or ovarian cancer at any age, where the index case constituted one of the cases. Amongst the 1,629 patients with negative BRCA1 and BRCA2 screening, 122 had at least one-first or second degree relative with leukaemia or lymphoma, and this relative belonged to the branch of the family in which breast cancers were segregating. Informed consent for breast cancer genetic studies was obtained from each woman at the family cancer clinic.

DNA preparation

Two DNA extraction methods from whole blood were used:

(1) a modified perchlorate/chloroform procedure [START_REF] Johns Mb | Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol[END_REF], or (2) a rapid method using the NucleoSpin Blood extraction kit (Macherey-Nagel; ref. http://www.mn-net.com). DNA concentrations and the quality of each sample were determined spectrophotometrically at 260/280 nm (NanoDrop Ò ND-1000, NanoDrop technologies).

EMMA

The enhanced mismatch mutation analysis, EMMA (Fluigent, France), is a method of mutation screening based on heteroduplex analysis (HDA). Heteroduplexes and homoduplexes are separated by their different electrophoretic mobility due to their conformational differences. The resolution of EMMA is considerably better than that of standard HDA due to a combination of high resolution blockcopolymer sieving matrix, and nucleosides as additives in the electrophoretic medium [START_REF] Weber | A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis[END_REF][START_REF] Weber | Improving sensitivity of electrophoretic heteroduplex analysis using nucleosides as additives: application to the breast cancer predisposition gene BRCA2[END_REF]. The medium was loaded on the ABI3130XL sequencing system (Applied Biosystems 2006). EMMA also allows simultaneous analysis of numerous amplicons by multiplex analysis. This method allows the simultaneous detection of both small mutations and large gene rearrangements (LGR) by applying semiquantitative PCR conditions and an external control amplicon [START_REF] Weber | High-throughput simultaneous detection of point mutations and large-scale rearrangements by CE[END_REF]. As a result of multiplex analysis and LGR detection, EMMA is a high throughput screening mutation method. Data analysis was performed with Emmalys Ó software (Fluigent, France) for both point and small mutations and Excel Visual Basic macro for LGR analysis.

Amplicon and multiplex design

Primers were designed using the web-based Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www. cgi) to cover the 62 coding exons of the ATM gene and the splice site junctions. A series of 36 multiplexes of 2-4 amplicons, including the external gene control, was prepared. Amplicon sizes ranged between 217 and 567 bp (basepair). In a given multiplex, the length difference between two adjacent amplicons was at least 50 bp. Primer sequences and multiplex amplicon PCR conditions are reported in Table S1 (supplementary data).

EMMA PCR conditions PCR conditions were identical for all multiplexes. About 100 ng of DNA were amplified using Taq ABgene 0.06 U/ ll (ABgene UK, ref: www.abgene.com), 5 mM MgCl 2 , 4 9 0.2 mM dNTPs, and 0.08-0.3 lM of each primer in a final reaction volume of 20 ll. Cycling conditions were as follows: 94°C for 5 min, followed by 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by two steps of 72°C for 7 min and 98°C for 10 min. Heteroduplex formation consisted of 73 cycles of 98°C for 30 s, with temperature decreasing by 1°at each cycle, and a final step at 25°C for 10 min.

DNAs for EMMA validation study

As small insertions or deletions have been shown to be easily detected by EMMA, we focused our validation study on nucleotide substitutions previously detected by direct sequence analysis [START_REF] Weber | Improving sensitivity of electrophoretic heteroduplex analysis using nucleosides as additives: application to the breast cancer predisposition gene BRCA2[END_REF]. DNAs from A-T children or their ATM heterozygote carrier relatives, previously referred to our laboratory, were selected for nucleotide substitutions. DNAs carrying a substitution were available for 49 amplicons. The substitutions studied are reported in Table S2 (supplementary data).

A heterozygote ATM deletion from exons 26-65 was also used to validate LGR detection. An amplicon containing exon 8 or 11 of the RB1 gene was used as external control [START_REF] Houdayer | Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF[END_REF]. DNAs from these A-T patients were used as experimental controls in the study of the 122 BC patients.

Sequencing of EMMA variant profiles

Each DNA corresponding to a different EMMA profile as compared with the majority of profiles was sequenced. 100 ng of DNAs were amplified using Taq ABgene 0.025 U/ll (ABgene UK, ref: www.abgene.com), 4 9 0.2 mM dNTPs, 1.5 mM MgCl 2 , and 0.3 lM of each primer in a final reaction volume of 50 ll. Amplification was performed with a touchdown PCR protocol consisting of an initial denaturation step at 94°C for 5 min followed by 35 PCR cycles: denaturation at 94°C for 30 s, annealing for 30 s with temperature gradually decreasing from 60°C to 52°C for 35 cycles, and elongation at 72°C for 30 s.

PCR products were separated by agarose gel electrophoresis, purified (Macherey Nagel), and sequenced, using one of the PCR primers (usually the forward primer, except in the case of poor sequence quality), Big Dye Cycle Sequencing Reactions and an ABI3130XL automated sequencer (Applied Biosystems). Seqscape Ò (Applied Biosystems) software was used for sequence analysis.

The ATM coding sequence reference is U33841. The first translated nucleotide is the reference nucleotide.

Control DNAs for analysing the population frequency of sequence variants identified in the 122 BC cases

The frequency of DNA variants detected in the series of 122 BC women was determined in a series of 93-186 unrelated controls. Controls were children tested for RB1 gene mutation due to the presence of retinoblastoma, a disease unrelated to the ATM gene and for which our laboratory routinely performs mutation screening.

The arbitrary threshold of 1% was selected according to the definition of polymorphism in population genetics: a DNA variant with a frequency of 1% or above was rather considered with no functional effect.

Splice site score predictions

The genomic sequence environment of a DNA variant was analysed using Splice Site Prediction by Neural Network (NNSPLICE available at http://www.fruitfly.org/seq_tools/ splice.html), Splice Site Finder (SSF available at http:// violin.genet.sickkids.on.ca/*ali/splicesitefinder.html), Max EntScan (MES available at http://genes.mit.edu/burgelab/ maxent/Xmaxentscan_scoreseq.html) [START_REF] Houdayer | Evaluation of in silico splice tools for decision-making in molecular diagnosis[END_REF].

Protein conservation and A-GVGD score

The A-GVGD algorithm developed by Tavtigian et al. for the analysis of various genes, including the ATM gene, was used to contribute to discriminate disease-causing missense mutations from neutral changes (http://agvgd.iarc.fr/) [START_REF] Tavtigian | An analysis of unclassified missense substitutions in human BRCA1[END_REF][START_REF] Tavtigian | In silico analysis of missense substitutions using sequence-alignment based methods[END_REF]. This algorithm combines the biophysical characteristics of amino acid changes (GV) and alignments of protein sequences of different and phylogenetically distant species (GD), from human to zebrafish. The output from Align-GVGD is an ordered series of grades ranging from C65 (most likely deleterious) to C0 (most likely neutral). Missense mutations with a score below C15 are considered as probably neutral. When the score is at or above C15 and below C35, missense mutations remain unclassified, at or above C35 they are likely deleterious [START_REF] Tavtigian | In silico analysis of missense substitutions using sequence-alignment based methods[END_REF].

Results

EMMA validation for the ATM gene study

All of the 49 nucleotide substitutions located in 49 different amplicons of the ATM gene and selected regardless of their biological significance were detected (Fig. 1 and data not shown, see supplementary data for the list of mutations studied, Table S2). The deletion of exons 26-65 was detected in all corresponding amplicons.

EMMA analysis in the series of 122 women with familial BC and one relative with haematological malignancy A total of 28 different monoallelic sequence variants were identified in 35 of the 122 BC women screened for ATM gene mutation. These variants were initially interpreted as follows: 1 nonsense mutation, 18 missense mutations, 2 silent mutations, 7 intronic variants (Table 1). No LGR was detected. Twenty-two heterozygous polymorphisms were also detected and were classified as polymorphisms on the basis of previous reports in the literature, in databases, especially the NCBI SNP database, or in A-T patients studied in our laboratory (data not shown) (Table 2).

A nonsense mutation: c.4852C[T/p.Arg1618X was identified in one patient. This mutation, leading to a putative truncated and non-functional protein was considered to be deleterious.

The biological function of the 18 missense mutations identified was unknown, as they have not been previously reported as deleterious in databases. The following five criteria were used to assess the significance of their functional effect: (1) a carrier frequency \1% in a series of controls, (2) identification of the missense mutation in an A-T child carrying no more than one deleterious ATM mutation, (3) location in an ATM protein domain known to be functionally important, especially the PIK3 and FAT domains, (4) the importance of the amino acid (aa) change and aa species conservation analysed by the A-GVGD algorithm, and (5) a putative splice effect by using in silico splice site score prediction models. These criteria are listed in order of increasing relevance with respect to a putative deleterious effect.

In one case, a splicing defect was suspected on in silico analyses, as the c.7789G[T mutation, initially interpreted as p.Asp2597Tyr, is actually a splicing mutation. c.7789G is the first nucleotide of exon 55. The G [ T substitution leads to disruption of the acceptor site of intron 54 and a putative truncated protein of 2,598 aa (c.7789_7927del139/ p.Asp2597_Lys2643[LysfsX3). This splicing defect was confirmed by RNA analysis of patient lymphoblasts showing loss of exon 55 (Fig. 2). This mutation was identified in only one patient.

The remaining 17 missense mutations were classified into three categories according to the above criteria: likely deleterious, unknown variants (UV), and likely neutral.

Six missense mutations were classified as likely deleterious: p.Arg250Gln, p.Gly2023Arg, p.Thr2396Ser, p.Asn 2435Lys, p.Arg2854Cys, and p.Arg3008His (Table 1). They were considered as ''likely deleterious'' according to the A-GVGD algorithm except for p.Thr2396Ser and p.Arg 3008His which were respectively ''neutral'' or ''unclassified''. These missense mutations were located in the FAT

G - IVS27 1.0 c.3993?48A[G - IVS28 1.3 c.4777-20A[G - IVS33 1.0 c.5497-8T[C - IVS38 1.5 c.5497-15G[C - IVS38 1.0 c.8786?8A[C - IVS62 1.1
Het. freq. Heterozygote frequency according to NCBI protein domain and were not detected in a set of control DNAs. They have also been previously reported in a T-PLL, a feature that may argue in favour of their deleterious effect as they could have been selected by the tumour process [START_REF] Vorechovsky | Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia[END_REF].

In addition, the p.Arg3008His missense mutation has been detected in an A-T child whose second mutation was introducing an ATM stop codon (Micol et al., in preparation). These six missense mutations were identified in six patients.

Eleven missense mutations were classified as UV: p.Leu651Pro, p.Ser707Pro, p.Leu752Val, p.Phe763Leu, p.Ile826Leu, p.Val1519Ala, p.Ser1601Asn, p.Ile1659Val, p.Ser1673Thr, p.Asn2282His, and p.Leu2307Phe (Table 1). The majority of these UV were not located in the PI3K or FAT domains, and the A-GVGD scores were in favour of unclassified or neutral effect except in two cases. Regarding the p.Leu651Pro and p.Val1519Ala missense mutations, although their A-GVGD score was in favour of a likely deleterious effect, they were classified as UV as their carrier frequency amongst controls was high, respectively above 2 and 4% (Table 1). These 11 missense mutations were identified in 14 patients.

Two silent and seven intronic variants were also detected. Only criteria (1), (2), and ( 5) quoted above can be used. The in silico analyses did not suggest a splicing defect in any of these nine variants. The frequency of the seven intronic variants was \1% in our series of controls; In summary, a total of eight patients carrying an ATM mutation, two patients with a truncating mutation and six patients with a likely deleterious ATM mutation, were identified in this series of 122 patients. The frequency of ATM heterozygote mutation carriers is 6.56% (95% CI: 2.16-10.95).

Discussion

Epidemiological studies designed to elucidate the role of ATM mutations in cancer risks are limited by the large size of the gene and the large number of mutations spread throughout the coding sequence. To simplify the extensive laboratory work required for ATM screening, a new DNA analysis method was applied. Using similar analysis conditions for each amplicon, all of the 49 different control mutations previously identified by direct sequencing in Fig. 3 Family history of the eight patients carrying a deleterious or likely deleterious ATM mutation. The BC women tested for ATM mutation are indicated by an arrow. ALL: acute lymphoid leukaemia A-T children and a large gene deletion were detected by EMMA. Multiplex analysis including the search for LGR allowed screening of the 10 kb ATM coding sequence in 3 weeks in a large set of 94 patients.

In the series of 122 BC women with a relative with haematological malignancy, two deleterious mutations, leading to a putative truncated protein were identified, a nonsense mutation, and a splicing mutation not previously reported and characterised. The remaining 17 missense mutations were classified into three categories: six likely deleterious mutations, nine mutations of unknown significance, and two likely neutral mutations. The main criterion used to classify these missense mutations was the A-GVGD score. Moreover, p.Gly2023Arg, p.Thr2396Ser, and p.Arg3008His variants have been reported as somatic events in T-PLL or B-CLL and associated with loss of the second ATM allele by 11q deletion, features that argue strongly in favour of their deleterious effect as they could have been selected by the tumour process [START_REF] Vorechovsky | Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia[END_REF][START_REF] Austen | Mutation status of the residual ATM allele is an important determinant of the cellular response to chemotherapy and survival in patients with chronic lymphocytic leukemia containing an 11q deletion[END_REF]. The multiplicity of criteria to assess the significance of the functional effect of a DNA variant underlines that none a criterion alone is sufficient and that they have to be altogether taken into account with in some cases when they are leading to divergent conclusions the need to make some arbitrary decisions. The availability of functional testing and in vitro functional assays allowing characterisation of missense mutations would be very useful [START_REF] Taylor | Molecular pathology of ataxia telangiectasia[END_REF].

For each of the eight BC women carrying a deleterious or likely deleterious ATM mutation, only one relative presented a haematological malignancy. In six cases, this relative was a first degree relative and in two cases a second degree relative (Fig. 3). The disease was leukaemia in seven cases and Kaposi sarcoma occurring at age 55 in the remaining case. Three acute lymphoid leukaemia cases occurred during childhood at the ages of 3, 8, and 10 years; one leukaemia case occurred at 44 years and the three other cases were late-onset leukaemia, occurring after the age of 80. No blood samples which would have been required for ATM mutation co-segregation study from both cases with haematological malignancies and with breast cancer were obtained, most of the cases being deceased or lost of follow-up.

The estimated frequency of A-T heterozygotes in our series is 6.56% (95% CI: 2.16-10.95), a significantly higher figure than that observed in the general population which is estimated to be between 0.3 and 0.6% [START_REF] Thompson | Cancer risks and mortality in heterozygous ATM mutation carriers[END_REF][START_REF] Renwick | ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles[END_REF]. Although a trend towards an increased frequency of ATM carriers was observed in our series, it was not different from that observed in a population of familial BC women not selected for haematological malignancy in relatives, as the frequency of ATM carriers was 2.70%, a value situated in the confidence interval 2.16-10.95 estimated in our study [START_REF] Renwick | ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles[END_REF]. It is noteworthy that, although the RR of haematological malignancies in ATM heterozygote carriers may be high-SIR 37 in Olsen's study- [START_REF] Olsen | Cancer in patients with ataxia-telangiectasia and in their relatives in the nordic countries[END_REF], the absolute risk remains low, as the incidence of haematological malignancies is low in the general population. Thus the availability of familial aggregations of both breast cancer and haematological malignancies is low, limiting the power of such study. Finally, although the expected final classification of some UVs in the deleterious mutation group and that of some rare polymorphic allele will increase this frequency, a larger set of patients with a control group of BC women with no family history of haematological malignancy needs to be studied to determine whether the presence of relatives with leukaemia and/or lymphoma in BC families is a predictive factor for ATM mutation.

Fig. 1

 1 Fig. 1 Emma analysis of the multiplex amplified ATM exons 24, 28, and RB1 exon 8. a The profiles of 93 EMMA analysed DNAs were superimposed with Emmalys Ó software. Each peak corresponds to a single amplified exon or amplicon. Fragment 1 is ATM exon 24. Two profiles were identified: (1) a single peak profile detected in 90 DNAs, and (2) a triple peak profile detected in three DNAs with a heterozygous c.3161C[G/p.Pro1054Arg polymorphism. Fragment 2 is RB1 exon 8, used as external control for LGR detection. Only one peak profile was detected. Fragment 3 is ATM exon 28. Two profiles were identified: (1) a single peak profile detected in 92 DNAs, and (2)

  they were therefore classified as UV: IVS14, c1899-24A[G; IVS28, c.3993?40G[A; IVS30, c.4237-54T[C; IVS31, c.4437-7A[C; IVS48, c.6807?39C[T; IVS52, c.7516-32T[G; IVS55, c.7927?69T[C. The p.Lys1114Lys variant was also classified as UV, as its frequency in the control group was \1%. The p.Leu229Leu silent variant was classified as likely neutral, since its frequency in controls was[1%.

Fig. 2

 2 Fig. 2 cDNA analysis of the c.7789G[T mutation. a Initially interpreted as a missense mutation, the c.7789G[T substitution is a splicing mutation as shown by cDNA analysis of a lymphoblast cell line established from the patient carrying this mutation. Exons 54-59 were amplified from primers located in the coding sequence (sequence available on request). The amplified product was analysed by 1.5% agarose gel electrophoresis. One 756 bp long fragment was

Table 1

 1 ATM germline variants and their characteristics in 122 BC patients with a family history of breast and haemotological malignancy

	Nucleotide	Amino acid	Exon/	Freq. in	Freq. in	FAT or PI3K	GV	GD	A-GVGD combined	Signification
	change	change	IVS	patients	controls	domain	score	score	score and prediction	proposed
	c.4852C[T	p.Arg1618X	exon 34 1/122	0/186	-	-	-	-	Deleterious
	c.7789G[T	p.Asp2597_Lys2643	exon 55 1/122	0/186	-	-	-	-	Deleterious
		[LysfsX3
	c.749G[A	p.Arg250Gln	exon 9 1/122	0/186	-	0.00	42.81 C35	Likely deleterious
	c.6067G[A	p.Gly2023Arg	exon 43 1/122	1/186	FAT	0.00	125.13 C65	Likely deleterious
	c.7187C[G	p.Thr2396Ser	exon 51 1/122	0/93	FAT	111.69 6.18	C0	Likely deleterious
	c.7305C[G	p.Asn2435Lys	exon 51 1/122	0/93	FAT	0.00	93.88 C65	Likely deleterious
	c.8560C[T	p.Arg2854Cys	exon 60 1/122	0/93	PI3K	42.81 154.23 C35	Likely deleterious
	c.9023G[A	p.Arg3008His	exon 65 1/122	0/186	PI3K	0.00	28.82 C25	Likely deleterious
	c.1952T[C	p.Leu651Pro	exon 15 1/122	4/186	-	0.00	97.78 C65	Unknown
	c.2119T[C	p.Ser707Pro	exon 15 3/122	0/186	-	73.35 0.00	C0	Unknown
	c.2254C[G	p.Leu752Val	exon 17 1/122	0/186	-	0.00	30.92 C25	Unknown
	c.2289T[A	p.Phe763Leu	exon 17 2/122	0/186	-	157.56 0.00	C0	Unknown
	c.2476A[C	p.Ile826Leu	exon 19 1/122	0/186	-	141.80 4.86	C0	Unknown
	c.4556T[C	p.Val1519Ala	exon 32 1/122	8/186	-	0.00	65.28 C65	Unknown
	c.4802G[A	p.Ser1601Asn	exon 34 1/122	0/186	-	115.58 0.00	C0	Unknown
	c.4975A[G	p.Ile1659Val	exon 35 1/122	0/93	-	93.66 0.00	C0	Unknown
	c.5018G[C	p.Ser1673Thr	exon 36 1/122	0/186	-	111.49 0.00	C0	Unknown
	c.6844A[C	p.Asn2282His	exon 49 1/122	1/186	FAT	64.77 36.57 C0	Unknown
	c.6919C[T	p.Leu2307Phe	exon 49 1/122	1/186	FAT	0.00	21.82 C15	Unknown
	c.1899-24A[G -	IVS14 1/122	0/186	-	-	-	-	Unknown
	c.3993?40G[A -	IVS28 1/122	0/186	-	-	-	-	Unknown
	c.4237-54T[C -	IVS30 4/122	1/186	-	-	-	-	Unknown
	c.4437-7A[C	-	IVS31 1/122	0/186	-	-	-	-	Unknown
	c.6807?39C[T -	IVS48 2/122	0/185	-	-	-	-	Unknown
	c.7516-32T[G -	IVS52 1/122	0/93	-	-	-	-	Unknown
	c.7927?69T[C -	IVS55 1/122	0/186	-	-	-	-	Unknown
	c.3342G[A	p.Lys1114Lys	exon 25 1/122	1/149	-	-	-	-	Unknown
	c.685C[T	p.Leu229Leu	exon 9 1/122	5/186	-	-	-	-	Likely neutral

Table 2

 2 Identified ATM variants classifoed as polymorphisms in 122 BC patients with a family history of breast and haematological malignancy

	Nucleotide change Amino acid change Exon/IVS Het. freq. (%)
	c.544G[C	p.Val182Leu	exon 8	2.0
	c.2442C[A	p.Asp 814Glu	exon 18	1.3
	c.2572T[C	p.Phe858Leu	exon 19	1.5
	c.3161C[G	p.Pro1054Arg	exon 24	1.9
	c.4258C[T	p.Leu1420Phe	exon 31	3.1
	c.4578C[T	p.Pro1526Pro	exon 32	4.4
	c.5557G[A	p.Asp1853Asn	exon 39	10.6
	c.5558A[T	p.Asp1853Val	exon 39	1.1
	c.1899-55T[G	-	IVS14	2.4
	c.2125-68T[C	-	IVS15	3.7
	c.2250?22A[C	-	IVS16	1.2
	c.2250?34A[C	-	IVS16	1.2
	c.2250?78G[A	-	IVS16	1.9
	c.2839-90T[G	-	IVS20	38.0
	c.3403-14insA	-	IVS25	49.0
	c.3285-9del	-	IVS24	11.0
	c.3747-34A[

Breast Cancer Res Treat (2010) 119:443-452

Acknowledgments We would like to thank the patients for their participation in this study. We thank Patricia Legoix-Ne ´for her help in sequencing analyses, Catherine Dubois d'Enghien for her help in the selection of A-T DNA controls, and Nadine Andrieu for her critical reading of the manuscript. This work received financial support from ANR (Agence Nationale pour la Recherche), RNTS program.


Uffe Birk Jensen

Lone Sunde

Susanne Timshel

Britta Halvarsson

Anja Nissen

Inge Bernstein

Mef Nilbert

Uffe Birk

Jensen Ae

A E Susanne

Timshel Ae

A E Anja

Nissen Ae

Ae Mef Nilbert

U B Jensen

Á M Nilbert

Á I Bernstein

Mismatch repair defective breast cancer in the hereditary nonpolyposis colorectal cancer syndrome

Keywords: HNPCC, MMR, MLH1, MSH2, MSH6, Hereditary cancer

Introduction

Heredity has been recognized as a major cause of breast cancer with twin studies suggesting that hereditary components cause up to one-third of the cases [START_REF] Lichtenstein | Environmental and heritable factors in the causation of canceranalyses of cohorts of twins from Sweden, Denmark, and Finland[END_REF]. The complexity of the genetic background of hereditary breast cancer is increasingly recognized, and the susceptibility alleles may broadly be classified into three groups with respect to their frequency in the population and the associated risk of cancer [START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF][START_REF] Foulkes | Inherited susceptibility to common cancers[END_REF]. Rare high-penetrance alleles, e.g., BRCA1, BRCA2, TP53, PTEN, CDH1, and STK11 are estimated to be responsible for 2-3% of the cases. Moderate-penetrance alleles that confer a 1.5-to 2-fold risk increase have been found in CHEK2, ATM, BRIP1, and PALB2 and could through interaction with other environmental or genetic factors contribute to a substantial proportion of the hereditary cases. Finally, an increasing number of low-penetrance alleles have been identified and could through multiplicative effects confer high absolute risk in individuals with inappropriate combinations of such alleles [START_REF] Antoniou | Evidence for further breast cancer susceptibility genes in addition to BRCA1 and BRCA2 in a population-based study[END_REF].

The hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is a major cause of hereditary colorectal cancer, predominantly within the proximal colon, with 50-80% cumulative life time risk in mutation carriers, and in addition female carriers are at a 40-60% risk of endometrial cancer and a 10-15% risk of ovarian cancer [START_REF] Barrow | Colorectal cancer in HNPCC: cumulative lifetime incidence, survival and tumour distribution. A report of 121 families with proven mutations[END_REF][START_REF] Aarnio | Cancer risk in mutation carriers of DNA-mismatch-repair genes[END_REF][START_REF] Barrow | Cumulative lifetime incidence of extracolonic cancers in Lynch syndrome: a report of 121 families with proven mutations[END_REF]. A number of other tumors types, i.e., gastric cancer, cancer of the small intestine and the upper urinary skin tumors, brain tumors, and soft tissue sarcomas, have also been linked to HNPCC, albeit at lower frequencies. Clinical criteria, the so-called Amsterdam criteria, consider development of colon cancer, endometrial cancer, cancer of the small intestine, and cancer of the upper urinary tract and are used for standardized classification of these families [START_REF] Vasen | New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC[END_REF]. Defective mismatch repair (MMR) has been identified as causative and families in which mutations have been identified are referred to as having Lynch syndrome. Identification of HNPCC/Lynch syndrome families is crucial since surveillance programs, primarily for colorectal tumors, have proven highly effective.

There are conflicting reports on a possible increased risk of breast cancer in families with HNPCC/Lynch syndrome [START_REF] Aarnio | Cancer risk in mutation carriers of DNA-mismatch-repair genes[END_REF][START_REF] Watson | The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome[END_REF][START_REF] Watson | Extracolonic cancer in hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Vasen | Is breast cancer part of the tumor spectrum of hereditary nonpolyposis colorectal cancer?[END_REF][START_REF] Scott | Hereditary nonpolyposis colorectal cancer in 95 families: differences and similarities between mutation-positive and mutation-negative kindreds[END_REF][START_REF] Muller | Exclusion of breast cancer as an integral tumor of hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Itoh | Risk of cancer death in first-degree relatives of patients with hereditary nonpolyposis cancer syndrome (Lynch type II): a study of 130 kindreds in the United Kingdom[END_REF]. Though breast cancer is not considered part of the tumor spectrum, MMR defective breast cancer has been described in a number of mutation carriers [START_REF] Watson | Extracolonic cancer in hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Vasen | Is breast cancer part of the tumor spectrum of hereditary nonpolyposis colorectal cancer?[END_REF][START_REF] Westenend | Breast cancer in an MSH2 gene mutation carrier[END_REF][START_REF] Risinger | Molecular genetic evidence of the occurrence of breast cancer as an integral tumor in patients with the hereditary nonpolyposis colorectal carcinoma syndrome[END_REF][START_REF] Nelson | Familial clustering of colon, breast, uterine, and ovarian cancers as assessed by family history[END_REF][START_REF] De Leeuw | Exclusion of breast cancer as an integral tumor of hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Boyd | Male breast cancer in the hereditary nonpolyposis colorectal cancer syndrome[END_REF]. In order to characterize the role of MMR in breast cancers that have developed in HNPCC families, we identified such cases in the national Danish HNPCC register and herein evaluated tumor morphology and MMR status.

Materials

The national Danish HNPCC-registry was initiated in 1991, covers a population of 5 million and contains clinical, molecular, and surveillance information on families diagnosed with HNPCC. Pedigree information is collected via the family history, and family members are traced by their unique civic registration numbers in the central population registry. Malignancies are verified in the national pathology register, in medical records and death certificates

Methods

For MMR protein immunostaining, 4-lm sections were deparaffinized in Tissue-Clear Ò (Sakura Finetek, Hoge Rijndijk, the Netherlands) and rehydrated through decreasing concentrations of ethanol. Antigen retrieval was achieved by microwave treatment in 1 mM EDTA/10 mM Tris buffer (pH 9.0) boiling for 15 min followed by a cooling for 15 min. Immunohistochemical staining was performed manually using antibodies to MLH1 (G168-15, dilution 1:100) and PMS2 (A16-4, 1:500) from BD Biosciences (San Jose, CA, USA), MSH2 (25D12, 1:200) from Novocastra (Newcastle, UK), and MSH6 (Clone 44, 1:200) from BD Biosciences. The EnVisionTM detection kit (DAKO Cytomation) was used and modified using an extra enhancing step after diaminobenzidine treatment and 0.1% hematoxyline staining. Retained staining was defined as nuclear staining in neoplastic cells. Nuclear staining in stromal components and lymphocytes served as internal controls and was required in order to classify a tumor as having loss of one or several MMR proteins in the tumor cells.

The microsatellite instability (MSI) analysis included the mononucleotide repeats BAT25, BAT26, BAT40 and the dinucleotide repeats D2S123, D5S346, BAT34, and D17S250 and was performed according to standard Fig. 1 MMR protein immunostaining of a breast cancer (case 3-14) demonstrating loss of expression for MLH1 and PMS2 in the tumor cells with retained staining in the stroma (top). The staining for MSH2 and MSH6 was retained in both components (bottom). In concordance with this observation, the patient carries a germline mutation in the MLH1 gene procedures using fluorescently labeled primers and size on an ABI310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). A detailed protocol is available from the authors upon request.

Results

The 20 breast cancers in mutation carriers were diagnosed at mean 50 (range 33-67) years of age. All but one of the diagnoses were histopathologically confirmed and revealed ductal carcinomas in 16 cases, medullary differentiation in one case and mucinous differentiation in one, whereas the last tumor was classified as ductal carcinoma in situ (DCIS). The disease-predisposing MMR gene mutations affected MSH6 in eight cases, MSH2 in seven cases, and MLH1 in five cases. Lymphocytic reactions were classified by pathologic review of 15 tumors and identified tumorinfiltrating lymphocytes in eight breast cancers and peritumoral lymphocytes in seven (Table 1; Fig. 2). MMR protein immunostaining was performed in 16 tumors and showed normal, retained expression in nine tumors and loss of at least one MMR protein in seven tumors. The losses affected MLH1/PMS2 in one case, MSH2/MSH6 in four, MSH2 only in one, and MSH6 only in one tumor. The losses were in all cases consistent with the underlying germline MMR gene mutation (Table 1; Fig. 1). The seven MMR defective tumors were diagnosed at mean age 50 (33-66) years and included five ductal carcinomas and one medullary cancer (Fig. 2).

Discussion

Studies on the role of MMR defects in breast cancer have reached different conclusions regarding a possible link to HNPCC/Lynch syndrome. Since no increased risk has been demonstrated, breast cancer is not considered part of the HNPCC/Lynch syndrome, and surveillance for breast cancer is not called for. A number of case reports have, however, described breast cancer in individuals with HNPCC (Table 2). This observation led us to investigate the frequency of breast cancer in the national Danish HNPCC register and to assess MMR status in tumors from mutation carriers. In the 164 families with disease-predisposing MMR gene mutations, we identified 20 mutation carriers affected with breast cancer at mean 51 years of age. Loss of MMR protein expression was found in 7/16 breast cancers evaluated, which demonstrates that the defective MMR plays a role in a substantial proportion of these cases. These seven tumors were linked to three MMR gene mutations, MLH1 c.1732-2A[T, MSH2 c.942? 3A[T, and MSH6 c.4001?2T[C. The MSH2 mutation is recognized as internationally recurrent, and the breast cancers occurred in individuals from 3 of the 16 Danish families with this mutation. The MSH6 mutation has been identified in four families in Denmark, but strikingly the breast cancers developed in three females from the same family (Fig. 3). MMR defective colorectal cancers are characterized by frequent lymphocytic reactions, most commonly expressed as peritumoral lymphocytes and as tumor-infiltrating lymphocytes [START_REF] Jass | Pathology of hereditary nonpolyposis colorectal cancer[END_REF]. Their overrepresentation in colorectal cancers in HNPCC has been clearly established, whereas a possible role in breast cancer remains to be clarified. Lymphocytic reactions in HNPCCassociated breast cancer have previously only been reported in one of the case studies, which identified extensive lymphocytic infiltrates [START_REF] Westenend | Breast cancer in an MSH2 gene mutation carrier[END_REF]. Half of the cancers here investigated showed lymphocytic reactions and if indeed present at the high frequency suggested here, it could represent a potential marker for MMR defective breast cancer in individuals with HNPCC/Lynch syndrome. 2). These tumors were diagnosed at an early age, mean 50 years, which is 10-20 years earlier than the sporadic cases are diagnosed and at about the same age as the more common colorectal and endometrial cancers typically are diagnosed in HNPCC. This age also concurs with the mean age for breast cancer in BRCA mutation carriers [START_REF] Risch | Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer[END_REF]. Though the route for breast cancer development in HNPCC remains unknown, it is possible that the MMR defect may also accelerate the accumulation of mutations in breast cancerassociated genes, and thereby enhance tumor progression. In summary, the demonstration of defective MMR in concordance with the gene mutated in about half of the breast cancers from female HNPCC/Lynch syndrome patients suggests that this tumor type can be added to the list of tumors, e.g., hepatobiliary cancer, gliomas, sebaceous carcinomas of the skin, and soft tissue sarcomas that occur at low frequency in this syndrome. From a clinical perspective, this observation serves as a reminder not to dismiss breast cancers in these families as sporadic, but to consider that development hereof may indicate mutation carrier status.

Fig. 2

 2 Fig. 2 Hematoxylin and Eosin staining of a MMR defective breast cancer showing a 910 magnification with abundant peritumoral lymphocytes and b 940 magnification with tumor-infiltrating lymphocytes

Fig. 3

 3 Fig. 3 Pedigree from a Lynch syndrome family with a MSH6 c.4001C[T mutation. The three females indicated with arrows developed breast cancers with immunohistochemical loss of MSH6

Table 1

 1 Summary of breast cancers in the Danish Lynch syndrome cohort [90% of the diagnoses confirmed. number of families are identified today and a selection bias toward the most explicit families registered. The register contains 164 families with disease-predisposing MMR gene mutations, 97 of which fulfill the Amsterdam I/II criteria[START_REF] Nilbert | Broad phenotypic spectrum in familial adenomatous polyposis; from early onset and severe phenotypes to late onset of attenuated polyposis with the first manifestation at age 72[END_REF]. Totally, 47 breast cancers were diagnosed in mutation carriers (n = 20) and first-degree relatives (n = 27), but due to uncertain carrier status the latter were not investigated. Of the 20 mutation carriers, 11 developed a metachronous, HNPCC-associated cancer, most commonly colon cancer and/or endometrial cancer. In order to assess MMR function, we retrieved breast cancer specimens from proven mutation carriers and could hereby study 16/20 tumors.

	Individual	Age at	Histopathology Tumor-	Peritumoral	Gene	Mutation	Immunohistochemical	Other cancer
	number	diagnosis		infiltrating	lymphocytes	mutated		expression	type/age
				lymphocytes
	B3-14	33	Ductal	-	-	MLH1 c.1732-2A[T	mlh1-/pms2-	Colon/36
	B1036-8 47	Medullary	?	?	MSH2 b c.942?3A[T	msh2-/msh6-	Endometrial/44
	B501-8	66	Ductal	?	?	MSH2 c.942?3A[T	msh2-/msh6-	Endometrial/43
	B1905-8 39	Ductal	NA	NA	MSH2 c.942?3A[T	msh2-/msh6-	-
	B748-36 44	Ductal	-	-	MSH6 c.4001?2T[C	msh2-/msh6-	-
	B748-28 58/63	Ductal a	-	-	MSH6 c.4001?2T[C	msh6-	-
	B748-30 66	Ductal	?	?	MSH6 b c.4001?2T[C	msh2-	Ureter/66,
									cardia/68
	B13-4	52/67	Ductal a	?	?	MLH1 c.1276C[T	4 MMR proteins normal -
	B19-46	59	Ductal/	-	-	MLH1 c.1165C[T	4 MMR proteins normal Colon/50
			mucinous
	B19-144 40	Ductal	-	-	MLH1 c.1165C[T	4 MMR proteins normal Colon/59
	B992-80 45	Ductal	?	?	MLH1 c.1852_1854delAAG 4 MMR proteins normal -
	B8-14	45	DCIS	?	?	MSH2 c.1387-?_1661??del 4 MMR proteins normal Colon/35,
									ovarian/44
	B669-8	53	Ductal	?	?	MSH2 c.3647-1G[A	4 MMR proteins normal -
	B172-10 48	Ductal	?	-	MSH2 c.888_889delC	4 MMR proteins normal Rectal/20,
									colon/38
	B678-60 60	Ductal	-	-	MSH6 c.3647-1G[A	4 MMR proteins normal Endometrial/58
	B1338-54 52	Ductal	NA	NA	MSH6 c.3647-1G[A	4 MMR proteins normal -
	B259-8	58	NA	NA	NA	MSH2 c.367-?_1076??del NA	-
	B748-12 59/61	Ductal a	NA	NA	MSH6 c.4001?2T[C	NA	Endometrial/55,
									urethral/79
	B1480-60 45	Ductal	NA	NA	MSH6 c.1444C[T	NA	Endometrial/45,
									urethral/69
	B1388-8 53	Ductal	NA	NA	MSH6 c.3647-1G[A	N A	-

a Refers to the second primary cancer, which was studied b MSI-high phenotype demonstrated in breast cancer tissue with a

Table 2

 2 Summary of present and published breast cancers linked to HNPCC/Lynch syndrome Including the cases here identified, 17 breast cancers that developed in individuals with Lynch syndrome have demonstrated MMR defects (Table

Breast Cancer Res Treat (2010) 120:777-782

Breast Cancer Res Treat (2010) 120:777-782

Acknowledgments Financial support was granted from the Danish Cancer Fund and the Lundbeck Foundation. We acknowledge Eva Rambech, Lund University, Sweden for performing the MSI analyses.


Marie Klintman
email: marie.klintman@med.lu.se

Ib Sidse Ørnbjerg Würtz

Pernille Jarle Christensen

Braemer Hertel

Mårten Fernö

Martin Malmberg

Henning Mouridsen

Frederik Cold

Anne-Sofie Schrohl

John A Foekens

A E Sidse

Ørnbjerg Wu

A E Ib

Ae Pernille Braemer

Hertel Ae

Ma ˚rten

Ferno ¨ae

A E Henning

Mouridsen Ae

Frederik Cold Ae

Anne-Sofie Schrohl

A E Per

Malmstro ¨m Ae

Nils Bru

Á M Ferno

¨á M Malmberg

Á P Malmstro

S Ørnbjerg Wu

Á P Braemer

A.-S Schrohl

Á N Bru

I J Christensen

Association between tumor tissue TIMP-1 levels and objective response to first-line chemotherapy in metastatic breast cancer

Keywords: TIMP-1, Predictive marker, Breast cancer, Resistance, Chemotherapy, Tumor extracts

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

The majority of women diagnosed with breast cancer are offered adjuvant systemic treatment, i.e., chemotherapy and/or endocrine therapy, following surgical removal of the primary tumor. However, a large proportion of these women do not benefit from the treatment, but they may still suffer from the toxic side effects caused by the drugs. Especially, anthracyclines can cause irreversible cardiac toxicity. Today, only few predictive markers are available in breast cancer, including the presence of estrogen (ER) and progesterone receptors (PR) for predicting response to endocrine treatment and HER-2 expression for predicting the response to trastuzumab [START_REF] Goldhirsch | Thresholds for therapies: highlights of the St Gallen international expert consensus on the primary therapy of early breast cancer[END_REF][START_REF] Thomssen | Do we need better prognostic factors in node-negative breast cancer?[END_REF][START_REF] Goldhirsch | First-select the target: better choice of adjuvant treatments for breast cancer patients[END_REF]. At present, no markers to predict the response to chemotherapy in general or to specific types of chemotherapy are in routine clinical use, although amplification/deletion of topoisomerase II alpha (topo IIa) seems promising in relation to treatment with anthracyclines [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][START_REF] Nielsen | The value of TOP2A gene copy number variation as a biomarker in breast cancer: Update of DBCG trial 89D[END_REF][START_REF] Arriola | Topoisomerase II alpha amplification may predict benefit from adjuvant anthracyclines in HER2 positive early breast cancer[END_REF]. Therefore, additional predictive markers need to be identified to enable individualized treatment of the patients.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has proven to be a promising marker in breast cancer carrying both prognostic [START_REF] Ree | High levels of messenger RNAs for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in primary breast carcinomas are associated with development of distant metastases[END_REF][START_REF] Mccarthy | High levels of tissue inhibitor of metalloproteinase-1 predict poor outcome in patients with breast cancer[END_REF][START_REF] Schrohl | Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer[END_REF][START_REF] Schrohl | Tumor tissue levels of tissue inhibitor of metalloproteinase-1 as a prognostic marker in primary breast cancer[END_REF][START_REF] Wu ¨rtz | Plasma and serum levels of tissue inhibitor of metalloprrteinases-1 are associated with prognosis in node-negative breast cancer-a prospective study[END_REF] and, as shown more recently, predictive information [START_REF] Lipton | Elevated plasma tissue inhibitor of metalloproteinase-1 level predicts decreased response and survival in metastatic breast cancer[END_REF][START_REF] Lipton | Serum TIMP-1 and response to the aromatase inhibitor letrozole versus tamoxifen in metastatic breast cancer[END_REF][START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF][START_REF] Willemoe | Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study[END_REF]. For example, our laboratory has previously demonstrated that high primary tumor tissue levels of TIMP-1 were significantly associated with a poor response to the most frequently used chemotherapeutic drugs in metastatic breast cancer [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF]. More recently, we have shown that TIMP-1 immunoreactivity in the cancer cells is significantly associated with benefit from adjuvant anthracycline treatment in patients with primary breast cancer [START_REF] Willemoe | Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study[END_REF]. These clinical findings have also been supported by in vitro cell studies [START_REF] Davidsen | TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapyinduced apoptosis[END_REF][START_REF] Li | Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells[END_REF]. For example, murine fibrosarcoma cells deficient of the TIMP-1 gene were shown to be considerably more sensitive to chemotherapy compared with their genetically identical wild-type controls [START_REF] Davidsen | TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapyinduced apoptosis[END_REF]. Likewise, transfected MCF10A cells overexpressing TIMP-1 were less sensitive to adriamycin compared with non-transfected cells [START_REF] Li | Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells[END_REF].

The aim of the present study was to further investigate the association between tumor tissue TIMP-1 and the effect of chemotherapy in patients with metastatic breast cancer. Therefore, using ELISA, we measured TIMP-1 in primary tumor tissue extracts from 162 patients with metastatic breast cancer and related the measurements to the objective response to first-line chemotherapy (primarily anthracycline-containing regimens). Whenever applicable, the REMARK recommendations for reporting of tumor marker studies were followed [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies (REMARK)[END_REF].

Materials and methods

Patients and treatment. This study included two retrospective patient populations: (1) a patient population with available detergent-extracted tumor tissue extracts (n = 43) and (2) a patient population with available cytosolic tumor tissue extracts (n = 119). These two patient populations were pooled resulting in a total population of 162 patients. Patients were included in the present study provided that (1) tumor tissue extracts from the primary tumor were available, (2) they had had recurrence of disease (recurrence was defined as appearance of metastatic cancer lesions after primary surgery as determined by biopsy and/or other relevant diagnostic procedures), (3) they had received chemotherapy (C3 cycles) for this recurrence, and (4) hospital medical records were available including data on objective response. The study was approved by the local ethical committees.

Patients with detergent-extracted tissues: This patient population consisted of a cohort of 43 Danish patients who underwent surgery for primary breast cancer from 1989 to 1993. Patient characteristics and pathological findings were registered by the Danish Breast Cancer Cooperative Group (DBCG). Clinicopathological characteristics of these patients have been described previously [START_REF] Schrohl | Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer[END_REF][START_REF] Pedersen | The complex between urokinase and its inhibitor in primary breast cancer, relation to survival[END_REF] and are summarized in Table 1.

Patients with cytosolic extracts: This patient population consisted of both Swedish and Danish patients. The Swedish patients consisted of 110 women from the South Sweden Health Care Region, and they had all undergone surgery for primary breast cancer during the period 1987-2000. Nine Danish patients were operated during the period 1995-1999 and were enrolled in the Scandinavian Breast Group Trial, study 9403 [START_REF] Ejlertsen | Phase III study of intravenous vinorelbine in combination with epirubicin versus epirubicin alone in patients with advanced breast cancer: a Scandinavian Breast Group Trial (SBG9403)[END_REF]. Characteristics of these patients are also summarized in Table 1.

In general, surgical procedures consisted of modified radical mastectomy or breast conserving surgery and axillary lymph node dissection. Radiotherapy was offered to patients undergoing breast conserving surgery as well as patients with invasion of the deep resection margin after mastectomy and to patients with 4 or more positive lymph nodes. Median age at surgery was 53 years (range 33-74 years). Adjuvant therapy included cyclophosphamide, methotrexate, and 5-fluorouracil (CMF, 52 patients) or cyclophosphamide, epirubicin, and 5-fluorouracil (CEF, 4 patients), radiotherapy (77 patients), and/or endocrine treatment (60 patients). All patients received first-line chemotherapy for their metastatic disease: 146 received anthracycline-containing therapy, 9 patients received CMF, and 7 patients received other types of chemotherapy (vinorelbine, novantrone, cyclophosphamide, or 5-fluorouracil). Response to chemotherapy had been evaluated during treatment and was reassessed according to WHO response criteria [START_REF]WHO handbook for reporting results for cancer treatment[END_REF] by thorough examinations of the patients' hospital records.

Samples. At surgery, residual breast tumor tissue from biochemical ER analyses was frozen and stored in sealed containers at -80°C until tissue extraction. The tissue extraction procedure has previously been described in detail [START_REF] Pedersen | The complex between urokinase and its inhibitor in primary breast cancer, relation to survival[END_REF]. For preparation of the detergent-extracted tissue, proteins were extracted using an acetate-detergent low pH extraction buffer [START_REF] Pedersen | The complex between urokinase and its inhibitor in primary breast cancer, relation to survival[END_REF]. For the cytosolic extracts, proteins were extracted using a buffer containing Tris-HCl (10 mmol/ L), EDTA (1.5 mmol/L), Na 2 MoO 4 (5.0 mmol/L), and monothioglycerol (1.0 mmol/L), pH of 7.4.

TIMP-1 measurements. The total level of TIMP-1 was measured using a previously described validated in-house ELISA [START_REF] Schrohl | Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer[END_REF][START_REF] Holten-Andersen | Quantitation of TIMP-1 in plasma of healthy blood donors and patients with advanced cancer[END_REF]. For measurements, samples were diluted 1:101 in sample dilution buffer. As internal control, duplicates of a control plasma pool were included on every plate. All TIMP-1 measurements were normalized by relating the measurements to the total concentration of protein (as measured by the protein BCA Protein Assay kit (Pierce) according to the manufacturer's instructions) in individual samples.

Statistical analyses. Descriptive statistics for TIMP-1 are presented by the median as well as minimum and maximum and stratified by population and response. Comparisons of categorical data were tested using a chi-square test for independence. The association between response and TIMP-1 was found using the logistic regression model. TIMP-1 was scored as a continuous variable on the log scale [ln (TIMP-1 ? 1)]. For the cytosolic samples, TIMP-1 was also analyzed as a dichotomous variable. The cut-off chosen, 33.4 ngTIMP-1/ mg protein, was the optimized cut-off derived from our previous publication [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF]. Response was scored as complete response (CR) or partial response (PR) (response) versus stable disease (SD) or progressive disease (PD) (nonresponse). Pooled analyses were performed using a random effects model thereby taking into account the difference among populations. The effect of TIMP-1 on response is presented as odds ratios (OR) with 95% confidence intervals (CI). The relationship between TIMP-1 and progression-free survival (PFS) as well as overall survival (OS) has been analyzed using the Cox proportional hazards model. The effect is presented as hazard ratios (HR). Model assessment was accomplished using conventional techniques. All calculations were done using SAS (v9.1, SAS institute, Cary, NC, USA). P \ 5% were considered significant.

Results

Patient characteristics. Differences between the characteristics of the patients in the cytosol and detergent group were tested using a chi-square test for independence. Significant differences between the groups were found for nodal status (P = 0.007), tumor size (P = 0.019), adjuvant therapy (P = 0.0004), and metastasis site (P = 0.025), whereas no difference was found for age at surgery (P = 0.68), menopausal status (P = 0.44), steroid receptor status (P = 0.15), or first-line chemotherapy (P = 0.15). Detailed characteristics of all patients are found in Table 1.

TIMP-1 levels and associations between TIMP-1 and clinicopathological parameters. For the patient population with detergent extracts, the median TIMP-1 level was 21.6 ng TIMP-1/mg protein (range 6.32-190 ng TIMP-1/mg protein) and for the patient population with cytosolic extracts, it was 14.3 ng TIMP-1/mg protein (range 0-75.8 ng TIMP-1/mg protein). Detailed characteristics of the patients are shown in Table 1. Associations between TIMP-1 and other clinicopathological parameters are shown in Table 1 for both populations separately. As can be seen, no associations between the TIMP-1 levels and the clinicopathological parameters were found except for tumor size, where a correlation was found for patients with cytosolic extracts (P = 0.04).

Response to chemotherapy

TIMP-1-univariate analysis. Median time between primary surgery and distant recurrence was 29 months (0-139 months). After primary surgery, median survival time of all patients was 47 months (5-166 months) and 16 months after initiation of chemotherapy. Median time to progression was 8 months (1-129 months). At the time of analysis, 153 patients (95%) had died.

For patients with detergent extracts, the median TIMP-1 level in responders (n = 22) was 19.8 ng TIMP-1/mg protein (6.32-52.1 ng TIMP-1/mg protein) and for nonresponders (n = 21), it was 24.6 ng TIMP-1/mg protein (9.13-190 ng TIMP-1/mg protein), P = 0.27. For patients with cytosolic extracts, the median TIMP-1 level in responders (n = 54) was 13.9 ng TIMP-1/mg protein (2.95-75.8 ng TIMP-1/mg protein) and for non-responders (n = 65), it was 15.4 ng TIMP-1/mg protein (0-64.4 ng TIMP-1/mg protein), P = 0.07.

For analysis of the association between TIMP-1 and response, the two populations were pooled using a random effects model chosen in order to accommodate for the intrinsic heterogeneity between the studies. In the pooled present patient population, 14 (8.6%) patients had CR, 62 (38.3%) patients had PR, 46 (28.4%) patients had SD (27 patients had SD C6 months), and 40 (24. 7%) patients had PD. For analysis of association between the TIMP-1 levels, response was scored as CR or PR (response) versus SD or PD (no response). When analyzed as a continuous logtransformed variable, increasing tumor levels of TIMP-1, reaching borderline significance, were associated with a decreasing probability of response to CMF or anthracycline-containing chemotherapy (OR = 1.59, 95% CI: 0.97-2.62, P = 0.07, Fig. 1 top; Table 2). This means that when the TIMP-1 level increased 1 unit on the log scale, i.e., *2.7 times, the risk of belonging to the group of nonresponders increased 1.59 times. Although not significant, similar ORs were achieved when performing subgroup analyses for the detergent population (OR = 1.95, 95% CI: 0.59-6.44, P = 0.27) and the cytosol population (OR = 1.50 95% CI: 0.86-2.63, P = 0.15). We also grouped SD C6 months with CR and PR, i.e., ''clinical benefit'', resulting in an OR of 1.43 (95% CI: 0.87-2.37, P = 0.16).

For comparison, we included the previous data from Schrohl et al. [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF] (OR = 2.0, 95% CI: 1.1-3.3, P = 0.01, Fig. 1 middle). It can be seen that the odds ratio from the present study, although not reaching statistical significance at the 5% level, and the odds ratio from the Schrohl study are quite similar. We also performed a pooled analysis of the Schrohl patient population and the present patient population (n = 335) using the random effects model and found that the OR for response was 1.61 (95% CI: 1.14-2.29, P = 0.007, Fig. 1 bottom).

In the Schrohl study, an optimized cut-point for the cytosol samples of 33.4 ng TIMP-1/mg protein was identified, above which none of the patients responded to chemotherapy [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF]. When applying this cut-point on the cytosol population in the current study, a significant difference in response rates was found between the patients with high TIMP-1 levels (n = 18), and low levels (n = 101), with corresponding response rates of 22% and 50%, respectively (P = 0.032, chi-square test, OR = 3.43, 95% CI: 1.06-11.1).

Other clinicopathological factors-univariate analysis. In univariate logistic regression analysis, dominant metastatic site (visceral or soft tissue vs. bone) as well as disease-free interval ([12 vs. B12 months) were associated with a better response to treatment. Lymph node status, age, tumor size, and steroid receptor status showed no significant association with response to chemotherapy (Table 2).

Multivariate analysis. In a multivariate analysis, only steroid receptor status and disease-free interval had a significant association with a better response to chemotherapy. For TIMP-1, an OR similar to the OR in the univariate analysis was found (Table 2).

Association between TIMP-1 and survival. We performed survival analyses with PFS and OS as endpoints. No statistically significant associations were found between increasing levels of TIMP-1 (continuous and log-transformed) and PFS (HR = 1.23, 95% CI: 0.94-1.61, P = 0.13) or OS (HR = 1.17, 95% CI: 0.92-1.48, P = 0.19).

Discussion

Predictive markers for the treatment of breast cancer are sparse. Especially, markers to predict whether the patients will respond to chemotherapy in general or to specific types of chemotherapy are required, since a significant number of the patients do not benefit from the treatment although they may suffer from treatment-induced adverse events. Thus, research concerning the identification of predictive markers is highly warranted. Recently, primary tumor tissue levels of TIMP-1 have been demonstrated to carry predictive information of the response to chemotherapy in patients with metastatic breast cancer, as patients with high levels of TIMP-1 benefit less from first-line chemotherapy compared with patients with low levels [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF]. Interestingly, this association has also been demonstrated in metastatic colorectal cancer [START_REF] Sørensen | TIMP-1 is significantly associated with objective response and survival in metastatic colorectal cancer patients receiving combination of irinotecan, 5-fluorouracil, and folinic acid[END_REF]. Furthermore, Lipton et al. [START_REF] Lipton | Serum TIMP-1 and response to the aromatase inhibitor letrozole versus tamoxifen in metastatic breast cancer[END_REF] showed that high serum TIMP-1 levels were predictive of low objective response rate of endocrine therapy in metastatic breast cancer. Recently, we have also shown that TIMP-1 immunoreactivity in breast cancer is associated with benefit from adjuvant anthracycline treatment but not from adjuvant treatment with CMF [START_REF] Willemoe | Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study[END_REF]. The purpose of the present study was to further investigate the preliminary findings from metastatic breast cancer patients in an independent patient population. An independent patient population was obtained by pooling metastatic breast cancer patients for whom detergent-extracted or cytosol-extracted samples from their primary tumors had been stored. The difference in the patient populations was circumvented in the statistical analysis by using a random effects model thereby taking into account the difference in sampling and extraction procedures. Next, we measured the primary tumor levels of TIMP-1 in the collected samples from the present patient population and related the TIMP-1 levels to the objective response to first-line anthracyclines or CMF treatment. When analyzing the present data in the same way as we have analyzed the original Schrohl data [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF], i.e., responders versus non-responders in relation to the primary tumor TIMP-1 levels, we found that high TIMP-1 levels were associated with a poor response to treatment although not statistically significant. The odds ratio from the present study was very similar to the odds ratio from the Schrohl study [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF]. Using the random effects model to perform a pooled analysis of the patients from the present patient population and the Schrohl patient population, we found an odds ratio for response of 1.61 (range 1.14-2.29, P = 0.007). Thus, even though the results in the present study did not reach statistical significance they support the results from the original Schrohl study. In the initial study on cytosolic extracts, an optimized cut-point of 33.4 ng TIMP-1/mg protein was identified, above which none of the patients responded to chemotherapy [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF]. When applying this cut-point on the cytosolic extracts in the present study, a significant difference in response to chemotherapy was found between the patients with low TIMP-1 levels compared to those with high TIMP-1 levels. In the survival analyses, there was no statistically significant association between increasing levels of TIMP-1 and PFS and OS. The explanation for the lack of statistical significance could be an insufficient number of patients. It can also be noted that even though more patients had received first-line CMF in the Schrohl study (54 vs. 6% in the present study) [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF], where a lower predictive value of TIMP-1 could be expected [START_REF] Willemoe | Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study[END_REF], a larger proportion of patients had received adjuvant therapy in the present study (35% adjuvant chemotherapy and 37% adjuvant endocrine treatment) compared to the Schrohl study [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF] (21 and 14%, respectively). This could have obscured the ''true'' response to first-line chemotherapy thereby masking a possibly stronger predictive value of TIMP-1 in the present study.

If TIMP-1 could be used as a predictive marker in the metastatic setting, it would be of great importance since it could be used to ensure a tailored treatment for the individual patient. The biological explanation for the clinical findings mentioned earlier has been addressed in a number of studies. In this regard, TIMP-1 has been shown to inhibit apoptosis [START_REF] Davidsen | TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapyinduced apoptosis[END_REF][START_REF] Li | Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells[END_REF], and since chemotherapy works by inducing apoptosis in the cancer cells, we have raised the hypothesis that TIMP-1 protects cells expressing high levels of TIMP-1 against chemotherapy by inhibiting apoptosis. In support of this hypothesis, our laboratory has recently shown that murine fibrosarcoma cells deficient of the TIMP-1 gene were significantly more sensitive to chemotherapy-induced apoptosis compared to their wildtype controls [START_REF] Davidsen | TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapyinduced apoptosis[END_REF]. These results have recently been extended to a human breast carcinoma cell system [START_REF] Wu ¨rtz | Tissue inhibitor of metalloproteinases-1 protects the human breast carcinoma cell line MCF-7 S1 against anthracycline-induced cell death by activation of the akt survival pathway[END_REF]. In this study, it was shown that human breast carcinoma cells expressing high levels of TIMP-1 were significantly less sensitive to chemotherapy. Interestingly, the study suggested that TIMP-1 may selectively protect cancer cells against topo IIa inhibitors. It is interesting to note that of the patients included in the present study, *90% had received anthracycline-containing chemotherapy, i.e., combinations containing epirubicin. Epirubicin is a topo IIa inhibitor, and thus our study supports the hypothesis that TIMP-1 may predict the response to this group of drugs. This hypothesis is now supported by our findings in patients receiving adjuvant chemotherapy [START_REF] Willemoe | Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study[END_REF]. In this context, it would be of interest to investigate whether TIMP-1 also associates with the effect of other groups of drugs, e.g., taxanes, or if the predictive value only applies to topo IIa inhibitors. Such studies would be of great interest since TIMP-1 as such could be used to discriminate between drugs being more effective than others in individual patients.

As a final remark, it should be mentioned, as emphasized in the introduction, that a number of previous studies have demonstrated that tumor tissue levels of TIMP-1 as well as TIMP-1 measured in blood carry prognostic information in primary breast cancer [START_REF] Ree | High levels of messenger RNAs for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in primary breast carcinomas are associated with development of distant metastases[END_REF][START_REF] Mccarthy | High levels of tissue inhibitor of metalloproteinase-1 predict poor outcome in patients with breast cancer[END_REF][START_REF] Schrohl | Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer[END_REF][START_REF] Schrohl | Tumor tissue levels of tissue inhibitor of metalloproteinase-1 as a prognostic marker in primary breast cancer[END_REF][START_REF] Wu ¨rtz | Plasma and serum levels of tissue inhibitor of metalloprrteinases-1 are associated with prognosis in node-negative breast cancer-a prospective study[END_REF]. It is important to be aware of the various limitations in prognostic studies where endpoints such as recurrencefree survival and overall survival are used. These endpoints can be influenced by previously administered adjuvant and palliative treatments. In this regard, based on our previous study [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF] and the present study on the association between TIMP-1 and response to chemotherapy, it seems that the prognostic value of TIMP-1 found in several previous studies could in part be the result of the TIMP-1 related effect on response to therapy. In order to investigate this issue further the prognostic value of TIMP-1 in patients who have not received adjuvant systemic therapy should be evaluated.

In conclusion, the data available on TIMP-1 and response to chemotherapy suggest that TIMP-1 carries predictive information in metastatic breast cancer that may enable a more tailored chemotherapy for the individual patient. Future studies should be aimed at converting the current ELISA technology with the need for frozen tumor tissue to methods that can be applied on paraffin sections [START_REF] Willemoe | Lack of TIMP-1 tumor cell immunoreactivity predicts effect of adjuvant anthracycline based chemotherapy in patients (n = 647) with primary breast cancer. A Danish Breast Cancer Cooperative Group Study[END_REF] or serum or plasma [START_REF] Lipton | Serum TIMP-1 and response to the aromatase inhibitor letrozole versus tamoxifen in metastatic breast cancer[END_REF][START_REF] Holten-Andersen | Quantitation of TIMP-1 in plasma of healthy blood donors and patients with advanced cancer[END_REF]. The latter would allow for direct measurement of TIMP-1 levels at the time of treatment for metastatic disease.

Table 1

 1 Clinicopathological characteristics of the two patient populations and correlations between each parameter and TIMP-1 level Patients with detergent extracts (n = 43) Patients with cytosolic extracts (n = 119)

		N	ng TIMP-1/mg protein,	P	N	ng TIMP-1/mg protein,	P
			median (range)			median (range)
	Age at surgery			0.67			0.83
	B39 years	2	16.4 (12.6-20.2)		10	16.7 (6.94-40.7)
	40-49 years	13	19.9 (9.13-43.0)		31	15.4 (5.87-64.4)
	50-59 years	14	27.0 (13.4-190)		46	13.4 (0.00-58.7)
	C60 years	14	20.4 (6.32-52.1)		32	17.2 (2.64-75.8)
	Menopausal status			0.35			0.82
	Pre	20	20.6 (9.13-43.0)		57	14.0 (0.00-64.4)
	Post	23	24.2 (6.32-190)		44	14.1 (2.64-75.8)
	Peri				1	20.1
	Missing values				17	19.2 (5.87-35.4)
	Nodal status			0.68			0.21
	Node negative	7	25.1 (10.1-190)		50	13.3 (0.00-58.7)
	1-3 positive nodes	15	21.7 (10.6-52.1)		26	13.7 (4.01-48.9)
	C4 positive nodes	21	19.8 (6.32-39.2)		40	17.3 (2.95-75.8)
	Missing values				3	34.0 (22.5-63.4)
	Tumor size			0.58			0.04
	0-20 mm	7	21.7 (6.32-35.2)		38	11.6 (0.00-58.7)
	21-50 mm	29	21.7 (8.94-190)		71	16.7 (2.95-75.8)
	[50 mm	7	19.9 (16.8-37.2)		6	18.2 (13.6-34.0)
	Missing values				4	28.9 (11.9-63.4)
	Steroid receptor status			0.56			0.73
	Neg (ER-and PR-)	11	19.8 (6.32-190)		36	14.7 (0.00-75.8)
	Pos (ER? or PR?)	30	23.2 (8.94-44.0)		83	14.2 (2.64-64.4)
	Missing values	2	15.4 (10.6-20.2)
	Adjuvant therapy			0.058			0.62
	Yes	37	20.2 (6.32-52.1)		88	14.8 (0.00-75.8)
	No	6	30.4 (21.7-190)		31	14.3 (7.34-64.4)
	First-line chemotherapy			0.11			0.08
	Anthracycline	36	17.3 (17.2-34.6)		110	25.4 (9.61-34.3)
	CMF	3	21.1 (6.32-52.1)		6	14.3 (0.00-75.8)
	Other	4	34.2 (25.1-190)		3	9.20 (8.00-10.8)
	Evaluable metastatic site			0.44			0.31
	Bone	14	21.1 (9.13-44.0)		16	11.8 (6.53-27.7)
	Soft tissue	6	18.9 (10.6-52.1)		15	15.0 (5.50-63.4)
	Visceral	23	22.1 (6.32-190)		83	15.4 (0.00-75.8)
	Other				5	18.3 (12.9-42.6)

Table 2

 2 Results of univariate and multivariate logistic regression analyses modeling probability of no response (SD ? PD) to chemotherapy in relation to clinicopathological parameters (n = 162, 76 responses; in the final multivariate model: n = 139, 65 responses)

	Covariate	Univariate analysis		Multivariate analysis
		OR	95% CI	P	OR	95% CI	P
	Age (years)
	B39	1.00			1.00
	40-49	0.94	0.26-3.64		1.66	0.13-3.49
	50-59	1.88	0.54-6.58		1.65	0.27-10.1
	C60	0.79	0.22-2.83	0.14	0.87	0.11-6.99	0.34
	Menopausal status a	0.89	0.65-2.75	0.94	0.63	0.17-2.34	0.78
	Lymph node status b	1.14	0.78-1.65	0.50	0.93	0.59-1.47	0.74
	Steroid receptor status c	1.40	0.71-2.78	0.34	3.30	1.07-10.9	0.04
	Dominant metastatic site d	2.73	1.13-6.60	0.008	2.03	0.73-5.63	0.17
	Disease-free interval e	0.35	0.15-0.87	0.023	0.26	0.08-0.91	0.03
	TIMP-1 f	1.59	0.97-2.62	0.07	1.43	0.79-2.61	0.24

a n = 144 patients, postmenopausal vs. premenopausal b n = 160 patients, N1 vs. N0 c n = 160 patients, ER or PR positive vs. ER and PR negative d Visceral or soft tissue vs. bone e Disease-free interval: [12 vs. B12 months f Log TIMP-1 (ng/mg of total protein)

Breast Cancer Res Treat (2010) 121:365-371

Acknowledgments The present study was supported financially by The Swedish Cancer Society, The Swedish Research Council, The Gunnar, Arvid, and Elisabeth Nilsson Foundation, The Mrs Berta Kamprad Foundation, The University Hospital of Lund Research Foundation, Governmental Funding of Clinical Research within the National Health Service, The Danish Cancer Society, Danish Center for Translational Breast Cancer Research, A Race Against Breast Cancer, The Ministry for Health and Prevention, and The Danish Strategic Research Council for Food and Health and the Sino-Danish Breast Cancer Research Centre.

Odds Ratio 0 1 2 3

Present study population

Schrohl study population

Present study and Schrohl study population pooled

Fig. 1 Forest plot illustrating the logistic regression analysis with TIMP-1 scored as a continuous variable on the log scale. OR with 95% CI as a measure of effect is presented representing the ratio of odds between two patients differing by 1 natural log (*2.7 times) in TIMP-1 levels. ORs are presented for the present study population (top), the Schrohl study population [START_REF] Schrohl | Primary tumor levels of tissue inhibitor of metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer[END_REF] (middle), and the Schrohl study population and present study population pooled together (bottom). Pooled analyses were performed using a random effects model