Laura Ottini
email: laura.ottini@uniroma1.it

Piera Rizzolo

Ines Zanna

Mario Falchetti

Giovanna Masala

Ketty Ceccarelli

Vania Vezzosi

Alberto Gulino

Giuseppe Giannini

Simonetta Bianchi

A E Piera

Rizzolo Ae

A E Mario

Falchetti Ae

A E Ketty

Ceccarelli Ae

Vania Vezzosi Ae

A E Giuseppe

Giannini Ae

A E Francesco

Sera Ae

Domenico Palli

Á M Falchetti

Á K Ceccarelli

Á A Gulino

Á G Giannini

Á G Masala

Á F Sera

Á D Palli

Á S Bianchi

/ mutation status and clinical-pathologic features of 108 male breast cancer cases from Tuscany: a population-based study in central Italy

Keywords: Male breast cancer, BRCA1, BRCA2, Germ-line mutations, Clinical-pathologic features

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

Introduction

Male breast cancer (MBC) is a rare disease, accounting for \1% of all cancers in men. However, recent epidemiological studies indicate that its incidence is rising 1.1% annually [START_REF]) database[END_REF][START_REF] Speirs | The rising incidence of male breast cancer[END_REF]. In Europe, the incidence of MBC is 1 in 100,000 men per year, and \1% of all breast cancer (BC) patients are male [START_REF] Fentiman | Male breast cancer[END_REF]. In Italy, MBC accounts for 0.2% of all cancers in males and incidence rates, standardized on the European population, are about 1 new case 9 100,000 men per year [START_REF]Il cancro in Italia 3. I dati dei Registri Tumori[END_REF].

At the epidemiological level, male and female BC share many similarities with the main differences represented by a lower incidence and older age of occurrence of MBC [START_REF] Agrawal | Male breast cancer: a review of clinical management[END_REF]. Hormonal, environmental and genetic factors are involved in the pathogenesis of BC in women as well as in men. Major risk factors related to MBC predisposition include pathological conditions associated with primary and secondary hyper-estrogenism, radiation exposure, and, particularly, a family history (FH) positive for breast and/or ovarian cancer. About 20% of MBC patients have a first-degree relative affected by BC [START_REF] Fentiman | Male breast cancer[END_REF], which points to a relevant genetic component in MBC predisposition. Germ-line mutations of BRCA2 (OMIM # 600185) and, with lower frequency, of BRCA1 (OMIM # 113705) are implicated in MBC predisposition. However, the frequency of BRCA1 and BRCA2 mutations are quite different in ethnically diverse population-and clinic-based MBC series, ranging between 4% and 40% for BRCA2 and up to 4% for BRCA1, being higher in the presence of founder effects [START_REF] Giordano | A review of the diagnosis and management of male breast cancer[END_REF]. BRCA1 and BRCA2 founder mutations have been identified in specific countries or ethnic groups, particularly in genetically isolated populations such as the Icelanders and Ashkenazi Jews. In Italy, there is evidence of founder BRCA1 and BRCA2 mutations in regions that show a micro-homogeneity [START_REF] Ferla | Founder mutations in BRCA1 and BRCA2 genes[END_REF][START_REF] Baudi | Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer[END_REF][START_REF] Pisano | Identification of a founder BRCA2 mutation in Sardinia[END_REF][START_REF] Russo | Is BRCA1-5083del19, identified in breast cancer patients of Sicilian origin, a Calabrian founder mutation? Breast Cancer Res Treat[END_REF][START_REF] Malacrida | BRCA1 p.Val1688del is a deleterious mutation that recurs in breast and ovarian cancer families from Northeast Italy[END_REF]. As for the association between MBC and BRCA2 germ-line mutations, the presence of a male affected by BC seems to be the strongest predictor for the occurrence of BRCA2 genomic rearrangements in high-risk families. Indeed, BRCA2 rearrangements are observed in about 10% of MBC families negative for BRCA1/2 point mutations, whereas BRCA1 rearrangements are rarely found [START_REF] Tournier | Significant contribution of germline BRCA2 rearrangements in male breast cancer families[END_REF][START_REF] Woodward | kConFab investigators. Large genomic rearrangements of both BRCA2 and BRCA1 are a feature of the inherited breast/ovarian cancer phenotype in selected families[END_REF][START_REF] Gutierrez-Enriquez | Screening for large rearrangements of the BRCA2 gene in Spanish families with breast/ovarian cancer[END_REF][START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF][START_REF] Walsh | Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer[END_REF]. On the other hand, we recently reported that both BRCA2 and BRCA1 rearrangements are infrequent in MBC cases, negative for BRCA1/2 mutations, unselected by FH [START_REF] Falchetti | BRCA1/2 rearrangements and CHEK2 common mutations are infrequent in Italian male breast cancer cases[END_REF].

Male breast cancer and female breast cancer (FBC) appear to be biologically similar but they differ mainly in the frequency of histological types and in the expression of hormone receptors (estrogen/progesterone, ER/PR) and of epidermal growth factor receptor 2 (HER2). In MBC, the predominant histological type is invasive ductal carcinoma, which characterizes more than 90% of all MBC cases [START_REF] Nahleh | Male breast cancer in the veterans affairs population: a comparative analysis[END_REF]. MBC shows higher rates of ER/PR positivity (ER/PR?) and lesser rate of HER2 over-expression (HER2?), compared with FBC [START_REF] Fentiman | Male breast cancer[END_REF][START_REF] Agrawal | Male breast cancer: a review of clinical management[END_REF][START_REF] Giordano | A review of the diagnosis and management of male breast cancer[END_REF]. Accumulating evidence supports that BRCA1 and BRCA2-related FBC tend to manifest specific phenotypic profiles [START_REF] Honrado | Pathology and gene expression of hereditary breast tumors associated with BRCA1, BRCA2 and CHEK2 gene mutations[END_REF]. In particular, BRCA1tumors tend to be ER-, PR-and HER2-negative (triple negative) and BRCA2-tumors tend to be more often ER/ PR? and HER2- [START_REF] Bane | BRCA2 mutation-associated breast cancers exhibit a distinguishing phenotype based on morphology and molecular profiles from tissue microarrays[END_REF]. At present, very few data are available for BRCA1/2-related MBC [START_REF] Kwiatkowska | BRCA2 mutations and androgen receptor expression as independent predictors of outcome of male breast cancer patients[END_REF]. Indeed, due to its rarity, MBC is less investigated compared to FBC, but it is now receiving more attention, probably because of its increasing incidence and of the better knowledge on the role of BRCA genes mutations in MBC predisposition. Retrospective studies on large series of MBC have been recently performed to investigate the major clinical-pathologic characteristics of BC in men [START_REF] Nahleh | Male breast cancer in the veterans affairs population: a comparative analysis[END_REF][START_REF] Burga | Invasive carcinomas of the male breast: a morphologic study of the distribution of histologic subtypes and metastatic patterns in 778 cases[END_REF]; however, these studies did not report data on BRCA1/2 mutation status. On the other hand, studies concerning BRCA1/2 mutation screening are often limited in the number of cases analyzed. To our knowledge, very few studies have been performed in MBC series of more than 100 cases and the screening was limited to founder mutations [START_REF] Evans | BRCA1/2 mutation analysis in male breast cancer families from North West England[END_REF][START_REF] Syrja ¨koski | BRCA2 mutations in 154 finnish male breast cancer patients[END_REF].

The present study was carried out with the aim to characterize the genetic and phenotypic features of MBC from a large and epidemiologically well-characterized population-based series from Tuscany (Central Italy). In particular, we wished (i) to assess the prevalence and spectrum of BRCA1 and BRCA2 mutations and (ii) to investigate the phenotypic features of tumors in order to further characterize BRCA1/2-related MBCs.

Patients and methods

MBC patients

In the present study, we expanded the original populationbased series of 25 MBC cases diagnosed in the area of Florence (Tuscany, Central Italy) in the period 1990-1998, that we have previously described [START_REF] Ottini | BRCA1 and BRCA2 mutation status and tumor characteristics in male breast cancer: a population-based study in Italy[END_REF], based on all currently available local sources (including Pathology Departments and the Hospital Discharge database). We enrolled 83 additional MBC cases, diagnosed in the period 1991-2007, for a total of 108 MBCs residing in Eastern Tuscany. The recruitment of the new MBC cases was carried out according to the same protocol as used for the previous MBC series. Overall, after exclusion of deceased and migrated patients, 94 additional unrelated MBC were traced and invited to participate into the study. Eleven cases refused to participate, mostly because of advanced age or severe illness, thus confirming our previous high response rate (83/94, 88.3%). For each study participant we obtained: (i) a signed informed consent form with a detailed description of the study protocol, including the information about the mutational analysis of the BRCA1/BRCA2 genes; (ii) detailed information on his FH for cancer at any sites, including all first-and seconddegree relatives of both genders (all of this information was validated by available sources, mainly local Cancer and Mortality Registries); (iii) detailed information on his personal history of cancer at any site; (iv) information on life style habits and a detailed occupational history up to the date of BC diagnosis; (v) one or more blood sample(s). All information were collected by a genetician in the frame of the High-Risk Cancer Family Project. The a priori probability of carrying a mutation in the BRCA1/2 genes was estimated by using the BRCAPRO software version 4.2 [START_REF] Parmigiani | Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2[END_REF]. Pathological reports and histological slides for all of the MBC cases were identified and retrieved from the archival files of the Pathology Departments of the area for diagnostic confirmation. Procedures to maintain confidentiality for all the information collected were developed and strictly applied. The study was approved by the Florence Ethical Research Committee.

Mutational analysis

The entire BRCA1 and BRCA2 coding sequences were screened for germ-line mutations. After isolation of genomic DNA from buffy coats, PCR-amplified BRCA1 exons 2-10 and 12-24 and BRCA2 exons 2-9 and 12-27 were analyzed by combining single strand conformational polymorphism (SSCP) and automatic sequencing with the ABI Prism-310 Genetic Analyzer (Applied Biosystems, Warrington, UK). The larger exons (BRCA1 exon 11, BRCA2 exons 10 and 11) were rapidly screened for truncating mutations by the protein truncation test (PTT) or by automatic sequencing analysis in cases negative for truncating mutations within these exons. Mutations were verified on two independent blood samples. The DNA mutation nomenclature is as used in the Breast Information Core (BIC) database (http://research.nghgri.nih.gov/bic) according to GenBank, U14680.1 and U43746 for BRCA1 and BRCA2, respectively. Novel BRCA1/2 variant sequences were analysed in silico for predicted splicing defects by using SpliceSiteFinder (http://www.genet.sickkids.on.ca/* ali/spilcesitefinder.html) and ESEfinder (http://rulai.cshl. edu/tools/ESE3/). Intronic variants predicted to alter RNA splicing were further analysed by RT-PCR, carried out using Superscript II (Invitrogen, Carlsbad, CA, USA) reverse transcriptase with gene-specific primers for the cDNA. Total RNA was extracted from peripheral blood leukocytes by using Trizol Reagent and following manufacturer instructions (Invitrogen, Carlsbad, CA, USA).

Haplotype analysis

A total of 10 individuals belonging to the two families segregating the recurrent BRCA1 3347delAG mutation and 14 population controls were genotyped at six microsatellite markers, including three intragenic loci (D17S1323, D17S1322 and D17S855, located in BRCA1 intron 12, 19 and 20, respectively), one centromeric (D17S250) and two telomeric (D17S858 and D17S1183) markers. The haplotype analysis encompassed a distance of about 1,332 Kb between the D17S855 and D17S1183 markers. Alleles were numbered according to the size of each microsatellite repeat marker. The disease-associated haplotypes were deduced by inspection of segregating genotypes in the analyzed individuals.

Immunohistochemical analysis

Step sections from formalin-fixed, paraffin-embedded primary mammary tumor blocks were obtained for the majority of the cases in order to perform ad hoc immunohistochemical analyses. Overall, out of 110 tumors in 108 patients, we obtained tissue blocks for 96 tumors in 94 patients (including two blocks each for two bilateral MBCs and only one for the other bilateral case). Immunohistochemical studies were performed on 96 available formalinfixed paraffin-embedded tissue blocks of 94 patients (out of the original 108 cases; including 5 blocks of 3 patients with bilateral BC). Immuno-histochemical data could not be obtained for some of these MBCs because available tissue specimens were not sufficient for the whole study protocol. Markers were thus available for a variable number of tumors (range 90-96). The following primary antibodies were applied: anti-HER2, clone TAB 250 (Zymed, S. Francisco, CA), diluted 1:20; anti-ER, clone 6F11 (Ventana Medical Systems, Tucson, AZ) prediluted; anti-PR, clone 1A6 (Ventana Medical Systems, Tucson, AZ) prediluted and anti-Ki-67, clone MIB1 (Dako Cytomation, Glostrup, DK) diluted 1:60. Immunoistochemical protocols and scoring procedures have been described in detail elsewhere [START_REF] Ottini | BRCA1 and BRCA2 mutation status and tumor characteristics in male breast cancer: a population-based study in Italy[END_REF][START_REF] Bianchi | ErbB-receptors expression and survival in breast carcinoma: a 15-year follow-up study[END_REF].

Statistical analysis

The association between mutation carrier status and firstdegree FH of breast/ovarian cancer was measured by calculating the prevalence rate ratio and its 95% CI. The association between mutation carrier status and PR, ER, HER2 or MIB1 immuno-histochemical expression was tested by using the Fisher exact test. The association between mutation carrier status and histopathological grade was tested using the Wald test of the coefficient associated with histopathological grade and considered as continuous variables in a logistic regression analysis with mutation carrier status as outcome. A P value \0.05 was considered statistically significant. All the analyses were carried out by using the SAS statistical software (SAS/STAT version 9.1).

Results

Prevalence and spectrum of BRCA1 and BRCA2 mutation in a population-based series of Italian MBC cases

To evaluate the contribution of BRCA1 and BRCA2 mutations in MBC we enrolled a large and well-characterized population-based series of 108 MBC cases, diagnosed in the period 1991-2007 in the area of Florence (Tuscany, Central Italy), thus expanding our previous study based on [START_REF] Ottini | BRCA1 and BRCA2 mutation status and tumor characteristics in male breast cancer: a population-based study in Italy[END_REF] MBCs [START_REF] Ottini | BRCA1 and BRCA2 mutation status and tumor characteristics in male breast cancer: a population-based study in Italy[END_REF]. In the present population-based series, age at first BC diagnosis ranged between 24 and 90 years (mean: 63.2 years; median: 65.0 years). Overall, 25.9% of the patients reported a first-degree FH of breast/ ovarian cancer (Table 1), including one patient with a firstdegree FH of MBC. Furthermore, 37 patients (34.3%) had a first-degree FH of cancer at sites other than breast/ovary, including nine patients (8.3%) that reported both a firstdegree FH of breast/ovarian cancer and of cancer at other sites. A personal history of cancer(s) at sites other than breast was reported in 14.8% of MBC cases (Table 1). In these patients prostate and bladder carcinomas were the most frequently observed metachronous cancers. Notably, three out of 108 MBC patients had been diagnosed with bilateral BC. With regard to the pathological characteristics of the MBC cases (Table 1), the majority of the tumors was invasive ductal carcinomas (78.2%), expressed ER (ER?, 87.5%) and PR (PR?, 79.2%), showed low proliferative activity (MIB1-, 65.6%) and did not over-express HER2 (HER2-, 81.3%).

Overall, deleterious BRCA1/2 germ-line mutations were detected in 10 of the 108 MBC cases analyzed, with a mutation prevalence of 9.3% (95% CI, 4-15%). In particular, two cases (1.9%, 95% CI, 0-4%) carried BRCA1 and eight cases (7.4%, 95% CI, 2-12%) carried BRCA2 mutations (Table 2). The same BRCA1 mutation (3347delAG) was identified in two unrelated MBC cases (Fig. 1). Haplotype analysis allowed the identification of a common haplotype shared by all mutation carriers (seven individuals) but not by non carrier relatives (three individuals). The same analysis conducted in 14 healthy controls revealed strong allele variability and different allele pattern compared to the carriers belonging to the BRCA1 3347delAG families (data not shown). As shown in Table 2, of the eight BRCA2 mutations here identified three are novel mutations not reported in the BIC database (http://research. nghgri.nih.gov/bic). Two of them, the BRCA2 802insA and the BRCA2 7935delT, are frameshift mutations that create a stop at codons 205 and 2647, respectively (Table 2, Fig. 2). The third novel mutation, the BRCA2 IVS7-2 A[G, is an intronic variant. By in silico analysis, the BRCA2 IVS7-2 A[G is predicted to affect the splicing of exon 8 by disrupting the intron 7 acceptor site (Splice-SiteFinder wild type efficiency score = 93.4) and the exonic splice enhancer SRp40 motif (ESEfinder wild type efficiency score = 3.63). We evaluated the consequence of the BRCA2 IVS7-2 A[G variant on splicing in vivo by examining RNA isolated from peripheral lymphocytes of the MBC patient (MB 115) carrying the variant. RT-PCR, performed to amplify a region encompassing BRCA2 exons 6-9 produced two cDNA fragments: one of the predicted size (314 bp) for the wild type product and the other approximately 50 bp smaller than the wild type cDNA product (Fig. 3). Direct sequence analysis of the abnormal PCR product showed a transcript lacking exon 8 (BRCA2 860-909del) and containing a stop signal at codon 236.

By using the BRCAPRO software version 4.2 [START_REF] Parmigiani | Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2[END_REF], the a priori probability of carrying a mutation in BRCA1/2 was estimated for all cases (mean value: 23.8%; range: 0-100%).

All BRCA1/2 deleterious mutations were detected in the 65 cases with an a priori probability above 10% (expected versus observed: 36.4 and 15.4%, respectively) and none in a Frequencies were calculated excluding tumors with information not available for specific variables because of lack of tissue samples; two patients with bilateral BC (out of the three identified) were considered separately for each tumor the 43 cases with an a priori probability below 10% (expected versus observed: 4.9 and 0%, respectively). In particular, we identified BRCA1/2 mutations in 3 of the 6 MBC patients with an a priori probability above 80% (expected versus observed: 95.3 and 50.0%, respectively). Indeed, one MBC case with a BRCAPRO 4.2 of 100% was shown to carry a mutation in p53, the main gene associated with Li-Fraumeni syndrome (data not shown). With regard to pathologic characteristics, of the two BRCA1-tumors, one was an invasive ductal carcinoma, ER?, PR?, HER2-and MIB1-, and the other was a lobular carcinoma, ER-, PR-, HER2-and MIB1? (Table 2). Overall, of the two BRCA1-tumors one resulted with a triple negative (ER-, PR-, HER2-) phenotype.

The majority of the BRCA2-tumors were invasive ductal carcinomas (75%, 6/8) and were of high grade (75%, 6/8), 62.5% (5/8) were ER?, 62.5% (5/8) were PR-, 50% (3/6) were MIB? and 71.4% (5/7) were HER2? (Table 2). We further investigated whether BRCA2-related MBCs showed specific phenotypic profiles by analyzing associations between BRCA2 mutation status and pathological characteristics (Table 3). Statistically significant associations emerged between BRCA2 mutations and absence of PR expression (P = 0.008), positive HER2 immunostaining (P = 0.002) and high tumor grade (P = 0.005). No the phenotypic features of BRCA1/2-related and unrelated MBCs.

Overall, the BRCA1/2 mutations prevalence (9.3%) observed in our population-based series is comparable with those reported in MBCs unselected by FH from other different populations [START_REF] Evans | BRCA1/2 mutation analysis in male breast cancer families from North West England[END_REF][START_REF] Hache | Clinical and pathological correlations in male breast cancer: intratumoral aromatase expression via tissue microarray[END_REF][START_REF] Basham | BRCA1 and BRCA2 mutations in a population-based study of male breast cancer[END_REF][START_REF] Chodick | Similar prevalence of founder BRCA1 and BRCA2 mutations among Ashkenazi and non-Ashkenazi men with breast cancer: evidence from 261 cases in Israel, 1976-1999[END_REF]. Although BRCA2 mutations are currently considered as the major genetic risk factor for MBC, the association between MBC and BRCA1 mutations is not as well established [START_REF] Fentiman | Male breast cancer[END_REF]. BRCA1 mutations are quite rare in unselected MBC cases being more frequent in specific populations in which a founder effect is known to occur [START_REF] Giordano | A review of the diagnosis and management of male breast cancer[END_REF]. Notably, the same BRCA1 mutation (3347de-lAG), represented the overall BRCA1 mutation frequency (1.9%) in the present study. We recently reported that the BRCA1 3347delAG is a founder mutation in Tuscany [START_REF] Papi | Founder mutations account for the majority of BRCA1-attributable hereditary breast/ovarian cancer cases in a population from Tuscany, Central Italy[END_REF], thus confirming that unselected series of cases from specific geographic areas may lead to the identification of BRCA1/2 mutation with founder effect in genetically heterogeneous countries, such as Italy.

Of the 10 mutations identified in our MBC series, three, including two BRCA2 frameshift mutations (802insA and 7935delT) and a BRCA2 intronic variant (IVS7-2 A[G), were novel alterations not reported in the BIC database (http://research.nghgri.nih.gov/bic). We demonstrated, by characterizing the transcript produced from RNA of the MBC patient carrying the variant, that the BRCA2 IVS7-2 A[G is a novel splice site deleterious mutation causing loss of BRCA2 exon 8 and creating a premature stop codon. In this respect, it is noteworthy that about 4% of the genetic variants in the BIC database are reported as splice site alterations. Overall, in the present study, we enlarged the mutational spectrum of BRCA2 gene and provided further data to contribute to the discussion on the causal role and clinical relevance of intronic variants.

As we recently observed in FBC [START_REF] Capalbo | Does the search for large genomic rearrangements impact BRCAPRO carrier prediction?[END_REF], when considering the frequency of mutations in MBC cases stratified according to BRCAPRO 4.2, the prediction accuracy improves as the threshold probability increases. Indeed, we found that 66.6% of MBC patients with probability prediction C80% have mutations in the BRCA1/2 genes and in other genes, such as p53. However, a degree of over-estimation is evident. Despite the comprehensive scan of the coding sequence and the intron-exon boundaries there remains a substantial chance of missed BRCA1/2 genetic alterations possibly due to mutations in other regions of the genes or due to genomic rearrangements. In this respect, it is noteworthy that we recently shown that no BRCA1/2 rearrangements were found in a large series of Italian MBCs, including 72 mutation-negative MBC cases from the population-based series here analyzed [START_REF] Falchetti | BRCA1/2 rearrangements and CHEK2 common mutations are infrequent in Italian male breast cancer cases[END_REF]. Thus, the data reported in the present study reflect the overall population prevalence of BRCA1/2 mutations in Italian MBC cases. Given the reasonably high sensitivity of our screening and the sample size, our results indicate that, at population level, a large fraction of MBC cases are not accounted for by BRCA1/2 alterations. Collaborative studies are essential to better investigate the pathogenesis of MBC, and, in this context, MBC cases in our series may be useful in identifying new genes predisposing to moderate/high MBC risk. From a clinical point of view, our data suggest that probands with a high (C80%) carrier probability prediction have to be considered as carriers of a strongly predisposing genetic alteration either in BRCA1/2 genes or in other yet unknown BC predisposing genes, independently of our ability to detect such mutations.

Consistent with published data on MBC from diverse populations [START_REF] Fentiman | Male breast cancer[END_REF][START_REF] Evans | BRCA1/2 mutation analysis in male breast cancer families from North West England[END_REF][START_REF] Hache | Clinical and pathological correlations in male breast cancer: intratumoral aromatase expression via tissue microarray[END_REF], in our population-based series, the mean age of BC presentation in males was 63 years and about one-fourth of MBC cases reported a first-degree BC-FH, thus indicating that our series is representative of a standard MBC population and suggesting that data obtained analyzing the present series can be relevant for MBC in general. We observed that the median age at BC diagnosis among BRCA1/2 mutation carriers tended to be younger (median: 58.5 years) than that of negative cases (median: 66.3 years). BRCA1/2 mutations were more prevalent in men with a positive first-degree FH (17.9%) compared with those without (6.3%) and a threefold association emerged between a positive FH and carrier status, falling short of the level of statistical significance. It has been previously reported that BRCA1/2 mutations are more frequent in MBCs with a positive FH [START_REF] Evans | BRCA1/2 mutation analysis in male breast cancer families from North West England[END_REF][START_REF] Basham | BRCA1 and BRCA2 mutations in a population-based study of male breast cancer[END_REF][START_REF] Miolo | Phenotypic features and genetic characterization of male breast cancer families: identification of two recurrent BRCA2 mutations in North-east of Italy[END_REF]. However, in our series, 50% of the mutations were identified in MBC cases without FH, thus from a clinical point of view, our data indicate that mutation screening is beneficial also among isolated MBC cases.

With regard to pathological characteristics of the tumors, in our series the majority of breast tumors were invasive ductal carcinomas and express ER/PR, whereas only a minority over-expresses HER2. Overall, our results are consistent with retrospective studies on large MBC series performed to characterize the major phenotypic features of BC in males [START_REF] Nahleh | Male breast cancer in the veterans affairs population: a comparative analysis[END_REF][START_REF] Kwiatkowska | BRCA2 mutations and androgen receptor expression as independent predictors of outcome of male breast cancer patients[END_REF]. To further investigate the pathological features of BC in men, we also examined the proliferative activity of MBCs by immunohistochemical evaluation of MIB1. Proliferative activity is considered an important prognostic factor in FBC, but it has been scarcely investigated in MBC and data are often inconsistent, most likely because of the small number of cases examined [START_REF] Pich | Proliferative activity is a significant prognostic factor in male breast carcinoma[END_REF][START_REF] Mun ˜oz De Toro | Proliferative activity and steroid hormone receptor status in male breast carcinoma[END_REF]. Our results showed that about 34% of MBCs exhibit a high proliferative activity (MIB1?). At present, little is known of the immunophenotypic characteristics of MBCs stratified according to BRCA1/2 mutation status [START_REF] Kwiatkowska | BRCA2 mutations and androgen receptor expression as independent predictors of outcome of male breast cancer patients[END_REF]. Notably, one of the two BRCA1-related MBC, identified in our series, showed a triple negative phenotype (ER-, PR-, HER2-) consistent with what observed in BRCA1-related breast tumors in females [START_REF] Honrado | Pathology and gene expression of hereditary breast tumors associated with BRCA1, BRCA2 and CHEK2 gene mutations[END_REF]. Furthermore, we observed that BRCA2-related MBCs showed a significant association with HER2 over-expression and with a high tumor grade, thus confirming on a large number of cases what we have previously reported [START_REF] Ottini | BRCA1 and BRCA2 mutation status and tumor characteristics in male breast cancer: a population-based study in Italy[END_REF]. Intriguingly, we found that BRCA2related MBC tended to be negative for PR expression. Taking into account that MBCs are predominantly PR? and HER2-, our findings suggest that specific phenotypic characteristics are associated with BRCA2-linked MBCs. These findings may be relevant on a clinical ground considering that a tumor phenotype characterized by PR-, HER2? and high grade may be indicative of aggressive behavior. Furthermore, our results may be also relevant for the treatment of MBC, by providing data on biomarkers useful to determine the subset of MBC patients (PR-, HER2?) that will benefit from trastuzumab therapy. Overall, our observations may have important prognostic and therapeutic implications and warrant to be replicated in larger collaborative investigations of this rare disease.

Fig. 1

 1 Fig. 1 Pedigrees of the two families carrying the BRCA1 3347delAG germ-line mutation. Probands MB5 (a) and MB56 (b), are indicated by arrows; *: Mutation carrier; -: non carrier

Fig. 2 Fig. 3

 23 Fig. 2 Pedigrees of families carrying two novel BRCA2 germ-line mutations: BRCA2 802insA (a) and BRCA2 7935delT (b). Probands, MB83 (a) and MB89 (b), are indicated by arrows. *: Mutation carrier; -: non carrier

Table 1

 1 Clinico-pathological characteristics of 110 tumors from the population-based series of 108 Italian MBC cases(Florence 1990(Florence -2007) )

	Clinico-pathological characteristics	N (%) a
	Family history of breast/ovarian cancer
	Negative	80 (74.1)
	Positive	28 (25.9)
	Total	108
	Personal history of cancer
	Negative	92 (85.2)
	Positive	16 (14.8)
	Total	108
	Histology
	Invasive ductal carcinoma	86 (78.2)
	In situ ductal carcinoma	8 (7.3)
	Papillary carcinoma	6 (5.5)
	Mucinous (colloid)	1 (0.9)
	Lobular carcinoma	3 (2.7)
	Paget's	2 (1.8)
	Adenoid cystic carcinomas	2 (1.8)
	Cribriform carcinoma	2 (1.8)
	Total	110
	Grade
	G1	18 (19.0)
	G2	50 (52.6)
	G3	27 (28.4)
	Total	95
	Stage
	0	10 (12.0)
	I	31 (37.4)
	II	30 (36.1)
	III-IV	12 (14.5)
	Total	83
	ER
	Negative	12 (12.5)
	Positive	84 (87.5)
	Total	96
	PR
	Negative	20 (20.8)
	Positive	76 (79.2)
	Total	96
	MIB1
	Negative	59 (65.6)
	Positive	31 (34.4)
	Total	90
	HER2
	Negative	74 (81.3)
	Positive	17 (18.7)
	Total	91

Table 2

 2 Personal and family history of cancer, pathologic characteristics and immunohistochemical staining for ER, PR, HER2 and MIB1 in the BRCA1/2 MBC mutation carriers(Florence, Italy 1990-2007)

	Gene	Mutations a	Mutation effect Case code Personal history	Breast/ovarian cancer in	Histology/	Immunostaining
					of breast cancer (age at diagnosis,	first-degree relatives (age at diagnosis, year)	Grade	ER PR HER2 MIB1
					year)
	BRCA1 3347delAG R1076fs1084X	MB 5 b	60	2 Breast (45, 41)	IDC/3	?	? -	-
		3347delAG R1076fs1084X	MB 56	55		ILC	-	--	?
	BRCA2 802insA	M192fs205X	MB 83	70		IDC/2	?	? -	na
		IVS7-2A>G Exon 8 skipping MB 115	41		IDC/3	?	-?	-
		1003delA	R259fs276X	MB 16 b	64		Papillary/3 -	-?	?
		2041insA	I605fs615X	MB 103	44	Breast (54)	IDC/3	-	-?	-
		5301insA	W1691fs1694X MB 90	57		IDC/2	?	--	?
		6010G[T	E1928X	MB 31 b	53	3 Breast (48, 45, na)	Paget's/3	-	-?	?
		6696delTC	S2156fs2174X	MB 3 b	63	Ovary (62)	IDC/3	?	? ?	-
		7935delT	G2569fs2647X	MB 89 c	74-77	Breast (41), ovary (58)	IDC/3	?	? na	na

IDC invasive ductal carcinoma, ILC invasive lobular carcinoma; na not available a Novel mutations are reported in bold; b cases reported in our previous study

[START_REF] Ottini | BRCA1 and BRCA2 mutation status and tumor characteristics in male breast cancer: a population-based study in Italy[END_REF]

; c bilateral BC

Table 3

 3

	Association between
	selected pathological
	characteristics and BRCA2
	mutation status in MBCs
	(Florence, Italy 1990-2007)

Breast Cancer Res Treat (2009) 116:577-586

Acknowledgments Authors are grateful to all patients, their families and their caring physicians for participating in this study. Authors wish also to thank Mr. Massimo Zani and Mr. Sergio Ferraro for expert technical assistance, and all the colleagues of the Pathology Departments of several hospitals in the study area (Drs. Carmelo Urso, Augusto Giannini, Lavinia Puglisi, Tiziana Megha, Michela Stumpo, Federica Zolfanelli, Francesco Mirri, Lidia La Magra, and Mauro Biancalani) for their active collaboration in providing tissue samples. The study was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC) to Laura Ottini and by Regione Toscana in the frame of the High-Risk Cancer Family Project.


Niall Mcinerney

A E Gabrielle

Colleran Ae

Andrew Rowan

A E Axel

Walther Ae

Ella Barclay

A E Sarah

Spain Ae

Angela M Jones

A E Stephen

Tuohy Ae

Catherine Curran

Nicola Miller

A E Michael

Kerin Ae

Ian Tomlinson

Ae Elinor Sawyer

Á G Colleran

Á S Tuohy

Á C Curran

Á N Miller

Á M Kerin

Á A Rowan

Á A Walther

Á E Barclay

Á S Spain

Á A M Jones

Á I Tomlinson

Á E Sawyer

E Sawyer

Low penetrance breast cancer predisposition SNPs are site specific

Keywords: Breast cancer, Colorectal cancer, Prostate cancer, Genetic susceptibility, 8q24, West Ireland

Large scale association studies have identified low penetrance susceptibility alleles that predispose to breast cancer. A locus on chromosome 8q24.21 has been shown to harbour variants that predispose to breast, ovarian, colorectal and prostate cancer. The finding of risk variants clustering at 8q24 suggests that there may be common susceptibility alleles that predispose to more than one epithelial cancer. The aim of this study was firstly to determine whether previously identified breast cancer susceptibility alleles are associated with sporadic breast cancer in the West of Ireland and secondly to ascertain whether there are susceptibility alleles that predispose to all three common epithelial cancers (breast, prostate, colon). We genotyped a panel of 24 SNPs that have recently been shown to predispose to prostate, colorectal or breast cancer in 988 sporadic breast cancer cases and 1,016 controls from the West of Ireland. We then combined our data with publicly available datasets using standard techniques of meta-analysis. The known breast cancer SNPs rs13281615, rs2981582 and rs3803662 were confirmed as associated with breast cancer risk (P allelic test = 1.8 9 10 -2 , OR = 1.17; P allelic test = 2.2 9 10 -3 , OR = 1.22; P allelic test = 5.1 9 10 -2 , OR = 1.15, respectively) in the West of Ireland cohort. For the remaining five breast cancer SNPs that were studied there was no evidence of an association with breast cancer in the West Ireland population (P allelic test [ 6.5 9 10 -2 ). There was also no association between any of the prostate or colorectal susceptibility SNPs, whether at 8q24 or elsewhere, with breast cancer risk. Meta-analysis confirmed that all susceptibility SNPs were site specific, with the exception of rs6983269 which is known to predispose to both colorectal and prostate cancer. This study confirms that susceptibility loci at FGFR2, 8q24 and TNCR9 predispose to sporadic breast cancer in the West of Ireland. It also suggests that low penetrance susceptibility SNPs for breast, prostate and colorectal cancer are distinct. Although 8q24 harbours variants that predispose to all three cancers, the susceptibility loci within the region appear to be specific for the different cancer types with the exception of rs6983269 in colon and prostate cancer.

Introduction

The risk of breast cancer is raised about twofold in the sisters and mothers of affected women. Only a small proportion of this risk is accounted for by known breast cancer predisposition genes such as BRCA1 and BRCA2. The search for further high penetrance genes has failed to identify loci that predispose to breast cancer, suggesting that less penetrant genes may be important. Despite the identification of rare alleles that confer a modestly increased risk of breast cancer-such as CHEK2, ATM, BRIP and PALB2-about 75% of the familial risk remains unexplained [START_REF] Antoniou | Models of genetic susceptibility to breast cancer[END_REF]. A polygenic model for susceptibility to breast cancer and other common cancers, including colorectal and prostate cancer, has been suggested, where risk is conferred by a large number of alleles, each conferring a small risk, but which, when combined, confer a range of susceptibilities within the population [START_REF] Pharoah | Polygenic susceptibility to breast cancer and implications for prevention[END_REF].

In order to identify common, low penetrance cancer susceptibility alleles, large genome-wide association studies (GWAS) have been performed. Easton et al. identified five loci associated with increased risk of developing breast cancer. These were initially identified in breast cancer patients with a family history and verified in a much larger cohort of patients with both sporadic and familial breast cancer [START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF]. The SNPs with the strongest association with breast cancer in this study were rs2981582 (10q: FGFR2), rs3803662 (16q:TNRC9) and rs13281615 (chr8q24) with a per allele OR typically of 1.1-1. [START_REF] Tomlinson | A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3[END_REF]. Several other studies have also identified an association with SNPs within FGFR2 and TNRC9 and breast cancer [START_REF] Hunter | A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer[END_REF][START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF]. More recent studies have also identified SNPs in other regions that predispose to breast cancer. SNPs on 2q35 (rs13387042) and 5p12 (rs4415084 and rs10941679) have been shown to be associated with estrogen-receptor (ER) positive breast cancer [START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Stacey | Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF] and SNPs lying within cell cycle genes have been shown to be associated with breast cancer in the British population (rs997669 in CCNE1, rs3176336 in CDKN1A, rs34330 in CDKN1B and rs3731239 in CDKN2A/2B) [START_REF] Driver | Association of singlenucleotide polymorphisms in the cell cycle genes with breast cancer in the British population[END_REF]. These associations have yet to be confirmed in other studies or populations, with the exception of rs34330, which has been shown to be associated with breast cancer in the Chinese population [START_REF] Ma | Variant genotypes of CDKN1A and CDKN1B are associated with an increased risk of breast cancer in Chinese women[END_REF].

One of the breast cancer susceptibility SNPs identified by Easton et al. rs13281615 (chr8:128,424,800), lies at chromosome 8q24.21, a region that is also associated with colorectal and prostate cancer risk. Within 8q24.21, several GWASs have shown an association between prostate cancer and SNP rs1447295 (chr8:128,553,970) [START_REF] Amundadottir | A common variant associated with prostate cancer in European and African populations[END_REF][START_REF] Freedman | Admixture mapping identifies 8q24 as a prostate cancer risk locus in African-American men[END_REF][START_REF] Gudmundsson | Genomewide association study identifies a second prostate cancer susceptibility variant at 8q24[END_REF][START_REF] Haiman | Multiple regions within 8q24 independently affect risk for prostate cancer[END_REF][START_REF] Yeager | Genome-wide association study of prostate cancer identifies a second risk locus at 8q24[END_REF]. This locus has been studied in breast cancer cohorts, but no association found [START_REF] Schumacher | A common 8q24 variant in prostate and breast cancer from a large nested case-control study[END_REF]. A second independent locus within 8q24 conferring prostate cancer risk, rs16901979 (chr8:128,194,098), has been identified [START_REF] Gudmundsson | Genomewide association study identifies a second prostate cancer susceptibility variant at 8q24[END_REF][START_REF] Haiman | Multiple regions within 8q24 independently affect risk for prostate cancer[END_REF]. Another nearby SNP, rs6983267 (chr8:128,482,237), confers an increased risk of colorectal cancer [START_REF] Tomlinson | A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21[END_REF][START_REF] Zanke | Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24[END_REF] and also confers some independent prostate cancer risk and ovarian cancer risk [START_REF] Ghoussaini | Multiple loci with different cancer specificities within the 8q24 gene desert[END_REF]. The association with prostate cancer is weaker than that with rs1447295 [START_REF] Witte | Multiple prostate cancer risk variants on 8q24[END_REF], but again independent of both rs1447295 and rs16901979. All four SNPs are found within 400 kb of each other: rs13281615 is 130 kb proximal of rs1447295, 60 kb proximal of rs6983267 and 230 kb distal of rs16901979.

Many of the common cancers are associated with a twofold increase in familial relative risk across the different cancer types [START_REF] Houlston | Genetics and the common cancers[END_REF]. This together with the finding of risk variants for breast, prostate, colon and ovarian cancer clustering at 8q24 suggests that there may be low penetrance susceptibility alleles elsewhere in the genome predisposing to all of these cancers. Large genome wide association studies of prostate and colorectal cancer have now identified new prostate [START_REF] Eeles | Multiple newly identified loci associated with prostate cancer susceptibility[END_REF][START_REF] Thomas | Multiple loci identified in a genome-wide association study of prostate cancer[END_REF][START_REF] Gudmundsson | Common sequence variants on 2p15 and Xp11.22 confer susceptibility to prostate cancer[END_REF] and colorectal [START_REF] Jaeger | Common genetic variants at the CRAC1 (HMPS) locus on chromosome 15q13.3 influence colorectal cancer risk[END_REF][START_REF] Tenesa | Genomewide association scan identifies a colorectal cancer susceptibility locus on 11q23 and replicates risk loci at 8q24 and 18q21[END_REF][START_REF] Broderick | A genome-wide association study shows that common alleles of SMAD7 influence colorectal cancer risk[END_REF][START_REF] Tomlinson | A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3[END_REF][START_REF] Houlston | Meta-analysis of genome-wide association data identifies 4 novel susceptibility loci for colorectal cancer[END_REF] susceptibility alleles in addition to the alleles at 8q24. It is not known whether these alleles also predispose to breast cancer.

The aims of this study were:

1. To confirm whether recently identified breast cancer predisposition SNPs also predispose to breast cancer in the Irish population. 2. To determine whether any of the other known cancer SNPs, also predispose, perhaps more weakly, to other common cancer types.

Methods

A total of 988 breast cancer cases and 1,016 controls from the West of Ireland were collected with appropriate ethical approval as part of the Breast cancer in Galway Genetics Study (BIGGS). This population shares similar ethnic ancestry to the population in the UK, but has been subject to fewer demographic movements [START_REF] Hill | Y-chromosome variation and Irish origins[END_REF] The power of our study, based on reasonable assumptions from previous studies, for the breast cancer SNPs rs2981582, rs3803662 and rs13281615, was as follows. Using an allelic test, 988 cases and 1,016 controls provided [90% power at P \ 0.05 (two-tailed test) to detect a SNP allele with a frequency of 0.4 associated with an OR of 1.3; [60% power to detect an allele of frequency of 0.25 associated with an OR of 1.2; and *30% power to detect an allele of frequency 0.4 associated with an OR of 1.1.

DNA was extracted from blood using the Chemagic Magnetic Separation Module (Chemagen, Baesweiler, Germany) using the manufacturers reagents. A panel of 24 breast cancer, colorectal cancer and prostate cancer susceptibility loci were genotyped using the KASPar SNP genotyping system (KBiosciences), Table 1. Primers are available on request. A subset of samples were sequenced for each SNP to confirm the results. Sequencing was performed using the ABI 3100 automated sequencer and sequence analysis software (Applied Biosystems, CA).

Deviation of genotype frequencies from those expected under Hardy-Weinberg equilibrium (HWE) was assessed by v 2 test, or Fisher's exact test where an expected cell count was \5. The risk associated with each SNP was estimated by allelic, dominant and recessive odds ratio (OR) and associated 95% confidence intervals (CI). Genotypic ORs were determined by logistic regression.

Determination of a general cancer risk allele was performed by meta-analysis using publicly available SNP data for breast and prostate cancer (CGEMS, http://cgems. cancer.gov/: breast study-1,183 postmenopausal cases and 1,185 controls; prostate study-1,177 cases and 1,105 controls) and SNP data from the first phase of our colorectal cancer GWAS (930 familial colorectal tumour cases and 960 controls) [START_REF] Tomlinson | A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21[END_REF]. Data from these breast, prostate and colorectal cancer samples were combined with the West of Ireland cohort to derive an overall estimate of cancer risk using a fixed effects model with inverse variance weighting of individual studies. Reported predisposition SNPs not found on the Illumina platform data were imputed using IMPUTE [START_REF] Marchini | A new multipoint method for genome-wide association studies by imputation of genotypes[END_REF]. Bias was assessed using the I 2 and Q estimates. For any significant findings in the fixed effects model with values I 2 C 50% (considered moderate heterogeneity [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF]), the analysis was repeated using a random effects model. All statistical analyses were undertaken using STATA 9.2 (Stata Corp, College Station, TX).

Results

Verification of reported breast cancer SNPs in the BIGGS study

Samples that consistently failed in [20% of the SNPs studied were excluded from the analysis (25 cases, 20 We used a threshold of P B 0.05 (two-tailed) for replication of previous studies. Of the five breast cancer loci previously identified by Easton et al. only rs2981582 (FGFR2) and rs13281615 (8q24) showed an association with breast cancer in the West of Ireland population at P \ 0.05, Table 2a. There was a borderline association with rs3803662 (TNRC9) at P allelic test = 5.1 9 10 -2 . rs2981582 showed the strongest association with breast cancer, allelic test of association (P = 1.8 9 10 -2 , Table 2a) and the genotypic test (P = 2 9 10 -3 , Table 2a). The odds ratio (OR) for the TT homozygote was 1.489 (95% CI 1.153-1.922, P = 2 9 10 -3 ) and that for the CT heterozygote was 1.176 (95% CI 0.958-1.443, P = 1 9 10 -1 ), relative to the CC homozygote in each case. The association between rs13281615 and breast cancer was also confirmed using the allelic test of association (P = 1.8 9 10 -2 , Table 2a) and the genotypic test (P = 0.026, Table 2a). The odds ratio (OR) for the GG homozygote was 1.28 (95% CI 0.98-1.66, P = 7 9 10 -2 ) and that for the AG heterozygote was 1.34 (95% CI 1.09-1.64, P = 6 9 10 -3 ) relative to the AA homozygote. There was a borderline association between rs3803662 and breast cancer (allelic test of association, P = 1.8 9 10 -2 and the genotypic test, P = 4.7 9 10 -2 , Table 2a). The odds ratio (OR) for the TT homozygote was 1.554 (95% CI 1.09-2.23, P = 1.6 9 10 -2 ) and that for the CT heterozygote was 1.060 (95% CI 0.88-1.278, P = 5.7 9 10 -1 ), relative to the CC homozygote in each case. rs889312 (MAP3K1) and rs3817198 (LSP1) showed no association with breast cancer in this population (P allelic test = 2.4 9 10 -1 , P allelic test = 5.4 9 10 -1 , respectively). There was also no evidence of an association with the cell cycle gene SNPs (rs997669, rs3176336, rs34330) or rs4415084 (5p12) that have recently been suggested as breast cancer predisposition SNPs. It is not clear whether these differences are due to the different population studied or due to a lack of power in this study to detect these weaker associations. In order to answer this question we performed a meta-analysis on those SNPs where there was publicly available data in breast cancer, Table 3. This confirmed the association with rs4415084 (5p12) and rs3176336 (CDKN1A) also showed a significant association with breast cancer. However, the latter finding was based only on data from two studies (SEARCH and BIGGS) as there was no data available from CGEMS for this SNP. There was no evidence of an association between breast cancer and rs34330 (CDKN1B), rs997669 (CCNE1) or rs3817198 (LSP1), Table 3.

Association between breast cancer SNPs and clinicopatholgical features of breast cancer

Histological information was available on 795 (80%) cases and is summarised in Table 4. Univariate analysis showed evidence of an association between rs2981582 and PR positivity (P = 3.5 9 10 -2 ), but not ER positivity (P = 1.0 9 10 -1 ). Univariate analysis also showed a negative association between rs3803662 and her two positivity (P = 3.7 9 10 -2 ), but no evidence of an association with ER/PR status. There was no evidence of an association between the rs13281615 risk allele and ER status, Her2 positivity, grade, nodal status or bilaterality. There was however an association with age and rs13281615 with younger cases more likely to have the at risk allele (P = 4.1 9 10 -2 ).

Analysis of colorectal and prostate SNPs in the BIGGS study

The prostate and colorectal SNPs on 8q24 showed no association with breast cancer in our cohort and there was no association with any of the other low penetrance colorectal and prostate SNPs that were studied for any of the genetic models tested (Table 2b, c for allelic and genotypic tests). Logistic regression analysis provided no evidence for an independent effect of the colorectal or prostate SNPs studied on breast cancer risk (details not shown).

Meta-analysis to search for general cancer predisposition alleles for the three common cancers

We performed a meta-analysis in order to try and ascertain whether there was any evidence of a general cancer predisposition allele. A comprehensive list of SNPs known to confer an increased cancer risk of either breast, colorectal and prostate cancer was compiled (Table 5). As all publicly available data were based on the Illumina platform, reported predisposition SNPs not found on the Illumina platform data were imputed using IMPUTE [START_REF] Marchini | A new multipoint method for genome-wide association studies by imputation of genotypes[END_REF] (rs4666451, rs981782, rs2180341, rs13281615, rs1045485). In the case of rs2981582 (FGFR2), we used a previously identified Illumina SNP (rs1219648) at this locus which has also been shown to be associated with the breast cancer [START_REF] Hunter | A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer[END_REF]. Two SNPs (rs3176336 and rs889312) previously shown to predispose to breast cancer could not be imputed due to lack of HapMap data.

Data from publicly available sources (see ''Methods'') on genotype frequencies in cases and controls for these risk SNPs were combined using a fixed effects model for allelic, genotypic, recessive and dominant modes of risk. Only one SNP (rs6983267 on 8q24, P \ 5 9 10 -4 ) conferred risk to cancer in two tissue types (CRC and prostate cancer), as

Discussion

We have provided replication of the association between rs2981582, rs13281615, and rs3803662 and breast cancer susceptibility in a sporadic breast cancer cohort from the West of Ireland. Unlike many previous studies, cases were not selected for family history of breast cancer [START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF] or enriched for bilateral breast cancers [START_REF] Fletcher | Association of genetic variants at 8q24 with breast cancer risk[END_REF]. The allelic ORs detected in this study for both rs2981582 and rs3803662 were similar to that detected by Easton et al. For rs13281615 the OR of 1.17 (95% CI = 1.03-1.33) is higher than that estimated by Easton et al. (OR = 1.08, 95% CI = 1.05-1.11) in populations of European origin [START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF], but not as high as that estimated by Fletcher et al.

(OR = 1.24, 95% CI = 1.12-1.38) in a series enriched for bilateral breast cancer cases [START_REF] Fletcher | Association of genetic variants at 8q24 with breast cancer risk[END_REF], although there is overlap of the 95% CIs.

A previous study showed that the rs2981582 risk allele had a stronger association with ER and PR positive breast cancers than ER negative breast cancers [START_REF] Garcia-Closas | Heterogeneity of breast cancer associations with five susceptibility Loci by clinical and pathological characteristics[END_REF]. A similar but weaker association was found for rs13281615 and rs3803662 [START_REF] Garcia-Closas | Heterogeneity of breast cancer associations with five susceptibility Loci by clinical and pathological characteristics[END_REF]. We only found an association between PRpositive tumours and rs2981582 on univariate analysis. ER and PR status showed no association with rs13281615 and rs3803662 in this study. We did however find a negative association between rs3803662 and her two positivity and an association between age of breast cancer diagnosis and rs13281615, with younger breast cancer patients more likely to have the risk allele.

In the study by Easton et al. rs889312 (MAP3K1) and rs3817198 (LSP1) showed a weaker association with breast cancer than rs2981582, rs13281615, and rs3803662.

Neither of these SNPs showed evidence of an association in BIGGS. We also found no evidence of an association between SNPs found within cell cycle genes and the West of Ireland breast cancer population, and did not verify the findings of Stacey et al. of a breast cancer predisposition SNP on 5p12. The latter association was only found in ER positive breast cancers. When we confined our analysis to this subgroup of breast cancer, we found no association with the 5p12 SNP, rs4415084, but did find an association between rs3176336, rs4779584 and rs7931342 and ER positive breast cancer (P allelic test = 0.02, P allelic test = 0.03, P allelic test = 0.03, respectively), but this may represent modulation of phenotype, not susceptibility. The explanation for some of these differences is due to the power of our study as when we performed a meta-analysis combining our data with publicly available data we did confirm the association with rs4415084 (5p12), but not for rs34330, rs997669 or rs3817198.

We have performed a comprehensive meta-analysis of alleles that predispose to breast, prostate and colorectal cancer in order to test whether they are general carcinoma susceptibility loci. This showed that only one SNP (rs6983267 on 8q24) conferred risk to cancer in two tissue types (colorectal cancer and prostate cancer). Importantly, rs6983267 was not associated with an increase in breast cancer risk despite the known association of breast cancer with that region, overall, our data suggest that low penetrance susceptibility SNPs for breast, prostate and colorectal cancer are distinct leaving open the exploration for the increase in familial relative risk across the cancer types.

The most recent SNP found to predispose to colorectal cancer is rs9929218 which is located in intron1 of CDH1 (E-cadherin) [START_REF] Houlston | Meta-analysis of genome-wide association data identifies 4 novel susceptibility loci for colorectal cancer[END_REF]. A SNP in the E-cadherin promoter (-160C ? A) has previously been shown to be associated with prostate cancer [START_REF] Wang | The E-cadherin gene polymorphism 160C-[ A and cancer risk: a HuGE review and meta-analysis of 26 case-control studies[END_REF]. However, the CGEMS prostate cancer study showed no evidence of an association with rs9929218 and prostate cancer. Loss of E-cadherin expression is characteristic of invasive lobular breast cancers [START_REF] Qureshi | E-cadherin status in breast cancer correlates with histologic type but does not correlate with established prognostic parameters[END_REF] and germline mutations have been found in some familial lobular carcinomas [START_REF] Masciari | Germline E-cadherin mutations in familial lobular breast cancer[END_REF]. There was no evidence of an association between rs9929218 and lobular cancer in the BIGGS cohort, however the study does not have the power to detect such an association if it existed with only 106 invasive lobular cancers in this series.

The finding of risk variants clustering at 8q24 is intriguing, our data and that of Fletcher et al. suggest that these variants are organ specific. Telomeric to the rs16901979 and rs13281615 haplotype blocks on 8q24 lie the expressed transcript POU5F1P1 (128.50 Mb), that has potential to encode a homologue of the transcription factor OCT4, and the oncogene MYC (128.82 Mb). It remains possible that combinations of different genomic control regions involved in regulating MYC or POU5F1P1 expression are associated with susceptibility to different types of the common cancers. It has been proposed that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to cancer susceptibility [START_REF] Sole | Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility[END_REF].

In conclusion we have confirmed that SNPs close to FGFR2 and TNCR9 and at 8q24(128424800) predispose to sporadic breast cancer in the West of Ireland. However, we have failed to replicate the association of SNPs close to MAP3K1, LSP1, CCNE1, CDKN1A, CDKN1B and at 5p12 in this population. We have shown that there is no association between the recently identified colorectal and prostate cancer SNPs and breast cancer. In general, prostate and colorectal cancer susceptibility alleles are site specific, despite some clustering at 8q24.

Table 1

 1 Genotypes were scored in [94% of samples for each SNP; frequencies did not deviate significantly from Hardy-Weinberg equilibrium (P [ 0.01 for controls) at any of the 24 SNPs. There was no linkage disequilibrium (LD) between any of the four SNPs at 8q24, with all pairwise r 2 values being zero and D 0 values being \0.20.

	Panel of SNPs genotyped in the breast cancer	SNP	Association	Gene	Locus
	population from Galway, West Ireland	rs13281615 rs2981582	Breast [3] Breast [3]	POU5F1P1/MYC FGFR2	8q (128424800) 10q (123342307)
		rs889312	Breast [3]	MAP3K1	5q (56067641)
		rs3817198	Breast [3]	LSP1	11p (1865582)
		rs3803662	Breast [3]	TNRC9/LOC643714	16q (51143842)
		rs997669	Breast [7]	CCNE1	19q (34996323)
		rs3176336	Breast [7]	CDKN1A	6p (36756544)
		rs34330	Breast [7]	CDKN1B	12p (12761962)
		rs4415084	Breast [6]		5p (44698272)
		rs6983267	Colorectal/Prostate [15, 16]	POU5F1P1	8q (128482487)
		rs16892766	Colorectal [26]		8q (117699864)
		rs4779584	Colorectal [23]	GREM1, SGNE1	15q (30782048)
		rs4939827	Colorectal [25]	SMAD7	18q (44707461)
		rs3802842	Colorectal [24]	LOC120376	11q (110676919)
		rs9929218	Colorectal [27]	CDH1	16q (67378697)
		rs16901979	Prostate [11]	POU5F1P1	8q (128194098)
		rs6465657	Prostate [20]	LMTK2	7q (97654013)
		rs7920517	Prostate [20]	MSMB	10q (51202627)
		rs7931342	Prostate [20]		11q (68751073)
		rs2660753	Prostate [20]	LOC285232	3 (87193364)
		rs5945619	Prostate [20]	NUDT11	Xp (51074708)
		rs266849	Prostate [20]	KLK3 KLK15	19q (56040902)
		rs2659056	Prostate [20]	KLK1 KLK15	19q (56027755)
		rs10993994	Prostate [20]	MSMB	10q (51219502)

Table 2

 2 Genotype frequencies and tests of association for (a) Previously reported breast cancer association SNPs in BIGGS; (b) Colorectal cancer association SNPs in BIGGS; (c) Prostate cancer association SNPs in BIGGS

	SNP	Genotype		Frequency	Allelic test			Genotypic test
							P (2 tail)	OR	95% CI	P (2 tail)	OR	95% CI
	(a)
	rs2981582	CC	CT	TT	C	T
	Cases	269	458	214	0.53	0.47	2.2 9 10 -3	1.22	1.07-1.39	2.00 9 10 -3	1.21	1.07-1.38
	Controls	335	483	179	0.58	0.42
	rs13281615	AA	AG	GG	A	G
	Cases	272	467	178	0.55	0.45	1.8 9 10 -2	1.17	1.03-1.33	2.63 9 10 -2	1.16	1.02-1.31
	Controls	355	456	182	0.59	0.41
	rs3803662	CC	CT	TT	C	T
	Cases	486	382	82	0.71	0.29	5.1 9 10 -2	1.15	1.00 -1.32	4.70 9 10 -2	1.16	1.00-1.33
	Controls	532	396	58	0.74	0.26
	rs889312	AA	AC	CC	A	C
	Cases	449	400	88	0.69	0.31	2.38 9 10 -1	1.08	0.95 -1.25	2.34 9 10 -1	1.09	0.95-1.25
	Controls	496	411	81	0.71	0.29
	rs3817198	TT	TC	CC	T	C
	Cases	363	454	121	0.63	0.37	5.36 9 10 -1	1.04	0.91-1.19	5.18 9 10 -1	0.96	0.84-1.09
	Controls	379	453	116	0.64	0.36
	rs997669	AA	AG	GG	A	G
	Cases	282	497	152	0.57	0.43	3.20 9 10 -1	0.94	0.82-1.06	2.91 9 10 -1	0.93	0.82-1.06
	Controls	305	471	200	0.55	0.45
	rs3176336	AA	AT	TT	A	T
	Cases	365	455	115	0.63	0.37	6.54 9 10 -1	0.94	0.85-1.16	6.38 9 10 -1	0.97	0.85-1.11
	Controls	380	452	135	0.63	0.37
	rs34330	CC	CT	TT	C	T
	Cases	567	335	45	0.78	0.22	2.0 9 10 -1	0.91	0.78-1.05	2.15 9 10 -1	0.90	0.78-1.06
	Controls	558	355	57	0.76	0.24
	rs4415084	CC	TC	TT	C	T
	Cases	290	417	175	0.57	0.43	8.28 9 10 -1	1.02	0.89-1.15	9.02 9 10 -1	1.01	0.86-1.15
	Controls	323	488	186	0.57	0.43
	(b)
	rs6983267	GG	GT	TT	G	T
	Cases	251	464	230	0.51	0.49	5.56 9 10 -1	0.96	0.85-1.09	5.61 9 10 -1	0.96	0.85-1.09
	Controls	248	464	245	0.50	0.50
	rs16892766	AA	AC	CC	A	C
	Cases	839	115	3	0.94	0.06	4.74 9 10 -1	1.10	0.85-1.43	4.72 9 10 -1	1.10	0.85-1.43
	Controls	891	110	3	0.94	0.06
	rs4779584	CC	CT	TT	C	T
	Cases	671	262	30	0.83	0.17	1.16 9 10 -1	0.88	0.74-1.03	1.12 9 10 -1	0.89	0.74-1.03
	Controls	655	301	34	0.81	0.19
	rs4939827	TT	TC	CC	T	C
	Cases	264	473	212	0.53	0.47	7.33 9 10 -1	0.99	0.86-1.11	7.36 9 10 -1	1.02	0.90-1.16
	Controls	278	468	235	0.52	0.48
	rs3802842	AA	AC	CC	A	C
	Cases	412	447	70	0.75	0.25	3.65 9 10 -1	0.94	0.82-1.08	3.47 9 10 -1	0.93	0.81-1.08
	Controls	416	428	96	0.72	0.28

previously published

[START_REF] Tomlinson | A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21[END_REF][START_REF] Zanke | Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24[END_REF]

. Of the remaining 31 SNPs analysed (5 associated with CRC risk, 13 with breast cancer, 13 with prostate cancer), none were significantly associated with cancer risk in another tumour type, either on its own or in combination of those tissue types in which the SNP was not discovered (Supplementary Table

1

Table 3

 3

	Results of meta-analysis of breast cancer studies for rs3176336, rs34330,	SNP	Studies used in meta-analysis	Allelic test (P value-2 tailed)
	rs3817198, rs4415084, rs997669 using random effects model	rs3176336 rs34330 rs3817198	BIGGS SEARCH a BIGGS SEARCH a CGEMS BIGGS CGEMS	\5.00 9 10 -4 3.32 9 10 -1 2.88 9 10 -1
	a Study which found a positive association	rs4415084 rs997669	BIGGS CGEMS BIGGS SEARCH a CGEMS	4.00 9 10 -3 9.88 9 10 -1

Table 2

 2

	continued
	SNP	Genotype		Frequency	Allelic test			Genotypic test
							P (2 tail)	OR	95% CI	P (2 tail)	OR	95% CI
	rs9929218	GG	AG	AA	G	A
	Cases	488	399	77	0.72	0.28	1.31 9 10 -1	0.90	0.78-1.03	7.49 9 10 -2	0.85	0.71-1.02
	Controls	467	451	84	0.69	0.31
	(c)
	rs16901979	CC	AC	AA	C	A
	Cases	909	54	3	0.97	0.03	7.74 9 10 -1	0.95	0.66-1.36	7.76 9 10 -1	0.95	0.67-1.35
	Controls	930	65	0	0.97	0.03
	rs6465657	TT	CT	CC	TT	CC
		285	441	198	0.55	0.45	7.16 9 10 -1	0.89	0.76-1.05	7.95 9 10 -1	1.02	0.90-1.15
		319	457	217	0.55	0.45
	rs7920517	AA	AG	GG	A	G
	Cases	259	473	223	0.52	0.48	1.83 9 10 -1	0.92	0.81-1.04	1.88 9 10 -1	0.92	0.81-1.04
	Controls	251	472	256	0.50	0.50
	rs7931342	GG	GT	TT	G	T
	Cases	296	478	187	0.56	0.44	1.17 9 10 -1	0.90	0.80-1.03	1.16 9 10 -1	1.11	0.98-1.26
	Controls	281	494	218	0.53	0.47
	rs2660753	CC	CT	TT	C	T
	Cases	786	176	7	0.90	0.10	4.17 9 10 -1	0.92	0.75-1.12	4.16 9 10 -1	0.92	0.75-1.12
	Controls	787	182	13	0.89	0.11
	rs5945619	TT	CT	CC	T	C
	Cases	397	433	136	0.64	0.36	6.45 9 10 -2	1.13	0.99-1.30	1.06 9 10 -1	1.12	0.98-1.27
	Controls	394	396	103	0.66	0.34
	rs266849	AA	AG	GG	A	G
	Cases	598	316	39	0.79	0.21	1.17 9 10 -1	0.90	0.80-1.03	1.59 9 10 -1	0.89	0.76-1.05
	Controls	642	328	24	0.81	0.19
	rs2659056	AA	AG	GG	A	G
	Cases	522	368	56	0.75	0.25	7.40 9 10 -2	1.14	0.80-1.03	6.40 9 10 -2	0.87	0.75-1.01
	Controls	498	410	68	0.72	0.28
	rs10993994	CC	CT	TT	C	T
	Cases	335	470	150	0.60	0.40	2.33 9 10 -1	0.93	0.81-1.05	2.36 9 10 -1	0.93	0.82-1.05
	Controls	342	464	187	0.58	0.42
	rs16892766	AA	AC	CC	A	C
	Cases	839	115	3	0.94	0.06	4.74 9 10 -1	1.10	0.85-1.43	4.72 9 10 -1	1.10	0.85-1.43
	Controls	891	110	3	0.94	0.06

Table 4

 4 Histological characteristics in 794 cases

	Histological characteristics	Percentage of
		cases
	Ductal	86.5
	Lobular	13.5
	Grade 1	13
	Grade 2	39
	Grade 3	35
	Unknown grade	13
	ER positive	60
	ER negative	21
	ER unknown	19
	Node positive	46
	Node negative	54
	Bilateral cases	5
	Family history of first degree relative with breast	15
	cancer
	Family history of second degree relative with
	breast cancer

Table 5

 5 Predisposition SNPs used in meta-analysis

		Chromosome	Position	Gene
	Prostate predisposition SNPs
	rs6983267	8	128482487	POU5F1P1
	rs7920517	10	51202627	MSMB
	rs7931342	11	68751073
	rs2660753	3	87193364	LOC285232
	rs5945619	9	51074708	NUDT11
	rs266849	19	56040902	KLK3|KLK15
	rs10993994	10	51219502	MSMB
	rs9364554	6	160804075	SLC22A3
	rs6465657	7	97460978	LMTK2
	rs902774	12	51560171
	rs2659056	19	56027755	KLK1|KLK15
	rs2735839	19	56056435	KLK3|KLK2
	rs4430796	17	33172153	TCF2
	Breast predisposition SNPs
	rs13281615	8	128424800	POU5F1P1
	rs981782	5	45321225
	rs30099	5	52454339	MOCS2
	rs4666451	2	19150174
	rs3803662	16	51143842	TNRC9
	rs2180341	6	127642073	RNF146
	rs1219648	10	123336180	FGFR2
	rs1045485	2	201857584	casp8
	rs997669	19	34996323	CCNE1
	rs34330	12	12761962	CDKN1B
	rs3731239	9	21964218	CDKN2A
	rs13387042	2	217731338
	rs4415084	5	44698272
	Colorectal predisposition SNPs
	rs6983267	8	128482487	POU5F1P1
	rs16892766	8	117699864
	rs4779584	15	30782048	GREM1|SGNE1
	rs4939827	18	44707461	SMAD7
	rs3802842	11	110676919	FLJ45803
	rs9929218	16	67378697	CDH1

Breast Cancer Res Treat (2009) 117:151-159

Acknowledgments We are grateful to the patients in the study and colleagues involved in patient ascertainment and sample collection. Help was also kindly provided by the Equipment Park, London Research Institute, Cancer Research UK. NM, GC and ST were funded by the National Breast Cancer Research Institute, Ireland. ES and AJ are funded by a Department of Health Clinician Scientist Grant, UK.


E R Van Beek

Ae C W G M Lowik

Ae J Van Wijngaarden

Ae F H Ebetino

Ae S E Papapoulos

C W G M Lowik

Á J Van Wijngaarden

S E Papapoulos

Synergistic effect of bisphosphonate and docetaxel on the growth of bone metastasis in an animal model of established metastatic bone disease

Keywords: Bisphosphonates, Bone metastasis, Breast cancer, Docetaxel, Risedronate, Zoledronate

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Synergistic effect of bisphosphonate and docetaxel on the growth of bone metastasis in an animal model of established metastatic bone disease

Introduction Metastatic bone disease is a major cause of morbidity in patients with different cancers including those of the breast and the prostate [START_REF] Coleman | Metastatic bone disease: clinical features, pathophysiology and treatment strategies[END_REF]. Despite differences in the pathogenesis of bone metastases from different cancer types, increased osteoclast-mediated bone resorption is the major mechanism for tumor-induced bone destruction [START_REF] Roodman | Mechanisms of bone metastasis[END_REF][START_REF] ¨ko ¨nen | Mechanisms of osteolytic bone metastases in breast carcinoma[END_REF]. Bisphosphonates decrease bone resorption and reduce significantly the rate of skeletal complications in patients with metastatic bone disease [START_REF] Aapro | Guidance on the use of bisphosphonates in solid tumours: recommendations of an international expert panel[END_REF]. In addition, several in vitro studies reported that bisphosphonates have direct antiproliferative and proapoptotic effects on cancer cells and can inhibit the adhesion of cancer cells to mineralized matrices suggesting that these compounds may also have a favorable action on the growth and invasive behavior of cancer cells [START_REF] Green | Antitumor effects of bisphosphonates[END_REF][START_REF] Clezardin | Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity[END_REF][START_REF] Stresing | Bisphosphonates in cancer therapy[END_REF][START_REF] Van Der Pluijm | Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro[END_REF]. However, in vivo studies in animal models of bone metastasis have produced equivocal results [START_REF] Sasaki | Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice[END_REF][START_REF] Kostenuik | Increased growth rate and tumor burden of spontaneously metastatic Walker 256 cancer cells in the skeleton of bisphosphonate-treated rats[END_REF][11][START_REF] Krempien | Prophylactic treatment of skeletal metastases, tumor induced osteolysis, and hypercalcemia in rats with the bisphosphonate Cl2MBP[END_REF][START_REF] Krempien | Protective effect of a prophylactic treatment with the bisphosphonate 3-amino-1-hydroxypropane-1,1 bisphosphonic acid on the development of tumor osteopathies in rat: experimental studies with the Walker Carcinosarcoma 256[END_REF][START_REF] Hall | Effect of the bisphosphonate risedronate on bone metastases in a rat mammary adenocarcinoma model system[END_REF][START_REF] Van Der Pluijm | Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo[END_REF][START_REF] Daubine | Antitumour effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis[END_REF][START_REF] Zhang | A novel bisphosphonate minodronate (YM529) specifically inhibits osteolytic bone metastasis produced by human small cell lung cancer cells in NK-cell depleted SCID mice[END_REF]. These apparently discrepant results regarding an antitumor effect of bisphosphonates may be related to the timing of interference with bone turnover during the metastatic process. Decrease of bone turnover by bisphosphonates before colonization of bone by cancer cells, inhibits to a great extent the formation of bone metastases [START_REF] Van Der Pluijm | Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo[END_REF][START_REF] Daubine | Antitumour effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis[END_REF]. However, when bisphosphonate treatment is given after the establishment of bone metastases, it has a minimal effect on the progression of cancer growth despite a substantial reduction of osteolysis. It was hypothesized that cancer cells metastatic to bone after an initial growth phase that depends on their interaction with the local stroma, they become independent of microenvironment's growth support and progress autonomously [START_REF] Van Der Pluijm | Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo[END_REF]. For the arrest of growth of established metastases, compounds with mechanisms of action different from that of bisphosphonates will be needed. Previous studies with concomitant administration of bisphosphonates and chemotherapeutics have shown a reduction in metastatic growth to bone [START_REF] Heymann | Enhanced tumor regression and tissue repair when zoledronic acid is combined with ifosfamide in rat osteosarcoma[END_REF][START_REF] Kim | Modulation of bone microenvironment with zoledronate enhances the therapeutic effects of STI571 and paclitaxel against experimental bone metastasis of human prostate cancer[END_REF][START_REF] Inoue | Effect of combination therapy with a novel bisphosphonate, minodronate (YM529), and docetaxel on a model of bone metastasis by human transitional cell carcinoma[END_REF][START_REF] Yano | Combined therapy with a new bisphosphonate, minodronate (YM529), and chemotherapy for multiple organ metastases of small cell lung cancer cells in severe combined immunodeficient mice[END_REF][START_REF] Michigami | The effect of the bisphosphonate ibandronate on breast cancer metastasis to visceral organs[END_REF][START_REF] Stearns | Effects of alendronate and taxol on PC-3 ML cell bone metastases in SCID mice[END_REF]. However, toxicity of the latter precludes the application of fully effective doses. In the present study, we, therefore, tested the hypothesis that doses of a cytostatic that lack full antitumor efficacy when given alone, can act synergistically with bisphosphonates to reduce the growth of bone metastases from breast cancer cells.

Material and methods

Cell line and culture conditions

Luciferase positive human MDA-MB-231 breast cancer cells (MDA-231-B/luc?), were used for in vivo optical imaging as described previously [START_REF] Wetterwald | Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease[END_REF]. MDA-231-B/luc? cells were cultured in DMEM (Life Technologies, Breda, The Netherlands) containing 4.5 g/l glucose and supplemented with 10% FCS (Life Technologies) and 800 lg/ml geneticin/G418 (Life Technologies).

Animals

Female nude mice (BALB/c nu/nu) were purchased from Charles River (Charles River, Maastricht, The Netherlands). Animals were housed in individual ventilated cages under sterile condition, and sterile food and water were provided ad libitum. Animal experiments were approved by the local committee for animal health, ethics and research of Leiden University and carried out in accordance with European Communities Council Directive 86/ 609/EEC.

Experimental animal model

MDA-231-B/luc? cells were harvested at about 80% confluence after changing to geneticin-free medium 24 h before inoculation.

The animals were anesthetized using the isofluorane anesthesia system (XGI-8, Xenogen) and a single-cell suspensions of 1.5 9 10 5 MDA-231-B/Luc? cells/10 ll PBS were injected into the right tibiae of 6-week old mice as described previously [START_REF] Wetterwald | Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease[END_REF].

Treatment of the animals started 2 days after intraosseous inoculation of MDA-231-B/Luc? cells. From this time point (day 0) and during a subsequent period of 5 weeks, they received risedronate or zoledronate (5 times per week (100 ll by i.p. injection)) (dissolved in PBS), docetaxel (2 times per week (50 ll by i.p. injection) (dissolved in DMSO) or a combination of 5 times per week risedronate and 2 times per week docetaxel concurrently. The control animals received vehicle treatment. The different treatment schedules are illustrated in Fig. 1.

Bioluminescent reporter imaging (BLI)

Tumour progression of intraosseous growth was monitored weekly by BLI. For this, the mice were anesthetized as described above and injected i.p. with 2 mg D-luciferin sodium salt (Synchem OHG) dissolved in PBS, and measurements were done 5 min after the injection of D-luciferin. Bioluminescence imaging was acquired with a 15-cm FOV, a medium binning factor, and exposure times of 10-60 s. Imaging data were analyzed by using the program living image (Xenogen). Values are expressed as relative light units (RLU) in photons per seconds.

Radiographs

After the experimental periods, mice were sacrificed by cervical dislocation and the tumor bearing hind legs were removed and assessed for osteolytic lesions by radiography (Kodak X-OMAT TL film, Eastman Kodak Co.) using a Hewlett Packard X-ray system Faxitron 43805 and quantified using NIH Image 1.62b7 software as described earlier [START_REF] Van Der Pluijm | Urokinasereceptor/integrin complexes are functionally involved in adhesion and progression of human breast cancer in vivo[END_REF].

Histology

The skin of the dissected hind legs was removed and the bones were fixed for 24 h in PBS with 4% formaldehyde; subsequently, the bones were decalcified in water containing 10% EDTA, pH 6.4 and embedded in paraffin and submitted to Masson-Goldner staining as previously described [START_REF] Van Der Eerden | Expression of estrogen receptors and enzymes involved in sex steroid metabolism in the rat tibia during sexual maturation[END_REF].

Effects of bisphosphonate treatment on metaphysal dry weight of the tibia

To determine the effectiveness of bisphosphonate treatment, at the end of the experiment, the dry weight of the metaphysis of the right tibia (not inoculated with cancer cells) was measured as previously described [START_REF] Brown | Differential effects of aminosubstituted analogs of hydroxy bisphosphonates on the growth of Dictyostelium discoideum[END_REF].

Results

Tumor growth kinetics

Following inoculation of the left tibiae of athymic nude mice with MDA-231-B/Luc? cells there was a progressive increase in tumor size with an increase of the BLI signal of more than 100-fold (1.4 Á 10 5 ± 2.5 Á 10 5 to 2.1 Á 10 7 ± 2.3 Á 10 7 RLU) from day 7 to 35 (Fig. 2a). Figure 2b shows representative images of the BLI signal intensity in the tumor baring leg of a control mouse on day 7, 21 and 35, respectively.

Effects of bisphosphonates

We first examined the efficacy of bisphosphonates on normal bone resorption. For this, we measured the dry weight of the metaphyses of contralateral tibiae, which were not inoculated with cancer cells, of the animals after 5 weeks of bisphosphonate treatment. Compared to controls, zoledronate (37. ). The lack of a dose-dependent effect in the zoledronate treated animals is due probably to already maximal inhibition of osteoclastic resorption by the lowest dose of this bisphosphonate used. Metaphysal weight in the risedronatetreated mice increased to the same extent as that in the zoledronate treated animals, indicating that resorption in these mice, was also maximally inhibited.

Both bisphosphonates prevented destruction of the tumor-bearing tibiae, assessed radiologically and histologically, but had no effect on tumor growth.

Figure 3 depicts representative radiographs, histological sections and BLI pictures of the tumor bearing legs, of these mice. Radiographically, the proximal tibia of the control animal was destroyed, whereas those of the bisphosphonate treated animals were intact, indicating protection of osteoclast-induced osteolysis by the bisphosphonates. Goldner stained histological sections demonstrated the presence of tumor in the legs of control mice. In bisphosphonate-treated mice there was a clear apparent reduction in the tumor within the bone whereas treatment had no effect on tumor load outside the bone collar. The bone marrow cavity of treated and untreated mice was invaded by the tumor which expanded outside the bone collar. Obviously, as result of their antiresorptive action, the tibial metaphysis of the bisphosphonate treated mice contained significantly more trabecular bone than that of controls. The results of BLI were consistent with the histological findings, as also shown previously [START_REF] Van Der Pluijm | Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo[END_REF][START_REF] Wetterwald | Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease[END_REF], and showed no difference in signal intensity between control and bisphosphonate-treated animals and neither bisphosphonate at any dose had any effect on tumor growth (Fig. 3b).

Effects of docetaxel

Figure 4a shows the effect of systemically administered docetaxel (2, 4 or 8 mg/kg, 2 times/week) on tumor growth after 5 weeks of treatment. Docetaxel inhibited tumor growth dose-dependently, with no BLI signal being measurable at the highest dose tested. Histological examination of the tibiae corroborated BLI findings. In contrast to controls, the tibiae of mice treated with 8 mg/kg docetaxel were intact and there was no detectable tumor tissue (Fig. 4b). Qualitative evaluation of the metaphyses revealed further that the amount of metaphyseal trabecular bone of docetaxel-treated mice appeared similar to that of controls and less than that of bisphosphonate-treated animals after 5 weeks.

Effects of combined treatment with risedronate and docetaxel

To examine the effect of the concomitant administration of bisphosphonate and docetaxel on tumor growth, docetaxel was given at minimally effective concentrations (4 mg/kg, twice/week) and risedronate at a dose of 150 lg/kg (5 times/week). As shown in Fig. 5a, risedronate alone did not affect tumor growth, as expected, while docetaxel alone failed to reduce tumor growth in five of the seven mice.

Treatment with the combination of docetaxel risedronate, however, resulted in a total absence of BLI signal in six out of seven mice. Histological examination confirmed the optical imaging findings, and only in one animal in the combined treatment group a tumor was present whereas in the other six mice no cancer tissue could be detected. In addition, like in the mice treated with risedronate alone, the tibiae of the animals treated with the combination of docetaxel and risedronate showed no osteolysis and contained a large quantity of trabecular bone (Fig. 5b).

Discussion

We show here that combined treatment with a potent bisphosphonate and a cytostatic, at doses that have minimal effect on tumor growth when given alone, protects skeletal integrity and inhibits the growth of breast cancer cells in an animal model of metastatic bone disease. Animal and human studies have previously shown that increased bone resorption comprises the main mechanism responsible for bone destruction in metastatic disease and is related to the incidence and severity of skeletal complications in patients with malignancies [START_REF] Brown | Bone resorption predicts for skeletal complications in metastatic bone disease[END_REF][START_REF] Brown | Bone turnover markers as predictors of skeletal complications in prostate cancer, lung cancer and other solid tumours[END_REF]. Breast cancer cells secrete factors, such as PTHrP, which stimulate the formation and activity of osteoclasts leading to bone destruction which causes bone pain, pathological fractures and hypercalcemia [START_REF] Coleman | Metastatic bone disease: clinical features, pathophysiology and treatment strategies[END_REF][START_REF] Roodman | Mechanisms of bone metastasis[END_REF][START_REF] Liao | Skeletal metastasis: established and emerging roles of parathyroid hormone related protein (PTHrP)[END_REF][START_REF] Siclari | Molecular interactions between breast cancer cells and the bone microenvironment drive skeletal metastases[END_REF]. This pathogenetic mechanism provided the rationale for the use of bisphosphonates in the management of patients with various tumours which metastasize to the skeleton, including those of the breast. However, during bone resorption induced by the osteoclasts, factors stored in the matrix of bone are also released in the bone marrow microenvironment and can act on cancer cells and stimulate further their growth as well as the production of bone resorbing factors [START_REF] Roodman | Mechanisms of bone metastasis[END_REF][START_REF] Siclari | Molecular interactions between breast cancer cells and the bone microenvironment drive skeletal metastases[END_REF]. It was, therefore, thought that inhibitors of bone resorption, such as the bisphosphonates, may not only protect the integrity of bone at metastatic sites but may also have a favourable effect on the local growth of bone metastases. In addition, several in vitro studies have shown that bisphosphonates have direct effects on tumour cells, increase their rate of apoptosis, decrease angiogenesis and prevent their attachment on bone matrices [START_REF] Green | Antitumor effects of bisphosphonates[END_REF][START_REF] Clezardin | Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity[END_REF]. Thus, bisphosphonates, in addition, to their bone protective effect, may also reduce the growth potential of cancer cells in the bone-bone marrow microenvorment. This attractive hypothesis has been, however, difficult to prove experimentally or clinically and appears to depend on the stage of the metastatic process as well as on the techniques used to assess cancer growth. For example, interference with the bone microenvironment with bisphosphonates before the establishment of bone metastases protects bone integrity and inhibits tumour growth. However, when bisphosphonates are given after the establishment of bone metastases, their effect on tumour growth is minimal as also shown in the present study. Furthermore, in studies reporting a beneficial effect on the tumour burden following bisphosphonate treatment, this is generally evaluated by histology of the area contained within the bones of animal models. However, it has been shown that tumour growth outside the bone collar was not affected by treatment and that the apparent decrease in tumour growth within bone was rather due to the decreased space available due to the preservation of the bone structure [START_REF] Van Der Pluijm | Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo[END_REF]. Such histological findings were supported by studies which assessed directly tumour growth by molecular imaging techniques and showed no effect in the overall growth of cancer cells [START_REF] Van Der Pluijm | Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo[END_REF]. We confirmed this in the present study and we showed that treatment with the two very potent bisphosphonates risedronate and zoledronate given at high doses with similar antiresorptive potencies to an animal model of established bone metastases were very effective in decreasing bone resorption and preventing bone destruction. However, bisphosphonate treatment given alone had only a minor effect on tumour growth assessed by histology and BLI once the tumour had been established in the bone marrow. In contrast, treatment with high doses of docetaxel did not only preserve the structure of bone but decreased also significantly the growth of the cancer cells within and outside the bone collar.

In recent years the significance of the interactions between tumour cells and cells of the bone marrow in the development of micrometastases to overt metastases has been increasingly recognized [START_REF] Roodman | Mechanisms of bone metastasis[END_REF][START_REF] Siclari | Molecular interactions between breast cancer cells and the bone microenvironment drive skeletal metastases[END_REF]. In this process, increased bone resorption plays an important role and promotes the initial growth of cancer cells. However, once these cells develop into macrometastases mechanisms other than bone resorption contribute to their growth potential, such as for example angiogenesis. This sequence of events explains why a bisphosphonate given to animals for prevention of bone metastases is effective whereas when given to models with established metastatic disease has minimal effect on the further growth of the tumour. The lack of an antitumour effect of bisphosphonates on bone metastases in vivo despite the demonstration of such effects in vitro is probably attributed to the specific pharmacokinetics of these compounds. Bisphosphonates are cleared rapidly from the circulation and are taken up preferentially by the skeleton at active remodelling sites where they bind strongly to bone [START_REF] Cremers | Pharmacokinetics/ pharmacodynamics of bisphosphonates: use for optimization of intermittent therapy for osteoporosis[END_REF][START_REF] Cremers | Skeletal retention of bisphosphonate (pamidronate) and its relation to the rate of bone resorption in patients with breast cancer and bone metastases[END_REF]. This action allows only very limited, if any, exposure of the cancer cells in the marrow to bisphosphonates [START_REF] Fournier | Lowering bone mineral affinity of bisphosphonates as a therapeutic strategy to optimize skeletal tumor growth inhibition in vivo[END_REF]. Therefore, for the adequate management of established metastatic disease in bone, bisphosphonates may have to be combined with other agents which specifically affect tumour growth and progression. Previous studies with bisphosphonates in combination with antitumor drugs were effective in decreasing tumour growth in relevant animal models and in vitro evidence of a synergism has been reported [START_REF] Fournier | Lowering bone mineral affinity of bisphosphonates as a therapeutic strategy to optimize skeletal tumor growth inhibition in vivo[END_REF][START_REF] Jagdev | The bisphosphonate zoledronic acid induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel[END_REF][START_REF] Jagdev | Zoledronic acid induces apoptosis of breast cancer cells in vitro: evidence for additive and synergistic effects with taxol and tamoxifene[END_REF][START_REF] Ottewell | Differential effect of doxorubicin and zoledronic acid on intraosseous versus extraosseous breast tumor growth in vivo[END_REF]. The question, therefore, addressed in this study was whether the combination of a bisphosphonate with a dose of a chemotherapeutic that has no effect on tumour growth when given alone, might act synergistically on tumour growth in vivo. Our results showed that a dose of docetaxel that affected tumour growth minimally, when dosed alone, had a profound effect on the growth of breast cancer cells in bone when dosed in combination with risedronate. In all but one of the treated animals with risedronate and the lower dose of docetaxel tumour cells were completely eliminated from bone. Thus, the combined treatment did not only preserve the structural integrity of bone but had a clear antitumour effect demonstrated both histologically and by BLI. Interestingly, trabecular bone of the animals treated with risedronate and docetaxel appeared to be better preserved than the bone of the animals which received the higher docetaxel dose. This should be attributed to the specific action of the bisphosphonate on bone.

In conclusion, bisphosphonates and chemotherapeutics act synergistically to protect bone and decrease tumour burden in an animal model of established bone metastases from breast cancer cells. This approach warrants further investigation in animal and human studies, as it may allow the use of less toxic dose of chemotherapeutics in the management of patients with bone metastases.

  E. R. Beek, C. W. G. M. Lowik, J. Wijngaarden, F. H. Ebetino, S. E. Papapoulos To cite this version: E. R. Beek, C. W. G. M. Lowik, J. Wijngaarden, F. H. Ebetino, S. E. Papapoulos. Synergistic effect of bisphosphonate and docetaxel on the growth of bone metastasis in an animal model of established metastatic bone disease. Breast Cancer Research and Treatment, 2008, 118 (2), pp.307-313. 10.1007/s10549-008-0236-6. hal-00478279

Fig. 1

 1 Fig. 1 Schematic representation of the treatment protocol. The left tibial bones of nude mice were injected with MDA-231-B/luc? cells (''day 0'') and 2 days later treatment with i.p. injections of bisphosphonate (5 times/week), docetaxel (2 times/week) or the combination was started. The mice were treated for a total of 35 days

Fig. 2 a

 2 Fig. 2 a BLI measurements of tumor growth in control mice, monitored weekly during the 5-week experimental period. Results are expressed as individual mouse values. b Representative bioluminescent images of a control mouse at week 1, 3, and 5 after intraosseous inoculation of MDA-231-B/luc? cells in the tibial bone

Fig. 3 a

 3 Fig. 3 a Representative radiographic, histological and bioluminescent images of a control, zoledronate (150 lg/kg) and risedronate (150 lg/kg) treated mouse after 5 weeks of treatment. b Effect of zoledronate (37.5, 75, and 150 lg/kg) and risedronate (150 lg/kg) on tumor growth after 5 weeks of treatment, monitored by BLI measurement

Fig. 4 a

 4 Fig. 4 a Effect of docetaxel (2, 4, and 8 mg/kg, 2 times/week) on tumor growth after 5 weeks of treatment, monitored by BLI measurement. b Histology of the tibiae of a control and a docetaxel (8 mg/kg) treated animal

Fig. 5 a

 5 Fig. 5 a Effect of risedronate (150 lg/kg, 5 times/week) and docetaxel (4 mg/kg, 2 times per/week) treatment, alone or in combination, on tumor growth after 5 weeks of treatment, monitored by BLI measurement. Difference among groups P \ 0.001 (one-way ANOVA); combination therapy (P \ 0.001) and docetaxel (P \ 0.05) different from risedronate alone; combination therapy different from docetaxel alone (P \ 0.05). b Representative bioluminescent, radiographic and histological images of a control, risedronate (150 g/kg), docetaxel (4 mg/kg) and risedronate ? docetaxel treated mouse after 5 weeks of treatment

Breast Cancer Res Treat (2009) 118:307-313

Acknowledgment The study was supported by a grant from Procter & Gamble Pharmaceuticals.