PhD Sara Gutiérrez-Enríquez

Teresa Ramón

Carmen Cajal

Anna Alonso

Pablo Corral

Mónica Carrasco

Judith Cornet

Montserrat Sanz

Montserrat Ribas

Orland Baiget

Diez

Teresa Ramón Y Cajal

Carmen Alonso

Anna Corral

Pablo Carrasco

Mónica Cornet

Judith Sanz

Montserrat Baiget

Orland Diez

Ionizing radiation or mitomycin-induced micronuclei in lymphocytes of or mutation carriers

Keywords: in vitro radiation, mitomycin c, micronuclei, BRCA1 and BRCA2

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

INTRODUCTION

Heterozygous germline mutations in BRCA1 or BRCA2 tumour suppressor genes (BRCA1/2) confer susceptibility to breast (BC) and ovarian cancers (OC) with high penetrance, and account for approximately 20% of BC cases associated with family history [reviewed in ref . 1].

BRCA1/2 genes are important in the maintenance of genome stability. The main role of BRCA2 appears to involve regulation of the RAD51 function in DNA repair by homologous recombination. BRCA1 is an E3 ubiquitin ligase that has a broader role upstream of BRCA2, participating in DNA repair, transcriptional regulation, cell cycle progression and meiotic sex chromosome inactivation [reviewed in refs. [START_REF] Boulton | Cellular functions of the BRCA tumour-suppressor proteins[END_REF][START_REF] Gudmundsdottir | The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability[END_REF]. BRCA1 -/- or BRCA2 -/-cells from chicken DT40 knockout cell lines, knockout mice and tumourderived human cell lines exhibit spontaneous chromosomal instability and are hypersensitive to DNA-damaging agents such as ionising radiation (IR) and DNA crosslinking agents [START_REF] Boulton | Cellular functions of the BRCA tumour-suppressor proteins[END_REF][START_REF] Gudmundsdottir | The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability[END_REF].

In contrast, whether some functions of the BRCA1/2 proteins are diminished or deregulated while still heterozygous or only after the loss of both functional alleles is not yet well established [START_REF] Cousineau | BRCA1 haploinsufficiency, but not heterozygosity for a BRCA1-truncating mutation, deregulates homologous recombination[END_REF][START_REF] Venkitaraman | Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment[END_REF]. The heterozygous status (BRCA1 +/-or BRCA2 +/-) might contribute to the impairment of genomic stability and increase the risk of cancer promoting mutations, contributing to the loss of the remaining wild-type BRCA1/2 allele [START_REF] Cousineau | BRCA1 haploinsufficiency, but not heterozygosity for a BRCA1-truncating mutation, deregulates homologous recombination[END_REF][START_REF] Venkitaraman | Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment[END_REF][START_REF] Arnold | Lower level of BRCA2 protein in heterozygous mutation carriers is correlated with an increase in DNA double strand breaks and an impaired DSB repair[END_REF]. Since BRCA1 and BRCA2 proteins are involved in DNA repair, BRCA1 +/- or BRCA2 +/-cells of mutation carriers may also have enhanced sensitivity to DNAdamaging agents, such as ionising radiation (IR) or mitomycin C (MMC); this phenotype may increase toxicity and the carcinogenic risk of chemotherapy, radiotherapy and mammography screening in BRCA1 or BRCA2 mutation carriers [START_REF] Cousineau | BRCA1 haploinsufficiency, but not heterozygosity for a BRCA1-truncating mutation, deregulates homologous recombination[END_REF][START_REF] Venkitaraman | Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment[END_REF]. If the functions of the BRCA1/2 proteins are affected by heterozygosity status, two mechanisms may explain this phenotype: haploinsufficiency or reduction in gene dosage, and/or dominant negative mutations. Several studies have claimed the existence of phenotypes associated with BRCA1/2 haploinsufficiency, whereas others have raised the possibility that BRCA1 or BRCA2 mutated proteins may block the function of the remaining wild-type BRCA1/2 allele [reviewed in ref . 4].

Several studies in blood lymphocytes to know whether the heterozygosity for BRCA1/2 mutations confers detectable sensitivity to genotoxic agents have been performed with contradictory results [reviewed in ref . 7]. Here, we have studied chromosomal sensitivity to ionising radiation or MMC treatments of peripheral blood lymphocytes from BRCA1 or BRCA2 germ-line mutation carriers with or without BC, familial BC patients without detected mutation in BRCA1/2, and controls with or without BC and no familial history of cancer. The chromosomal damage was measured by quantification of micronuclei (MN) and nucleoplasmic bridges (NPBs) using the cytokinesis-block micronucleus assay (CBMN). The in vitro proliferation capacity of the treated lymphocytes was also evaluated by the nuclear division index (NDI).

The MN in dividing cells are the result of fragments or whole acentric chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. These fragments can be originated from non-repaired or misrepaired DNA double-strand breaks (DSBs) [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. Misrepair of DSBs could also lead to the formation of asymmetrical chromosome rearrangements producing dicentrics or ring chromosomes and acentric fragments. The centromeres of the dicentric chromosomes, dicentric ring chromosomes or concatenated double rings can be pulled to opposite poles of the cells at anaphase resulting in the formation of a nucleoplasmic bridges (NPB) between the daughter nuclei [START_REF] Thomas | Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay[END_REF]. Therefore, the presence of MN or NPB could indirectly reflect the cell DNA repair capacity [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. In fact, increased frequencies of spontaneous and mutagen induced MN or NPBs have been observed in different cells deficient in DNA repair mechanisms such as non-homologous DNA end-joining [START_REF] Fell | Role for non-homologous end-joining in the repair of UVA-induced DNA damage[END_REF] or homologous recombination [START_REF] Ouyang | Loss of Ubr2, an E3 ubiquitin ligase, leads to chromosome fragility and impaired homologous recombinational repair[END_REF][START_REF] Yue | The cytoskeleton protein filamin-A is required for an efficient recombinational DNA double strand break repair[END_REF].

The cytokinesis-block micronucleus (CBMN) assay is the preferred method for measuring MN in cultured human cells because scoring is specifically restricted to once-divided binucleated (BN) cells, which are the cells that can show MN [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. The inhibition of cytokinesis by cytochalasin B allows to discriminate between cells that did not divide after treatment and cells that did divide, thus preventing the confounding effects caused by differences in cell division kinetics [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. Because cells are blocked in the BN stage, it is also possible to measure nucleoplasmic bridges [START_REF] Thomas | Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay[END_REF]. In addition to micronuclei in binucleated cells and NPB, the CBMN assay also allows for scoring of other critical events, such as cell division and cell cytotoxicity by scoring of proportion of mono-, bi-and multicucleated cells to calculate the nuclear division index (NDI) [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. To date the CBMN assay is one of the best-validated methods for measuring chromosome damage in human lymphocytes [START_REF] Fenech | The lymphocyte cytokinesis-block micronucleus cytome assay and its application in radiation biodosimetry[END_REF].

MATERIALS AND METHODS

Subjects and study design

Heparinized blood samples were collected from 76 women between 2004 to 2007: 60 with a family history of BC and OC and 16 without a family history of cancer. The ethical committee of the hospital approved the study and all women participating signed an informed consent.

Women with a family history of cancer were recruited from those attending the Cancer Genetics Clinic of Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Subjects were referred for genetic assessment because of familial BC/OC, early onset BC or previous identified BRCA1/2 mutation in the family. The mutation analysis of the BRCA1 and BRCA2 genes was performed in women fulfilling one of the following criteria: families with one or more cases of BC (at least 1 case diagnosed before age 50) and 1 or more cases of OC; site-specific female BC families with 2 or more cases (at least 1 diagnosed at 50 years of age); families with at least 1 case of BC or OC in addition to at least 1 male case of BC.

The entire coding sequence of BRCA1 and BRCA2 was analyzed for sequence variants using denaturing high-performance liquid chromatography (DHPLC) and for large deletions and duplications using Multiplex Ligation-dependent Probe Amplification (MLPA) (MRC Holland). In those women referred due to a BRCA1/2 mutation previously identified in the family, the analysis was performed by direct sequencing or MLPA.

The mutational status of the 60 individuals with family history was: 21 BRCA1 mutation carriers (12 with BC and 9 healthy), 24 BRCA2 mutation carriers (13 with BC and 11 healthy) and 15 females with familial BC/OC without detected mutation in BRCA1/2 (14 with BC and 1 with OC). The BRCA1/2 mutations are shown in Table 1.

As controls, blood samples were also collected from 16 women without family history of BC/OC: five with sporadic BC and 11 healthy women. The sporadic BC patients came from those attending the Medical Oncology Department and the healthy subjects were recruited from the laboratory staff of the Genetics Department of the Hospital.

Considering the disease status of the analysed subjects, 31 were healthy and 45 had BC/OC. Most of the BC/OC patients (n=31) had received different combinations of adjuvant chemotherapy: cyclophosphamide, methotrexate and 5-fluorouracil (CMF) (n=7); 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n=6); adriamycin and cyclophosphamide (n=3); FEC in a high-dose chemotherapy regimen (with autologous transplantation) (n=2); FEC, trastuzumab and taxanes (n=2); CMF, adriamycin, taxol and capecitabine (n=2). The rest of patients (n=9) received individually different regimens each: FEC, taxanes and carboplatin; cyclophosphamide, adriamycin and taxol; FAC (5-fluorouracil, adriamycin and cyclophosphamide); FEC and taxanes; FAC and taxanes; FEC and CMF; CMF, MMC and vinblastine; anthracyclines; cyclophosphamide. Four patients required neo-adjuvant chemotherapy (2 patients with CMF, 1 with FEC and 1 with epirubicin and doxorubicin). Three patients required chemotherapy because of BC recurrence (one patient was treated with CMF, one with CMF, MMC and vinblastine, and one with MMC and vinblastine).

Culture and treatment with mutagenic agents

Peripheral blood cultures were set up by adding 0.5 mL of whole blood to 4.5 mL of RPMI 1640 medium (PAA Laboratories GmbH, Pasching, Austria) supplemented with 15% heat-inactivated foetal calf serum (PAA Laboratories GmbH), 1% antibiotics (penicillin and streptomycin), 1% L-glutamine (provided by Gibco, Invitrogen Corporation, Paisley, United Kingdom) and 1% phytohaemagglutinin (PHA) to stimulate the division of T-lymphocytes (Gibco). Six cultures were set up for each subject immediately after drawing blood: 2 that will be treated in the G 0 phase with 2 Gy of gamma irradiation (before incubation at 37ºC with PHA), 2 that will be treated in the G 1 /S phase with 0.05 g/ml of MMC (24h after the beginning of the culture with PHA) and 2 replicates without any treatment.

Irradiation of the two respective cultures was done just after re-suspending the blood with the supplemented RPMI 1640 medium. All irradiations were performed at room temperature with 2 Gy of gamma irradiation from a source of Co-60 (Theratron 780 radiotherapy unit, Theratronics Ltd., Canada) at a high dose rate (HDR) of 0.6 -0.8 Gy/min. To minimize the dose gradient within the tubes, the cultures were irradiated at a large distance (80 cm from the cobalt source). A polymethyl methacrylate (PMMA) scattering slab was placed on the irradiated tubes to minimize the build-up effect and to guarantee uniform irradiation.

Immediately after irradiation the 6 cultures per individual were incubated at 37ºC. After 24h of incubation and stimulation with PHA, mitomycin-C (Sigma, St. Louis, Missouri, USA) was added to the 2 corresponding cultures to obtain a final concentration of 0.05 µg/mL and cultures were returned to the incubator.

After 44 h of incubation with PHA at 37ºC, cytochalasin-B (Cyt-B, Sigma, St. Louis, Missouri, USA) was added to the 6 cultures to a final concentration of 6 µg/mL to arrest cytokinesis. Seventy-two hours after incubation with PHA at 37ºC the cells were centrifuged (800 rpm for 8 min at room temperature), the supernatant was aspirated, and the cells were re-suspended in a hypotonic solution (0.075 M KCl) for 3 min at 4°C. The cells were re-centrifuged, and a 3:1 (v/v) methanol:acetic acid solution gently added. This fixation step was repeated twice and the cells were then re-suspended in a small volume of fixative solution and dropped onto clean slides. The air-dried slides were subsequently stained with 1 g/mL of 4',6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, Missouri, USA) and 2 g/mL of propidium iodide (PI; Sigma) in an antifade solution (Vectashield; Vector Laboratories, Burlingame, CA, USA). The slides were stored at 4°C until assessed by microscopy. All slides were randomized and coded before being scored by the same observer to minimize variability. The slides were scored under the 100x objective of a Nikon Eclipse E400 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a triple-band-pass filter to visualize the nuclei and MN, in bright blue (DAPI), and the cytoplasm, in red (PI). The presence of MN was confirmed using the filter to view only the DAPI stain.

To determine the presence of both micronuclei (MN) and nucleoplasmic bridges (NPB), a total of 1000 binucleated cells with well-preserved cytoplasm for each subject (500 per replicate) were blind scored on coded slides according to previously described criteria [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. In addition, a total of 500 lymphocytes (250 cells per replicate) were examined to determine the proportion of cells with different numbers of nuclei (1-4 nuclei) in order to calculate the nuclear division index (NDI) [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF].

Statistical methods

In the untreated cultures, the outcome variables analyzed were the basal values of MN, NPB and NDI. In the treated cultures, the cytogenetic endpoints evaluated were the number of radiation or mitomycin-C-induced MN (calculated by subtracting the basal yields from the yields obtained in the treated samples), the absolute values of NPB after the mutagenic treatments and, the NDI index values in untreated cultures less NDI values in treated cultures.

The analysis included 2 factors: carrier/non-carrier, with 4 levels (BRCA1 carrier, BRCA2 carrier, familial BC without detected mutations in BRCA1/2, and controls without familial history of BC/OC) and, disease with 2 levels (healthy women or cancer patients groups). Potential confounding factors of MN were also considered.

Age at time of experiments, smoking habit, and number of cigarettes were recorded for all subjects. Given that the blood samples were not obtained concurrently and that experimental conditions may have varied between groups, the days elapsed since the first blood culture were calculated (as a centered variable) for each analyzed subject. For cancer patients the age at cancer diagnosis and the years elapsed between the end of radiotherapy and chemotherapy and time of MN cultures were also recorded.

Basic descriptive statistics of outcome and continuous variables were computed for all subgroups. The means for between-group demographic and clinical variables were compared using the t-test. To quantify the potential association among continuous and outcome variables, correlation matrices analyses were performed using the Pearson correlation coefficient.

The between-group differences in basal and treatment-induced MN, NPB and NDI were analyzed using generalized linear models (GLZ). A logarithmic link function was applied to the basal level of MN and NPB to achieve a best fit. The continuous variables that were shown by the correlation analysis to have a possible effect were included in the analyses. In the GLZ analysis of NPB after mutagens treatment, the basal NPB values were considered as a covariable since the treatment-induced NPB values, obtained by subtracting the basal yields from yields obtained after treatment, encompassed negative values which would be difficult to adjust in the GLZ analyses. A backward selection method was used and potential confounding factors that did not reach statistical significance were not considered in the final model. Post hoc comparisons using the sequential Bonferroni correction for multiple comparisons were also carried out.

The figures illustrating the between-group differences in this study show the estimated marginal means, which correspond to the mean adjusted for the other terms in the model.

For all analyses, differences were regarded as statistically significant at P < 0.05. Statistical computations were performed using the SPSS v.15.0 software (SPSS, Chicago, IL, USA) and the STATISTICA v.8.0 statistical software package (StatSoft, Tulsa, OK, USA).

RESULTS

Demographic, clinical and experimental factors

The mean value of age, days elapsed since first experiment as well as the clinical characteristics of the cancer patient groups are presented in Table 2. The group of all healthy women was significantly younger than the overall group of cancer patients (P<0.001). The peripheral blood cultures for cancer patients were established earlier than the cultures for healthy women and this difference in time, measured as the days elapsed since the first blood culture, was statistically significant (P<0.05). The blood cultures of BRCA2 mutation carriers (with and without BC) were set up significantly earlier than the cultures of control group (BC patients and healthy women without familial history of BC) (P<0.01). The cultures of familial BC patients negative for BRCA1/2 mutations were established earlier than the cultures of the all other groups and this difference in time is statistically significant (P<0.001). The average of the years elapsed since last chemotherapy and radiotherapy sessions in the group of familial BC patients that had no detected mutation in BRCA1/2 is lower, although not statistically significant, than the average of the other BC patient groups.

Basal levels of MN, NPB and NDI

Table 3 shows the data (Mean ± SD) for the 3 endpoints evaluated in the untreated lymphocytes from all groups. The analysis of correlation matrices revealed higher basal MN levels when less time had elapsed since the last session of either chemotherapy or radiotherapy in BC patients. The decline in MN yield with time after cancer treatment showed a reasonably good fit to an inverse relationship (r= 0.799, P<0,001 and r=0.725, P<0.001 for chemotherapy and radiotherapy, respectively) (Fig. 1A). The mean lifetime of mature human T lymphocytes varies from 1.5-10 years [reviewed in ref . 14]. Therefore, the data showed in the Figure 1A are probably reflective of dying off of T lymphocytes with MN that were formed during the time of chemotherapy/radiotherapy. Given that the years elapsed since chemotherapy and radiotherapy were highly correlated, only the variable of chemotherapy was included in the subsequent analyses because it had a higher correlation coefficient with the basal MN level than the radiotherapy variable. The chemotherapy-years variable was inverse transformed in order to achieve a linear distribution. This allowed the assignment of a 0 value to the untreated healthy individuals, thereby including them in the analysis.

The results of the GLZ final model indicated that both cancer status (P=0.014) and the years since last sessions of chemotherapy (P<0.001) had a significant effect on the basal level of MN. Thus, the cancer patients showed higher basal MN frequencies than the healthy subjects irrespective of carrier status (20.16 versus 13.73 as adjusted means for cancer and healthy groups, respectively; P= 0.007) (Fig. 1B). The significant effect of the time since chemotherapy on basal MN indicates that the frequency of MN is higher when less time had elapsed since cancer therapy.

The GLZ analysis of basal NPB showed significant effects of cancer (P=0.027) and carrier status (P=0.018). The NPB in cancer patients were significantly higher than in healthy subjects (1.76 versus 0.81 as adjusted means for cancer and healthy groups, respectively; P= 0.025) (Fig. 1C). The carrier status affected the NPB basal level, since the levels of NPB in untreated cultures in both BRCA2 carriers (adjusted mean, 0.80) and familial-BC group without detected mutation in BRCA1/2 (0.63) were lower than the levels found in BRCA1 carriers (1.81) and in non-carrier controls (2.22) (Fig. 2); nevertheless, the differences between the estimated marginal means, evaluated using the Bonferroni post-hoc test, did not attain statistical significance.

In terms of NDI levels in the cultures without any treatment, the analysis of the GLZ final models indicated no differences between healthy and cancer groups or between mutation carriers and non-carriers. However, a slight non-significant increase (P= 0.062) in the basal NDI was observed in the group of BRCA2 carriers (adjusted mean, 1.85) compared to that in the BRCA1 (1.79), BRCA1/2 negative (1.75), and noncarrier control (1.77) groups. The final analysis model also showed that both subject age and time elapsed since chemotherapy or radiotherapy significantly affected the proliferation index values. Thus, lower NDI values appeared in older individuals (P= 0.002), and in those with less time elapsed since cancer treatment (P=0.006).

Effect of the radiation treatment in the G 0 phase

Table 4 shows the mean values of MN and NDI induced by radiation and the absolute values of NPB obtained after radiation treatment. The final model of the GLZ analysis did not indicate any effect of cancer or carrier status on MN and NPB after radiation with 2 Gy. Only days elapsed since first blood culture significantly affected both measures. The values of radioinduced MN were higher at the time of final experiments than those at the time of first experiments (P=0.002), and the frequency of NPB also increased with experimentation time (P<0.001).

To evaluate whether in vitro treatment with IR produced alterations in the capacity of cells to divide, we used the NDI value obtained by subtracting the values obtained after treatment from those obtained in the untreated cultures. The GLZ evaluation to assess the delay in the proliferation capacity after IR did not show any significant effect of carrier status, disease status or any confounding factor.

Effect of the mitomycin-C (MMC) treatment in the G 1 /S phase

Table 5 shows the means of MN and NDI induced by MMC treatment and the values of NPB obtained after MMC treatment. The GLZ final models showed that carrier status had a significant effect on MMC-induced MN (P<0.001). The lymphocytes of BRCA2 mutation carriers (irrespective to their disease status) presented higher levels of MN after exposure to MMC (adjusted mean, 135.48) than those of BRCA1 carriers (96.39), and non-carrier controls (88.45) (P<0.001) (Fig. 3). The MMCinduced MN of BRCA2 carriers was also higher (135.48) than that of familial cancer patients without detected mutation in BRCA1/2 (111.63), but this difference was not statistically significant (Fig. 3). Furthermore, the frequency of MMC-induced MN increased with the continuous variable of days elapsed since the first blood extraction (P<0.001). The NPB obtained after MMC treatment was not significantly affected by carrier and cancer status or by any covariable.

The evaluation of between-group differences after adjusting the values of MMCinduced NDI for the different confounding factors did not show any significant effect of carrier or disease status on cell proliferation capacity after adding MMC to the cultures. The NDI yielded by MMC was only significantly correlated to the years elapsed since treatment for cancer (P=0.010). This correlation indicated a lower effect of MMC on cell proliferation with less time elapsed since the last cancer treatment. This apparent resistance to MMC in lymphocytes from subjects just treated with chemotherapy or radiotherapy could be explained by the fact that the cellular proliferating potential has been surpassed by the already lower cell proliferation, observed in the non-treated cultures, in subjects after cancer treatments and the high cytostatic effect of MMC (which is stronger than that of the radiation treatment; see NDI values in Tables 4 and5).

DISCUSSION

Chromosomal sensitivity (CRS) to radiation and chemical mutagens, quantified by G 0 MN or G 2 chromosome aberrations assays, has been proposed as a marker for low-penetrance predisposition to several common cancers including breast cancer [START_REF] Speit | Chromosomal mutagen sensitivity associated with mutations in BRCA genes[END_REF][START_REF] Baria | Chromosomal radiosensitivity as a marker of predisposition to common cancers?[END_REF][START_REF] El-Zein | Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer risk[END_REF][START_REF] El-Zein | Cytokinesisblocked micronucleus cytome assay biomarkers identify lung cancer cases amongst smokers[END_REF]. Several independent studies have shown enhanced in vitro sensitivity to the chromosome-damaging effects of ionizing radiation and other mutagens in a significant number of BC patients compared to normal healthy controls [START_REF] Baeyens | Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition[END_REF][START_REF] Burrill | Heritability of chromosomal radiosensitivity in breast cancer patients: a pilot study with the lymphocyte micronucleus assay[END_REF][START_REF] Parshad | Deficient DNA repair capacity, a predisposing factor in breast cancer[END_REF][START_REF] Roberts | Heritability of cellular radiosensitivity: a marker of low-penetrance predisposition genes in breast cancer?[END_REF][START_REF] Scott | Increased chromosomal radiosensitivity in breast cancer patients: a comparison of two assays[END_REF]. It has been also reported that the enhanced CRS to different mutagens is a heritable trait [START_REF] Burrill | Heritability of chromosomal radiosensitivity in breast cancer patients: a pilot study with the lymphocyte micronucleus assay[END_REF][START_REF] Roberts | Heritability of cellular radiosensitivity: a marker of low-penetrance predisposition genes in breast cancer?[END_REF][START_REF] Wu | Mutagen sensitivity has high heritability: evidence from a twin study[END_REF].

The studies that have investigated CRS to mutagens in blood lymphocytes from patients with germline mutations in BRCA1 or BRCA2 have yielded inconsistent results [START_REF] Baeyens | Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition[END_REF][START_REF] Baeyens | Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers[END_REF][START_REF] Baria | Induced micronucleus frequencies in peripheral blood lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families[END_REF][START_REF] Barwell | Lymphocyte radiosensitivity in BRCA1 and BRCA2 mutation carriers and implications for breast cancer susceptibility[END_REF][START_REF] Buchholz | Evidence of haplotype insufficiency in human cells containing a germline mutation in BRCA1 or BRCA2[END_REF][28][29][30][31][32][33][34][35]. While it has been well documented that biallelic inactivation of BRCA1 o BRCA2 (BRCA1 -/-or BRCA2 -/-) genes leads to repair deficiency, and hence to significantly enhanced CRS after treatment with different mutagens, the situation in heterozygous cells (BRCA1 +/-or BRCA2 +/-) remains elusive.

In our study we assessed the chromosome sensitivity of BRCA1 +/-or BRCA2 +/- lymphocytes to ionising radiation or mitomycin-C treatment measuring MN and NPB with the MN G0 phase assay. Our results showed that, in both cancer and healthy women, heterozygosity for BRCA1/2 mutations did not associated with an increased sensitivity to ionising radiation exposure in the G 0 phase. This result contrasts with the findings in lymphocytes heterozygote for BRCA1/2 mutations showing evidence of an enhanced MN radiation induction [START_REF] Speit | Chromosomal mutagen sensitivity associated with mutations in BRCA genes[END_REF][START_REF] Baeyens | Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition[END_REF][32][33][34][35]. However, other studies have not reported a significant increase of MN after radiation in BRCA1/2 mutated lymphocytes [START_REF] Baeyens | Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition[END_REF][START_REF] Baeyens | Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers[END_REF]30]. These inconsistent results may be due to small population sizes, heterogeneous reference groups and different techniques used for both irradiation and evaluation of MN [START_REF] Speit | Chromosomal mutagen sensitivity associated with mutations in BRCA genes[END_REF]36].

We found that the frequency of radiation-induced MN as well as the MN induced by MMC increase with the days elapsed since first blood culture. This could not be related to a change in experimental conditions or to reported sources of MN variability such as storage time of the blood samples [37,38] and differences between scorers [39]. To reduce the inter-test variability, a direct comparison of cases with concurrent controls and/or automated counting by image analysis has been suggested [START_REF] Speit | Chromosomal mutagen sensitivity associated with mutations in BRCA genes[END_REF]32].

The lack of increased radiosensitivity of BRCA1 +/-or BRCA2 +/-lymphocytes shown in our study may also suggest that the BRCA1/2 proteins do not play a role in the repair mechanisms acting on the radiation-induced DNA damage in the G 0 /G 1 phase such as non-homologous DNA end-joining (NHEJ). NHEJ reseals DNA double-strand breaks (DSBs) efficiently throughout all cell cycle phases but especially in the G1 phase [START_REF] Burma | Role of non-homologous end joining (NHEJ) in maintaining genomic integrity[END_REF]. Homologous recombination (HR), the other mechanism that the cell employs to repair radiation induced DSBs, acts in the S and G 2 phase when a duplicated copy of DNA is available [START_REF] Frankenberg-Schwager | Single-strand annealing, conservative homologous recombination, nonhomologous DNA end joining, and the cell cycle-dependent repair of DNA double-strand breaks induced by sparsely or densely ionizing radiation[END_REF][START_REF] Koch | Impact of homologous recombination on individual cellular radiosensitivity[END_REF]. Despite some studies have described that BRCA1-deficient cells had significantly reduced NHEJ activity, it is not definitively clear whether BRCA1 or BRCA2 are required for efficient NHEJ of radiation-induced DNA breaks [START_REF] Baldeyron | A single mutated BRCA1 allele leads to impaired fidelity of double strand break end-joining[END_REF][START_REF] Bau | The role of BRCA1 in non-homologous end-joining[END_REF]. In contrast, it is well known the role of BRCA1/2 in the functioning of HR pathway [START_REF] Boulton | Cellular functions of the BRCA tumour-suppressor proteins[END_REF][START_REF] Gudmundsdottir | The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability[END_REF].

Given that HR is the most important mechanism for the repair of DSBs induced in the S and G 2 phases [START_REF] Frankenberg-Schwager | Single-strand annealing, conservative homologous recombination, nonhomologous DNA end joining, and the cell cycle-dependent repair of DNA double-strand breaks induced by sparsely or densely ionizing radiation[END_REF][START_REF] Koch | Impact of homologous recombination on individual cellular radiosensitivity[END_REF], impaired repair of radiation-induced DSBs may be expected at these phases in lymphocytes of BRCA1/2 mutation carriers. When analysing the metaphases after irradiation at S phase Barwell et al. [START_REF] Barwell | Lymphocyte radiosensitivity in BRCA1 and BRCA2 mutation carriers and implications for breast cancer susceptibility[END_REF] found that the lymphocytes of BRCA1/2 mutation carriers without cancer had increased chromosome breaks compared to age-matched unaffected controls. Blood cells of BRCA1 mutation carriers irradiated with 8 Gy 24 hours after stimulation and cultured 6 days following irradiation exhibited a significantly higher level of chromosomal damage than those of non-carrier controls [29]. However, using the G 2 assay, in which metaphases are analysed after irradiation at the G 2 phase of the cell cycle, some authors have described elevated levels of chromosomal aberrations in BRCA1/2 lymphocytes [START_REF] Baeyens | Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition[END_REF][START_REF] Barwell | Lymphocyte radiosensitivity in BRCA1 and BRCA2 mutation carriers and implications for breast cancer susceptibility[END_REF][START_REF] Buchholz | Evidence of haplotype insufficiency in human cells containing a germline mutation in BRCA1 or BRCA2[END_REF][28][START_REF] Ernestos | Increased chromosomal radiosensitivity in women carrying BRCA1/BRCA2 mutations assessed with the G2 assay[END_REF], while other authors have not [START_REF] Baeyens | Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers[END_REF][START_REF] Baria | Induced micronucleus frequencies in peripheral blood lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families[END_REF].

Knowing whether the normal cells of BRCA1/2 mutation carriers exhibit a phenotype of in vitro enhanced radiosensitivity has implications for their cancer screening and treatment using modalities that involve irradiation. Epidemiological studies that have examined whether BRCA1/2 mutation carriers have an increased risk of developing BC related to screening mammography have yield inconsistent results [reviewed in ref . 46]. Clinical studies of BC women with BRCA1/2 mutations have not demonstrated increased acute or late toxicity after radiotherapy [START_REF] Shanley | Late toxicity is not increased in BRCA1/BRCA2 mutation carriers undergoing breast radiotherapy in the United Kingdom[END_REF]. However, Broeks et al. [START_REF] Broeks | Identification of women with an increased risk of developing radiation-induced breast cancer: a case only study[END_REF] suggested that pathogenic alleles in BRCA1/2, CHEK2 or ATM genes increase the risk of radiation-induced contralateral BC after radiotherapy for a first BC. It is clear that more and larger studies are warranted to determine the in vitro and in vivo radiosensitivity phenotype of BRCA1/2 normal cells.

In contrast to the lack of increased in vitro sensitivity to radiation observed in our study, BRCA2 mutations confer enhanced mitomycin-C susceptibility on blood lymphocytes of both cancer and healthy women (Fig 3). Hence, the heterozygosity status for BRCA2 mutations probably affects the DNA repair capacity for MMCinduced damage in the G 1 /S phase of lymphocytes. To our knowledge, there are only 2 published studies on the effects of MMC on normal cells heterozygous for BRCA1/2 . Shorrocks et al. [START_REF] Shorrocks | Primary fibroblasts from BRCA1 heterozygotes display an abnormal G1/S cell cycle checkpoint following UVA irradiation but show normal levels of micronuclei following oxidative stress or mitomycin C treatment[END_REF] found no differences in MN response between BRCA1 +/-and +/+ fibroblasts. Arnold et al. [START_REF] Arnold | Lower level of BRCA2 protein in heterozygous mutation carriers is correlated with an increase in DNA double strand breaks and an impaired DSB repair[END_REF] measured by the Comet assay the repair of MMC-induced crosslinks and observed no clear differences in repair capacity between BRCA2+/-and BRCA2 +/+ lymphoblastoid cell lines.

Although the repair of MMC-induced DNA damage in mammals has not been fully delineated, MMC induces several types of DNA damage that are repaired by multiple repair mechanisms such as nucleotide excision repair, HR and trans-lesion bypass repair [reviewed in ref . 50]. Under the experimental conditions used in our study, our results indicate a relevant role for BRCA2 in the repair of MMC-induced lesions in the G 1 /S phase but not for BRCA1. In this regard Venkitaraman [START_REF] Venkitaraman | Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment[END_REF] suggested that defects in DSBs repair in BRCA1/2 mutated cells arise from distinct roles for BRCA1 or BRCA2 in HR. The major role of BRCA2 protein in DSB repair is the regulation of HR, acting as a mediator of interactions with RAD51 protein, while the functions of BRCA1 in DNA repair are not restricted to HR, performing varied functions in the cellular response to DNA breakage or replication arrest.

Our results with MMC may concur with those of some studies reporting high sensitivity to in vitro and in vivo MMC treatment of a pancreatic cancer cell line defective in BRCA2 [START_REF] Van Der Heijden | In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor[END_REF] or the response to a third-line MMC-based chemotherapy of a metastasic pancreatic adenocarcinoma in a BRCA2 carrier patient [START_REF] Chalasani | Response to a third-line mitomycin C (MMC)-based chemotherapy in a patient with metastatic pancreatic adenocarcinoma carrying germline BRCA2 mutation[END_REF]. Moreover, treatment of blood lymphocytes with MMC may be used to distinguish BRCA2 mutation carriers and identify new BRCA2 interacting genes.

The endpoint of nucleoplasmic bridges (NPB) is an indicator of DNA misrepair and/or telomere end-fusions [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. With regard to this endpoint, the only significant finding we observed in our study was that BRCA2 carriers, irrespective of their cancer status, and the familial BC patients without detected mutation in BRCA1/2 had lower basal levels of NPB compared with those of BRCA1 and non-carrier control groups (Fig 2 ). A similar trend was also observed by Beetstra et al. [53], who found that, in folate deficient medium cultures, the lymphocytes of BRCA2 mutation carriers had a significantly lower frequency of NPB compared with controls. These authors suggested that the lower number of NPB may be due to an extended delay in cell division of BRCA2 carriers. An extended cell division would allow more time for the bridges to break during anaphase prior to cell harvesting. Beetstra et al. [53] based their hypothesis on data obtained by Daniels et al. [54], who reported that the period taken for cells to progress from anaphase onset to completion of cell division is significantly extended in cells with targeted gene disruption of BRCA2 or reduced transcription of BRCA2 by RNA interference when compared with controls. Likewise, Jonsdottir et al. [55] have demonstrated that BRCA2 mutation carrier fibroblasts had delayed cytokinesis, being the mean cell division time 6 min longer compared with BRCA2 wild type cells. However, in contrast to results from these two studies, Lekomtsev et al. [56] have recently indicated that BRCA2 does not regulate cytokinesis in human cells. Thus, further research is required to verify a role of BRCA2 in the regulation of cell division. Interestingly, in the familial BC patients without detected mutation in BRCA1/2 a decrease in the basal NPB formation was also detected. It is tempting to speculate that BRCA2 and other genes related to cell division might be associated with cancer susceptibility in familial BC patients negative for BRCA1/2 mutations.

It should be mentioned that the group of familial BC patients who tested negative for BRCA1/2 mutations did not present a different chromosomal sensitivity (CRS) to either radiation or MMC treatment compared with control non-carriers. These data do not support the findings of the studies carried out by Baeyens et al. [START_REF] Baeyens | Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition[END_REF][START_REF] Baeyens | Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers[END_REF]. They showed that the lymphocytes of non-BRCA1/2 familial BC patients had a higher CRS to radiation, measured by G 0 MN and G 2 assays, than the control group. Further research will be needed to more precisely determine the CRS in the group of familial BC with no BRCA1/2 mutations.

The nuclear division index (NDI), provides a measure of the proliferative status of the viable lymphocytes, indicating both the lymphocyte mitogenic response and the cytostatic effects of agents examined in the assay [START_REF] Fenech | Cytokinesis-block micronucleus cytome assay[END_REF]. We did not find any significant difference in either basal or mutagen-induced levels of this proliferation index between BRCA1/2 carriers and non carrier groups; this would indicate that the lymphocytes from heterozygous BRCA1/2 mutation carriers have no important defects in the mitogenic response or cellular growth in culture after mutagenic treatments. However, it is interesting to note that, although differences were not statistically significant, the BRCA2 carriers showed higher basal levels of NDI values than the other groups analysed. A similar finding has been reported by Beetstra et al. [53], who detected that the NDI of BRCA2 mutation carriers in lymphocytes cultured with folate deficiency tended to be higher than in controls. Thus the BRCA2 carriers may have a defect in cytokinesis that entails an increased sensitivity to the cytokinesis-blocking action of cytochalasin-B [53]. This may be attributed to an extended time for these cells to progress from anaphase onset to completed cell division, that allows the accumulation of binucleated cells in culture [54,55].

In conclusion, the BRCA1 or BRCA2 heterozygote status in peripheral blood lymphocytes is not related to an increased radiosensitivity. But the monoallelic BRCA2 mutations are associated with a higher level of chromosomal damage induced by MMC, probably due to an impaired DNA repair capacity. This finding suggests that the MMCinduced MN analysis may be used in the identification of individuals with a deficiency in BRCA2 or genes related to BRCA2. Given that MMC can be used as an anti-cancer drug, these data may be relevant for the management and follow-up of BRCA2 mutation carriers.

FIGURE CAPTIONS

FIG. 3

Adjusted means and 95% confidence limits illustrating the differences of MN levels after MMC treatment between carrier and non-carrier groups. BRCA negative: familial breast cancer patients without detected mutation in BRCA1 or BRCA2. Control non carriers: healthy women and sporadic BC patients without history of familial cancer.

FIG. 1

 1 FIG. 1 Effect of previous chemotherapy and radiotherapy of BC/OC patients on MN and NPB endpoints (A)Relationship between the basal values of MN of the cancer patients and the time passed since last session of chemotherapy. (B) Means adjusted for the other terms in the model and 95% confidence limits showing the differences of levels of MN in the non-treated cultures between cancer and healthy groups. (C) Means adjusted for the other terms in the model and 95% confidence limits illustrating the differences of basal levels of NPB in the non-treated cultures between cancer and healthy groups.

FIG. 2

 2 FIG.2Adjusted means and 95% confidence limits showing the differences of basal levels of NPB in the non-treated cultures between carrier and non-carrier groups. BRCA negative: familial breast cancer patients without detected mutation in BRCA1 or BRCA2. Control non carriers: healthy women and sporadic BC patients without history of familial cancer.

a

  Calculated by subtracting the spontaneous yields from the yields obtained in the treated samples. b Absolute values of NPB obtained in the cultures treated with radiation c NDI index values in untreated cultures less NDI values in treated cultures.

Table 1 .

 1 Description of BRCA1 and BRCA2 mutations amongst carriers with or without cancer

	28. Febrer E, Mestres M, Caballin MR, Barrios L, Ribas M, Gutierrez-Enriquez S,
	Alonso C, Cajal T, Francesc BJ (2008) Mitotic delay in lymphocytes from
	BRCA1 heterozygotes unable to reduce the radiation-induced chromosomal
	damage. DNA Repair (Amst) 7:1907-1911
	29. Kote-Jarai Z, Salmon A, Mengitsu T et al (2006) Increased level of
	chromosomal damage after irradiation of lymphocytes from BRCA1 mutation
	carriers. Br J Cancer 94:308-310
	30. Kotsopoulos J, Chen Z, Vallis KA, Poll A, Ainsworth P, Narod SA (2007) DNA
	repair capacity as a possible biomarker of breast cancer risk in female BRCA1
	mutation carriers. Br J Cancer 96:118-125
	31. Kowalska E, Narod SA, Huzarski T, Zajaczek S, Huzarska J, Gorski B, Lubinski
	J (2005) Increased rates of chromosome breakage in BRCA1 carriers are
	normalized by oral selenium supplementation. Cancer Epidemiol Biomarkers
	Prev 14:1302-1306
	32. Rothfuss A, Schutz P, Bochum S, Volm T, Eberhardt E, Kreienberg R, Vogel
	W, Speit G (2000) Induced micronucleus frequencies in peripheral lymphocytes
	as a screening test for carriers of a BRCA1 mutation in breast cancer families.
	Cancer Res 60:390-394
	33. Trenz K, Rothfuss A, Schutz P, Speit G (2002) Mutagen sensitivity of peripheral
	blood from women carrying a BRCA1 or BRCA2 mutation. Mutat Res 500:89-
	96
	34. Trenz K, Lugowski S, Jahrsdorfer U, Jainta S, Vogel W, Speit G (2003)
	Enhanced sensitivity of peripheral blood lymphocytes from women carrying a
	BRCA1 mutation towards the mutagenic effects of various cytostatics. Mutat
	Res 544:279-288
	35. Varga D, Hoegel J, Maier C et al (2006) On the difference of micronucleus
	frequencies in peripheral blood lymphocytes between breast cancer patients and
	controls. Mutagenesis 21:313-320
	36. Vogel W, Surowy H (2007) Reduced DNA repair in BRCA1 mutation carriers
	undetectable before onset of breast cancer? Br J Cancer 97:1184-1186
	37. Bonassi S, Fenech M, Lando C et al (2001) HUman MicroNucleus project:
	international database comparison for results with the cytokinesis-block
	micronucleus assay in human lymphocytes: I. Effect of laboratory protocol,
	scoring criteria, and host factors on the frequency of micronuclei. Environ Mol
	Mutagen 37:31-45
	38. Patino-Garcia B, Hoegel J, Varga D, Hoehne M, Michel I, Jainta S, Kreienberg
	R, Maier C, Vogel W (2006) Scoring variability of micronuclei in binucleated
	human lymphocytes in a case-control study. Mutagenesis 21:191-197
	39. Fenech M, Bonassi S, Turner J et al (2003) Intra-and inter-laboratory variation
	in the scoring of micronuclei and nucleoplasmic bridges in binucleated human
	lymphocytes. Results of an international slide-scoring exercise by the HUMN
	project. Mutat Res 534:45-64

Table 2 .

 2 Mean values and SD of subject age, age of cancer onset, years elapsed since radiotherapy and chemotherapy treatment and days since first experiment.  10.89 574.62  316.73 40.22  9.46 7.87  7.07 7.53  7.03

	Group	Nº	Age	Days since first experiment	Cancer onset age	Chemotherapy years a	Radiotherapy years a
	Healthy women	11	40.91  9.03 777.73  223.04
	BC non-familial or sporadic 5	52.6  7.50	1051.8  9.39	42.2  8.17	10.0  8.37	9.4  7.09
	Total group of control	16	44.56  10.03 863.38  224.50
	BRCA1 healthy	9	35.67  10.25 822.89 285.12
	BRCA1 with cancer	12	48.5  10.66 639.50  334.85	36.83 7.71	8.09  6.79	8.68  7.67
	Total group of BRCA1	21	43.0  12.12 718.10  320.68
	mutation carriers
	BRCA2 healthy	11	39.64  11.42 654.18  270.74
	BRCA2 with cancer	13	52.31  9.56 620.15 110.49 38.69  6.41	10.42  8.32	8.83  7.12
	Total group of BRCA2	24	46.50  12.10 635.75  196.32
	mutation carriers
	Familial BC group	15	50.53  13.45 324.20  250.31 43.60  12.44	4.42  4.53	3.76  5.66
	without detected mutation
	in BRCA1/2
	Total healthy subjects	31	38.94  10.17 747.0  260.63
	Total cancer patients	45	50.73

a years since last exposure. BC: breast cancer. BRCA1/2: BRCA1 or BRCA2 genes

Table 3 .

 3 Mean values and SD of the different endpoints recorded between all analysed groups in the non-treated cultures.

	Group	Nº	Basal MN	Basal BPN	Basal NDI
	Normal non-carriers	11	14.09  3.4	1.36  1.91	1.84  0.14
	Sporadic cancer	5	12.6  4.28	3.8  1.92	1.71  0.15
	Total group of control	16	13.63  3.61	2.13  2.19	1.80  0.15
	BRCA1 healthy	9	8.89  3.10	0.89  1.36	1.89  0.11
	BRCA1 with cancer	12	27.75  15.24	3.08  3.29	1.68  0.15
	Total group of BRCA1 carriers	21	19.67  14.94	2.14  2.82	1.77  0.17
	BRCA2 healthy	11	14.27  8.10	0.82  1.17	1.85  0.15
	BRCA2 with cancer	13	19.69  14.76	0.85  0.99	1.88  0.15
	Total group of BRCA2 carriers	24	17.21  12.24	0.83  1.05	1.86  0.15
	Familial BC group without detected	15	27.33  21.56	0.93  1.58	1.72  0.16
	mutation in BRCA1/2
	Total healthy subjects	31	12.65  5.85	1.03  1.49	1.86  0.13
	Total cancer patients	45	23.6  17.17	1.80  2.35	1.75  0.17

BC: breast cancer. BRCA1/2: BRCA1 or BRCA2 genes

Table 4 .

 4 Mean  SD values of MN, NBPs and NDI obtained in the blood cultures treated with 2 Gy of gamma-radiation.

	Group	Nº	2 Gy MN a	2 Gy BPN b	2 Gy NDI c
	Normal non-carriers	11 227.64  25.47	18.18  6.62	0.20  0.13
	Sporadic cancer	5	253.4  23.73	42.4  11.63	0.22  0.10
	Total group of control	16 235.69  27.10	25.75  14.13	0.20  0.12
	BRCA1 healthy	9 221.56  39.81	26.56  19.06	0.20  0.08
	BRCA1 with cancer	12 224.67  47.03	18  15.13	0.19  0.07
	Total group of BRCA1 carriers	21 223.33  43.05	21.67  17.03	0.20  0.07
	BRCA2 healthy	11 245.91  60.61	20.91  14.47	0.22  0.07
	BRCA2 with cancer	13 213.23  57.25	9.39  6.32	0.23  0.11
	Total group of BRCA2 carriers	24 228.21  59.86	14.67  12.1	0.22  0.09
	Familial BC group without detected	15	184.0  42.67	14  7.33	0.15  0.07
	mutation in BRCA1/2
	Total healthy subjects	31 232.36  44.43	21.58  13.9	0.21  0.09
	Total cancer patients	45	211.0  50.81	16.9 13.83	0.19  0.09

BC: breast cancer. BRCA1/2: BRCA1 or BRCA2 genes.

Table 5 .

 5 Mean  SD values of MN, NBPs and NDI in the blood cultures treated with MMC

	Group	Nº	MMC MN a	MMC BPN b	MMC NDI c
	Normal non-carriers	11	108.36  26.41	4.82  3.49	0.29  0.14
	Sporadic cancer	5	105.60  23.29	11  3.81	0.28  0.08
	Total group of control	16	107.63  24.87	6.75  4.6	0.29  0.12
	BRCA1 healthy	9	105.78  39.13	5.89  4.29	0.26  0.11
	BRCA1 with cancer	12	97.5  17.39	7.25  5.15	0.28  0.10
	Total group of BRCA1 carriers	21	101.05  28.22	6.67  4.74	0.27  0.10
	BRCA2 healthy	11	145.82  60.49	6.64 4.65	0.26  0.09
	BRCA2 with cancer	13	123.85  43.99	5.31  7.29	0.30  0.12
	Total group of BRCA2 carriers	24	133.92  52.20	5.92  6.13	0.28  0.11
	Familial BC group without	14	80.14  26.16	3.14  1.96	0.24  0.09
	mutations in BRCA1/2
	Total healthy subjects	31	120.90  47.06	5.77  4.1	0.27  0.11
	Total cancer patients	44	100.73  34.40	5.8  5.5	0.27  0.10
	BC: breast cancer. BRCA1/2: BRCA1 or BRCA2 genes

a Calculated by subtracting the spontaneous yields from the yields obtained in the treated samples. b Absolute values of NPB obtained in the cultures treated with MMC c NDI index values in untreated cultures less NDI values in treated cultures.

ACKNOWLEDGMENTS

This study was supported by two grants of the "Fondo de Investigación Sanitaria" from the Spanish Health Ministry: FIS 04/1832 and FIS 05/2181 and by a grant of the "Fundación Mutua Madrileña" (2008-2011).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.


Julie Richardson

Abeer M Shaaban

Mohamed Kamal

Rawiah Alisary

Clare Walker

Ian O Ellis

Valerie Speirs

Andrew R Green

Dr Sandra M Bell

Microcephalin is a new novel prognostic indicator in breast cancer associated with BRCA1 inactivation

Keywords: Microcephalin, breast cancer, immunohistochemistry, HER2, triple negative, BRCA1

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

Introduction

Previously, we identified mutations in MCPH1 a DNA damage response gene, as a cause of primary microcephaly [START_REF] Jackson | Identification of Microcephalin, a protein implicated in determining the size of the human brain[END_REF]. The MCPH1 protein called microcephalin is also known as BRIT1 (BRCT-repeat inhibitor of hTERT expression) which was initially identified as a transcriptional repressor of human telomerase reverse transcriptase [START_REF] Lin | Multiple tumor suppressor pathways negatively regulate telomerase[END_REF]. Microcephalin contains three BRCA1 carboxyl-terminal (BRCT) motifs which were originally identified in the BRCA1 gene and have been identified in other proteins involved in DNA repair and cell cycle checkpoints [START_REF] Koonin | BRCA1 protein products… Functional motifs[END_REF]. The DNA damage response is complex and MCPH1 appears to have a number of important roles in this process. In response to ionizing radiation and ultra-violet light, MCPH1 co-localises to DNA damage response foci with other DNA repair proteins such as γH2AX [START_REF] Wood | MCPH1 functions in a H2AX-dependent but MDC1 independent pathway in response to DNA damage[END_REF]. MCPH1 knockdown reduces the formation of foci by the DNA repair proteins MDC1, 53BP1 and ATM [START_REF] Ria | BRIT1 regulates early damage response, chromosomal integrity and cancer[END_REF]. MCPH1 interacts directly with BRACA2 and regulates the amount of both BRCA2 and Rad51 at DNA repair sites [START_REF] Wu | Microcephalin regulates BRCA2 and Rad51-associated DNA doublestrand break repair[END_REF]. SiRNA knockdown of MCPH1 expression decreases BRCA1 and CHK1 expression and impairs the ionizing radiation induced S and G2/M checkpoints [START_REF] Xu | Microcephalin is a DNA damage response protein involved in regulation of CHK1 and BRCA1[END_REF][START_REF] Lin | BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly[END_REF]. MCPH1 plays a role in G2-M checkpoint control by regulating CHK1 localization [START_REF] Tibelius | Microcephalin and pericentrin regulate mitotic entry via centrosomal-associated Chk1[END_REF].

These studies strongly implicated MCPH1 as an early mediator in the DNA damage response, regulating the recruitment of repair proteins to the site of damage and triggering both the ATM/ATR damage response signalling cascades. Recently microcephalin has been shown to interact with the transcription factor E2F1, leading to alterations in the regulation of CHK1, BRCA1, RAD51, p73 and caspases [START_REF] Yang | MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis[END_REF].

The function of microcephalin in DNA repair and checkpoint control makes it a potential tumour suppressor gene [START_REF] Ria | BRIT1 regulates early damage response, chromosomal integrity and cancer[END_REF][START_REF] Lin | Multiple roles of BRIT1/MCPH1 in DNA damage response, DNA repair and cancer suppression[END_REF]. Deletions at the MCPH1 loci (8p23) are associated with tumour development and poor prognosis in a wide range of cancers including breast cancer [START_REF] Qin | Chromosomal aberrations related to metastasis of human solid tumours[END_REF]. Previously comparative genomic hybridization (CGH) studies have identified decreased MCPH1 DNA copy number in 72% (39/54) of breast cancer cell lines and in 40% (35/87) of ovarian cancers. Decreased MCPH1 mRNA levels were also identified in 63% (19/30) of ovarian cancers. These findings support the hypothesis that MCPH1 is a tumor suppressor gene [START_REF] Ria | BRIT1 regulates early damage response, chromosomal integrity and cancer[END_REF].

Breast cancer is a heterogeneous disease both clinically and genetically. Recently breast cancer has been classified into molecular sub groups based on their gene expression profiles, these include luminal A and B, HER2 amplified tumours, basal-like tumours and the normal-breast like tumours [START_REF] Sorlie | Repeated observation of breast cancer sub types in independent gene expression data sets[END_REF]. These subgroups could also be identified using immunohistochemistry for hormone receptor, HER2 and basal markers [START_REF] El-Reihm | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Rakha | Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression[END_REF]. Interestingly dysfunction of the BRCA1 pathway is frequently observed in basal-like breast cancers [START_REF] Turner | Basal-like breast cancer and the BRCA1 phenotype[END_REF]. At least three quarters of breast cancers arising in germ line BRCA1 mutation carriers have a basal-like phenotype by immunohistochemistry [START_REF] Foulks | Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer[END_REF] or gene expression microarray [START_REF] Sorlie | Repeated observation of breast cancer sub types in independent gene expression data sets[END_REF]. Basal-like breast cancers express basal/myoepithelial markers such as cytokeratin 5/6, 14, 17 or vimentin and EGFR (HER1) [START_REF] Rakha | Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression[END_REF][START_REF] Rakha | Basal-like breast cancer: A critical review[END_REF]. This sub type of breast cancer has an aggressive phenotype, poor prognosis and lack expression of the receptors estrogen alpha (ER), progesterone (PR) and HER2 [START_REF] Rakha | Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression[END_REF][START_REF] Rakha | Basal-like breast cancer: A critical review[END_REF], often referred to as triple negative. Potentially due to the function of microcephalin in DNA repair, particularly the BRCA1 pathway, reduced microcephalin expression may be associated with the basal-like phenotype.

In this study we have investigated microcephalin expression in a large well-characterised series of breast cancer samples with long term follow-up to determine its clinical and biological relevance. The association of microcephalin expression with the biomarkers ER, PR, HER2, EGFR, CK5/6, CK14, and BRCA1 has also been investigated.

Materials and Methods

Patients

This study involves two independent cohorts of breast cancer cases. The first cohort containing 65 cases from Leeds Teaching Hospitals Histopathology archives were designated as the training set and the second cohort containing 319 cases from Nottingham University Hospitals NHS Trust Histopathology archives were used as the validation set. The Leeds cases were unselected from patients presenting between 1987 and 2004. The Nottingham cases were unselected from patients presenting between 1988 and 1998. Clinical history and tumour characteristics (age, tumour type, size, histological grade, lymph node status, and NPI) were available for both series. In both sample cohorts DFS was defined as the interval (months) from primary surgical treatment to the first loco regional or distant recurrence. BCSS was taken as time (months) from primary surgical treatment to time of death from breast cancer. Patients received Tamoxifen or classical cyclophosphamide, methotrexate and 5-fluorouracil chemotherapy dependent on their ER status. The Nottingham series is well characterised with data available for other biomarkers involved in breast cancer including ER, PR, HER2, EGFR, BRCA1 and basal cytokeratins (CK) 5/6 and 14 [START_REF] El-Reihm | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Rakha | Expression of BRCA1 protein in breast cancer and its prognostic significance[END_REF]. Ethical approval for this study has been obtained from the Local Research Ethics Committee of the Leeds Teaching Hospitals NHS Trust and from the Nottingham Research Ethics Committee 2. The reporting recommendations for tumour marker prognostic studies (REMARK) criteria, recommended by Mc Shane et al [START_REF] Mcshane | Reporting recommendations for tumour marker prognostic studies[END_REF] have been followed throughout this study.

Immunohistochemistry

To optimize the MCPH1 staining protocol, full sections of paraffin embedded tissue from both normal and breast tumour samples were stained using a range of primary antibody dilutions, incubation times and antigen retrieval methods. MCPH1 was stained on full sections of patient's samples in the training set, while in the validation set TMAs were constructed as previously described [START_REF] El-Reihm | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF]. Immunohistochemical analysis of microcephalin expression was performed with the rabbit anti-microcephalin antibody (BL1610, Bethyl Laboratories) at a 1:50 dilution on full sections and at 1:100 on the TMA. To avoid reduction in immunoreactivity of tissue sections over time the sections were cut just before staining.

Four m TMA sections were deparaffinised in graded alcohols. Endogenous peroxide activity was inhibited by immersing the sections in 0.5% H2O2. Antigen retrieval was carried out by pressure cooking slides for 2 minutes at full pressure in 1% Antigen Unmasking Solution (Vector Laboratories Ltd, Peterborough, UK).

A casein incubation (1/2 dilution) was also carried out to reduce non-specific staining (Vector Laboratories).

Sections were incubated with the antibody for 2 hours at room temperature. Bound antibody was detected with EnVision™ polymer (DAKO, Ely, Cambridgeshire, UK), using diaminobenzine as the substrate (Sigma, Poole, Dorset, UK). Sections were counterstained with Mayer's haematoxylin (VWR International Ltd, Poole, England). Negative controls, without primary antibody and positive controls of normal breast tissue were included in each batch of immunohistochemistry.

Immunohistochemical evaluation

The TMA sections were scored using high resolution digital images (NanoZoomer, Hamamatsu Photonics, Welwyn Garden City, UK), at 20X magnification, using a web-based interface (Distiller, Slidepath Ltd., Dublin, Ireland). Nuclear and cytoplasmic staining was scored as a percentage of the positive cells in relation to the total number of tumour cells present. All samples were scored by one observer (SMB) and a representative sample was counter-scored by a specialist consultant breast histopathologist (AMS) to ensure reproducibility. The immunoreactivity, scoring and categorisation of ER, PR, HER2, EGFR, CK5/6, CK14, BRCA1 and triple negative phenotype were defined in this series as previously described [START_REF] El-Reihm | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Rakha | Expression of BRCA1 protein in breast cancer and its prognostic significance[END_REF].

Statistical analysis

The percentage of cells with nuclear microcephalin staining was evaluated as a continuous variable to avoid the loss of information that results from categorization of continuous data [START_REF] Breslow | Fitting models to continuous data[END_REF][START_REF] Altman | methodological challenges in the evaluation of prognostic factors in breast cancer[END_REF]. However, for completeness the percentage of nuclear microcephalin staining was also dichotomized by sequentially testing every cut-off value versus BCSS using the Kaplan-Meier curves. The cut-off point which showed the highest significance between patient groups with regard to BCSS was used for subsequent analyses. Statistical analysis was performed using SPSS version 16.0, (SPSS Inc., Chicago, IL, USA). Pearson correlation was used to correlate microcephalin staining as a percentage of the positive cells versus tumour size, age of patient, number of positive nodes, vascular invasion, BCSS duration and DFS duration as continuous variables. The Chi-square test/Fisher exact test or Mann-Whitney U-test (non parametric) was used for comparisons among groups/categorical data as appropriate. Survival curves were generated using the Kaplan-Meier method, and differences among groups were analyzed by the log-rank test and confirmed by applying the Cox regression model. Previously published cut-off values were used for the established prognostic factors in this series of patients [START_REF] El-Reihm | High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses[END_REF][START_REF] Rakha | Expression of BRCA1 protein in breast cancer and its prognostic significance[END_REF]. For systematic modeling, a forward stepwise multivariable Cox regression model was used, testing the independent prognostic relevance of microcephalin immunoreactivity. All statistical tests were two-sided and a p ≤ 0.05 value was considered statistically significant.

RESULTS

Patient's characteristics

This study included two groups of patients after excluding core loss and unscoreable cores. The first group had 65 patients and is considered as a training set, while the second group had 319 patients and is considered as a validation set. Detailed patient characteristics of the validation set are summarized in Table 1.

Since this is the first large immunohistochemistry study on microcephalin expression in breast cancer no prior cut off point was available therefore two types of analysis were performed. Initially the percentage of cells with nuclear microcephalin staining was treated as continuous data. In addition we dichotomized the percentage of nuclear microcephalin staining. A cut off point of 35% positively stained cells showed the most significant difference between the two groups of patients (low and high microcephalin) in BCSS in the validation study.

There was insufficient follow up information to meet the statistical analysis requirements in the training set.

However, in the validation set, during a median follow-up period of 124 months (range 5-229 months), death occurred in 101/293 patients (34.5%) and metastasis or/and recurrence occurred in 133/293 patients (45%).

Microcephalin expression in normal and tumour breast tissues

Initially the expression of microcephalin was evaluated in whole breast cancer sections (n = 65). Strong nuclear staining was identified in normal breast samples (Fig. 1a). In the tumour samples, microcephalin was expressed in both the nucleus and cytoplasm of tumour cells (Fig. 1b). In many cases strong nucleolar staining was also detected in the nuclei (Fig. 1c). Interestingly in a small number of tumour samples (3/65) only cytoplasmic microcephalin staining was detected (Fig. 1d). 1 (f andg). The adjacent normal epithelium which was used as an internal control showed high microcephalin nuclear staining in the majority of cases.

Dichotomous analysis in

Relation of microcephalin expression to histological parameters

In each patient sample, the level of microcephalin expression was correlated with the severity of the disease.

Continuous data analysis of the validation set identified a highly significant association between low expression of microcephalin and high tumour grade (p < 0.003). Mean microcephalin expression was 61% in grade 2 tumours compared to 50% in the high grade 3 tumours. Microcephalin expression did not differ significantly in the distribution of other clinicopathological variables. Similar results were obtained after dichotomous analysis into low and high microcephalin expression however the statistical significance was reduced (Table 2).

Association of microcephalin expression with biomarkers

Continuous data analysis of microcephalin nuclear expression identified significant correlations with the ER, PR and HER2. Microcephalin expression was significantly associated with the negative expression of ER and PR (p < 0.001). Mean microcephalin expression was 45.2% in ER negative cases compared to 57% in positive cases.

Similarly mean microcephalin expression was 46.5% in PR negative cases compared to 59% in positive cases.

In the triple negative phenotype (ER, PR, and HER2) mean microcephalin expression was 55.8% compared to 43.7% (p < 0.004). Conversely mean microcephalin expression was significantly lower (43.8) in HER2 positive tumours than HER2 negative ones (55.4%) (p < 0.034).

Continuous data analysis of microcephalin nuclear expression identified a significant difference with absent or reduced nuclear BRCA1 staining (p < 0.001). Mean microcephalin expression was 30.6% in BRCA1 negative cases compared to 52.7% in positive cases with reduced BRCA staining and 63.3% with strong nuclear BRCA1 staining. No significant correlation was identified between microcephalin expression and cytoplasmic BRCA1 staining or the basal markers CK14, CK5/6 and EGFR. Similar results were obtained when the data was dichotomized into low and high microcephalin expression however the statistical significance was reduced. The correlation of microcephalin expression and other biomarkers is shown in Table 3.

Univariate and Multivariate analyses

In the validation set, continuous data analysis of microcephalin expression identified no correlation with BCSS.

However after dichotomization a weak association with reduced microcephalin expression and shorter BCSS was identified. In patients with low microcephalin expression the mean BCSS was 146.6 months (95%CI: 128-164) compared to 154.8 months (95%CI: 143.9-165.7) in patients with high microcephalin expression (p = 0.103) (Fig. 2a). This reached statistical significance in invasive ductal carcinomas alone (HR = 0.6, 95%CI: 0.4-1, p = 0.05) (Table 4). Mean BCSS of patients with low expression of microcephalin was128.7 months compared to 146.7 months in patients with high microcephalin expression (Fig. 2b). There was no significant association between microcephalin expression and DFS (Fig. 3).

A multivariate Cox regression model was carried out to examine the independent prognostic impact of microcephalin expression on BCSS and DFS in ductal carcinomas in relation to other established clinicopathological and tumour-biological factors such as tumour grade, size, lymph node status, NPI, vascular invasion, adjuvant hormonal and chemotherapy. In a systematic model, microcephalin, together with lymph node involvement and tumour size were independent predictors of BCSS in invasive ductal carcinomas alone (HR = 0.6, 95%CI: 0.4-1, p = 0.037) (Table 4).

In the small cohort of patients treated with chemotherapy (59) the mean BCSS of patients with tumours expressing microcephalin was 145 months compared to 112 months in patients with tumours having reduced microcephalin expression. This difference however was not statistically significant. No significant correlation was identified between microcephalin expression and response to hormone therapy.

Discussion

In this study, we have undertaken the first large scale investigation of the expression of the MCPH1 protein microcephalin in breast cancer samples. In normal breast duct epithelial cells nuclear and nucleolar microcephalin staining was observed. The nuclear localization is consistent with the role of microcephalin in DNA repair, checkpoint and transcription regulation. Nucleolus localization is often seen in DNA repair proteins such as BRCA1, which are stored in the nucleolus prior to relocalization to DNA damage foci when required [START_REF] Dejmek | DNA-dependent protein kinase (DNA-PK) dependent cisplatin induced loss of nucleolar facilitator of chromatin transcription (FACT) and regulation of cisplatin sensitivity by DNA-PK and FACT[END_REF]. In the tumour cells nuclear (including nucleolar) and/or cytoplasmic microcephalin staining was identified. Interestingly in a small number of high grade tumour samples only cytoplasmic microcephalin staining was detected.

Initially, we recorded low microcephalin expression in 34% (22/65) of the training set. Our larger validation set also showed a reduction in microcephalin expression of 29% (93/319). Our results agree with another studies which report reduced MCPH1 DNA copy number in 72% (39/54) of breast cancer cell lines and also reported reduced microcephalin staining in 7/10 breast cancer samples [START_REF] Ria | BRIT1 regulates early damage response, chromosomal integrity and cancer[END_REF][START_REF] Rai | Differential regulation of centrosome integrity by DNA damage response proteins[END_REF].

Our results revealed that low microcephalin expression was significantly associated with high tumour grade in our validation set. In the validation set, reduction of microcephalin expression was weakly associated with shorter BCSS. This association with shorter BCSS was even more obvious among invasive ductal carcinomas, which is the main histological subtype of breast cancer with a highly variable prognosis and, therefore, more accurate prognostic factors are needed especially for this histological breast cancer subtype. The multivariate Cox regression model showed that microcephalin levels and lymph node involvement and tumour size are independent predictors of BCSS in invasive ductal breast cancers. This finding is similar to a recent breast cancer study which identified an association between decreased MCPH1 transcript levels and reduced time to metastasis [START_REF] Ria | BRIT1 regulates early damage response, chromosomal integrity and cancer[END_REF].

In this study, low microcephalin immunohistochemical expression correlated with the triple negative phenotype and conversely positive HER-2 status. Morphologically the triple negative and HER2 positive cancers tend to be aggressive high grade cancers. Since reduced microcephalin expression is significantly associated with increasing tumour grade it is likely that microcephalin expression may be a marker of poor differentiation rather than of a particular subtype.

Interestingly reduced microcephalin expression also correlated with no or reduced nuclear BRCA1 expression. This finding is in agreement with previous DNA repair studies have reported MCPH1 siRNA knockdown causing reduced BRCA1 expression in a range of cell lines [START_REF] Xu | Microcephalin is a DNA damage response protein involved in regulation of CHK1 and BRCA1[END_REF][START_REF] Lin | BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly[END_REF][START_REF] Yang | MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis[END_REF][START_REF] Lin | Multiple roles of BRIT1/MCPH1 in DNA damage response, DNA repair and cancer suppression[END_REF]. To our knowledge this is the first study to confirm the association between reduced microcephalin and BRCA1 expression in breast tumour samples. A potential explanation for this association is that microcephalin is a positive regulator of BRCA1 due to its interact with the transcription factor E2F1 on the promoter of the BRCA1 gene increasing transcription [START_REF] Yang | MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis[END_REF], consequently reduced microcephalin expression would result in reduced BRCA1 expression.

Many sporadic breast cancers many show decreased BRCA1 expression and an aggressive BRCA1-like phenotype, however this can only be partially accounted for by methylation, mutations and deletions [START_REF] Turner | Basal-like breast cancer and the BRCA1 phenotype[END_REF][START_REF] Mueller | Regulation of BRCA1 expression and its relationship to sporadic breast cancer[END_REF], reduced expression of positive regulators like microcephalin could be another mechanism.

Recently the use of Parp inhibitors has proved very effective in the treatment of patients with breast and ovarian cancers containing BRCA1/2 mutations [START_REF] Fong | Inhibition of poly(ADP-ribose) polymerase in tumours from BRCA mutation carriers[END_REF]. The Parp-1 enzyme plays an important role in base excision DNA repair, when this function is inhibited in cells with compromised homologous recombination DNA repair , such as BRCA1/2 deficient cells , the cells are more sensitive to the increased DNA damage and apoptosis occurs [START_REF] Fong | Inhibition of poly(ADP-ribose) polymerase in tumours from BRCA mutation carriers[END_REF]. Potentially Parp inhibitors may prove useful in treating microcephalin deficient cancers due to the association between loss of microcephalin expression and decreased BRCA1 expression and the requirement for microcephalin in BRCA2/RAD51 mediated repair [START_REF] Wu | Microcephalin regulates BRCA2 and Rad51-associated DNA doublestrand break repair[END_REF].

Reduced expression of microcephalin was associated with aggressive high grade tumours and poor patient outcome particularly in invasive ductal breast cancers. Potentially reduced expression of the DNA repair protein microcephalin could cause resistance to chemotherapy. Unfortunately, the number of patients used in this study was not large enough to determine whether or not microcephalin had an effect on chemotherapy response. However the 33 months lower survival in patients with low microcephalin expression identified in our study indicates further studies are warranted. In addition to regulating BRCA1 expression through binding E2F1 microcephalin also alters the expression of p73, caspase 3 and 7, CHK1 and RAD51 which may also influence response to chemotherapy [START_REF] Yang | MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis[END_REF].

Recently two BRIT1/MCPH1 knockout mice models have been developed. In one deregulated mitosis and premature chromosome condensation was identified and in the other deficient DNA repair [START_REF] Trimborn | Establishment of a mouse model with misregulated chromosome condensation due to defective Mcph1 function[END_REF][START_REF] Liang | BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice[END_REF]. While to date no signs of cancer development have been detected in either model, Liang et al reported that when the DNA repair deficient BRIT1/MCPH1 mice were crossed with p53 knockout mice this resulted in a significant increase in susceptibility to cancer. The authors also reported that low dosages of irradiation induced breast tumors in mice with conditional knockout of BRIT1 in the mammary glands but not in control littermates [START_REF] Liang | BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice[END_REF].

Our results identifying reduced microcephalin expression in HER2 amplified and BRCA1 negative breast cancers suggest it would be very interesting to cross the BRIT1/MCPH1 deficient mice with amplified HER2 and BRCA1 mice to determine the influence of microcephalin expression on tumour development.

Further large confirmatory studies are warranted to determine the potential use of microcephalin expression assessment in clinical practice in breast and other cancer types. In summary, reduction of microcephalin expression was identified in almost a third of breast carcinoma cases particularly in higher grade tumours and BRCA1 negative cases. Although reduced microcephalin expression was identified in triple negative and HER2 positive cancers, this association may simply reflect the aggressive nature of these subtypes.

Multivariate analysis which identified microcephalin as an independent prognostic factor for BCSS provides clinical evidence to support the idea that microcephalin is a tumour suppressor gene, which may prove to be a new prognostic biomarker in aggressive breast cancers.

  the training set, identified low microcephalin expression in 22/65 (34%) samples. Fig 1e shows a grade 3 invasive ductal carcinoma showing loss of microcephalin expression. In the validation set, 93/319 (29%) of cases showed low microcephalin expression. Low and high microcephalin expression in TMA samples with IDC are shown in Fig.

Fig. 1 Fig. 2 Fig. 3

 123 Fig. 1 Immunohistochemical analysis of microcephalin expression in normal and malignant breast tissue samples a) Normal mammary ducts showing strong nuclear expression of microcephalin (x20). b) Grade 1 invasive ductal carcinoma of no special type (NST) showing nuclear and cytoplasmic microcephalin staining (x20). c) Invasive ductal carcinoma NST showing nucleolar expression of microcephalin (arrows) (x40). d) Grade 3 invasive ductal carcinoma NST with no nuclear microcephalin expression. Cytoplasmic staining is however present (x40). e) Grade 3 invasive ductal carcinoma NST, with no microcephalin expression (x40). f) Grade 1 invasive ductal carcinoma TMA showing high level microcephalin expression (x20). g) Grade 3 invasive ductal carcinoma TMA showing no nuclear microcephalin expression and low level cytoplasmic staining (x20)

Table 1 Patients' characteristics

 1

	Variables

a A 35% cut off was used to categorize microcephalin expression into low and high groups.

b Includes mucoid and invasive papillary carcinoma.

c Includes ductal/NST mixed with lobular or special types.

d Statistical analysis was only performed for most common sub types P 0.05 is significant and are shown in bold

Table 3 Correlation of microcephalin expression with other biomarkers Microcephalin Expression

 3

		Continuous		Categorised a
	Parameter	Mean	p value	High	Low	p value
		(%)		(%)	(%)
	ER
		Negative 45.2	0.001	55 (27)	33 (35) 0.117

Acknowledgements

This study was supported by grants from Yorkshire Cancer Research grant number L317 (SMB & VS) and the Breast Cancer Campaign 2007NovPR53 (SMB & VS). MK is supported by a scholarship from the Egyptian government. RA is supported by a scholarship from the Saudi Arabian government.

a Includes mucoid and invasive papillary carcinoma.

b Includes ductal/NST mixed with lobular or special types.