E Bastiaannet

A J M Craen

P J K Kuppen

M J Aarts

L G M Geest

C J H Velde

R G J Westendorp

MD G J Liefers
email: g.j.liefers@lumc.nl

A J M De Craen

L G M Van Der Geest

C J H Van De Velde

Socioeconomic differences in survival among breast cancer patients in the Netherlands not

come

INTRODUCTION

Breast cancer is the most commonly diagnosed cancer among females in developed countries. In the Netherlands, more than 12000 new cases of breast cancer are diagnosed each year and incidence is still increasing. Breast cancer is one of the few cancers that is more common in affluent women. [START_REF] Clegg | Impact of socioeconomic status on cancer incidence and stage at diagnosis: selected findings from the surveillance, epidemiology, and end results: National Longitudinal Mortality Study[END_REF] However, women with a lower socioeconomic status seem to have a decreased survival. [START_REF] Clegg | Impact of socioeconomic status on cancer incidence and stage at diagnosis: selected findings from the surveillance, epidemiology, and end results: National Longitudinal Mortality Study[END_REF][START_REF] Taylor | Social deprivation and breast cancer[END_REF][START_REF] Thomson | Prognostic factors in women with breast cancer: distribution by socioeconomic status and effect on differences in survival[END_REF][START_REF] Schrijvers | Deprivation and survival from breast cancer[END_REF][START_REF] Pollock | Deprivation and emergency admissions for cancers of colorectum, lung, and breast in south east England: ecological study[END_REF] Socioeconomic differences in incidence have typically been attributed to life-style, particularly differences in distribution of known risk factors such as age at menarche, age at menopause, age at first pregnancy and use of hormone replacement therapy. [START_REF] Rutqvist | Socioeconomic gradients in clinical stage at presentation and survival among breast cancer patients in the Stockholm area 1977-1997[END_REF][START_REF] Faggiano | Socioeconomic differences in cancer incidence and mortality[END_REF][START_REF] Van Loon | Differences in cancer incidence and mortality among socio-economic groups[END_REF] Social gradients in survival have also been reported which appear to persist after adjustment for stage, suggesting an inequality regarding awareness of the disease, access to early detection, treatment or other yet unknown factors. [START_REF] Rutqvist | Socioeconomic gradients in clinical stage at presentation and survival among breast cancer patients in the Stockholm area 1977-1997[END_REF] Overall, survival of cancer is influenced by factors which can be classified into three groups: tumor biological characteristics, patient characteristics and treatment. [9;10] Consequently, SES differences in survival must originate from an unequal socioeconomic distribution of some of these factors. [START_REF] Woods | Origins of socio-economic inequalities in cancer survival: a review[END_REF] Until now, the reasons for social disparities in breast cancer prognosis are not completely known. Various studies have shown that reasons are probably multi-factorial, with possible explanations including health service factors, late stage at diagnosis, different tumor biology, treatment and levels of comorbidity. [2;5;11-14] Socioeconomic differences in tumor size may be related to the length of delay between the occurrence of the first symptoms and the time of diagnosis, which is shorter for women of higher classes. [START_REF] Norredam | Effect of social class on tumour size at diagnosis and surgical treatment in Danish women with breast cancer[END_REF] Besides, women from lower socio-economic strata are less likely to attend population screening programs and are also more likely to present with unfavorable stage at diagnosis, although not all studies confirm this. [4;16-18] In the Netherlands an equal-access health care system is provided; all residents have compulsory health insurance and for those who have insufficient income, an extra government allowance is paid to ensure overall coverage. A study from Wojcik et al showed that the type of health care system significantly affects disease outcome. [START_REF] Wojcik | Breast carcinoma survival analysis for African American and white women in an equal-access health care system[END_REF] African-American women with breast cancer treated in the military health care facilities from the U.S. department of defense had a better survival rate than African-American women represented in the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. [START_REF] Wojcik | Breast carcinoma survival analysis for African American and white women in an equal-access health care system[END_REF] This suggests that ready access to medical facilities and the full complement of treatment options improves survival rates.

The aim of this study was to analyze socioeconomic differences in breast cancer outcome in the Netherlands where treatment is guided by strict guidelines for all, without any financial restrictions.

METHODS

Patient selection

Adult female patients with their first primary breast cancer (invasive and in situ) diagnosed between 1995 and 2005 were selected from the Netherlands Cancer Registry (NCR). Patients with other tumors before their breast cancer were excluded. The nationwide Dutch network and registry of histo-and cytopathology, regularly submits reports of all diagnosed malignancies to the regional cancer registries. The national hospital discharge databank, which receives discharge diagnoses of admitted patients from all Dutch hospitals, completes case ascertainment. After notification, trained registry personnel collect data on diagnosis, staging, and treatment from the medical records, including pathology and surgery reports, using the registration and coding manual of the Dutch Association of Comprehensive Cancer Centers. Stage was divided according to TNM classification at the year of diagnosis.

Pathological T, N and M stage was used; clinical stage was used if pathology was missing.

Vital status was established either directly from the patient's medical record or through linkage of cancer registry data with the municipal population registries (follow-up until January 1 st , 2008) which record information on their inhabitant's vital status.

Socioeconomic status was assigned to each individual using an area-based measure according to place of residence at the time of diagnosis. The area-based SES was provided by the Netherlands Institute for Social Research and consists of data concerning income, employment and education. These data are provided to the institute by a private organization which collects information by telephone calls with one person per 6-digit postal code area; this person is seen as representative for his or her area. Next, numbers are aggregated to 4 digit postal code areas. Validation studies indicate that numbers at aggregated level approach the true situation. [START_REF] Tesser | Rapportage minderheden[END_REF] The range of SES in the cohort was from -3.60 to 5.01, with a higher score representing a high social deprivation (low SES) and a low score representing little social deprivation and consequently a high SES. Scores were divided in quintiles.

Statistical analysis

The association between stage (both T-stage and combined TNM stage) and socioeconomic status was assessed. Overall Survival (OS) was calculated using standard survival analysis with multivariable Cox Proportional Hazard Analysis, with death due to any cause defined as event. Relative survival was calculated by the Hakulinen method as the ratio of the survival observed among the cancer patients and the survival that would have been expected based on the corresponding (age and year) general population. National life tables were used to estimate expected survival. Relative Survival (10-years) was stratified according to stage and calculated for each socio-economic group. Relative Excess Risks of death (RER) with p-value were estimated using a multivariable generalized linear model with a Poisson distribution, based on collapsed relative survival data, using exact survival times.

Finally, the number of avoidable deaths for each SES group relative to the highest SES group was calculated by: avoidable deaths within 5 years = total number of patients x expected survival x difference in relative survival between deprivation category and most affluent category.

RESULTS

Overall, 127599 breast cancer patients were included in this study. As shown in Table 1, most of the patients had breast cancer with ductal histology (70.0%), usually T1c (33.3%) or T2 (32.4%); N-stage was almost equally divided (N0 47.7% and N1-2 46.8%) and most of the patients had no distant metastases (M0 67.1%).

Figure 1 shows the distribution of tumor size according to the SES groups. For in situ tumors the percentage of patients decreased as SES decreased: 7.9% for patients with a low SES versus 8.9% for patients with a very high SES (p<0.001). For T1 there was no statistically significant decrease (p=0.3). For the stage T2, T3 and T4 there was a clear increase in the percentage of patients as SES became lower (all p<0.001). For T2 the percentage of patients increased from 33.1% for patients with a very high SES to 35.1% in the very low SES; also for T4 this increase was shown (4.9% to 6.9%). SES was also significantly associated with grade, N-stage and M-stage. After adjusting for these factors, SES was still associated with tumor size; patients presented with larger tumors for lower SES groups (p<0.001).

As shown in Table 2, relative survival per T-stage of disease was statistically different in almost all stages. With the exception of in situ tumors, patients with a very low SES had a decreased survival as compared to the other SES groups. After adjustment for age, year of diagnosis, histology, grade, N-stage, M-stage and treatment there remained a statistically significant difference for most stages, especially the lower stages. Also, for the combined stage at diagnosis (stage I, II, III and IV), socioeconomic differences were shown. After adjustment for potential confounders, socioeconomic differences were no longer apparent in stage III and IV.

Table 3 shows the results of the multivariable survival analysis of both Overall Survival (death due to any cause) and Relative Survival. Adjusted for age, year of diagnosis, histology, grade, T-stage, N-stage, M-stage and treatment, the SES score remained a significant independent prognostic factor for Overall Survival (HR 1.04 (SES as continuous factor); p<0.001) and Relative Survival (RER 1.07; p<0.001). Overall and Relative Survival were significantly lower in the lower socioeconomic groups.

Figure 2 shows Relative Survival according to SES; the 10-years RS was 74.0% for the lowest SES and 79.0% for the highest SES (p<0.001). When stratified for age category, the difference in survival was most evident in the 61-75 years old patients with an RER of 1.26 (95%CI 1.14-1.40; p<0.001). In all stages of disease there were treatment disparities for the different SES groups; patients from a very low SES received less surgery and less adjuvant treatment. Table 4 shows the number of avoidable deaths if relative survival in the lower SES groups was the same as the highest SES group. For the lowest SES group 777 deaths could be avoided if the survival was equally high as the highest SES group.

DISCUSSION

The present study shows that, in an 11-year nationwide study in breast cancer patients in the Netherlands (n=127599), a low SES is associated with worse survival, even stratified for tumor size or stage of disease. Also in an equal-access health care system SES should be considered as an independent prognostic factor for breast cancer survival. Health inequalities between social classes were already recognized several centuries ago, but one would expect that due to an improvement of working and living conditions these differences would have been reduced or even disappeared. [START_REF] Bouchardy | Social class is an important and independent prognostic factor of breast cancer mortality[END_REF] However, this is not the case for breast cancer patients in the Netherlands.

In the present study, an association between size of the tumor and SES was shown where larger tumors were associated with a lower SES. In the literature several conclusions concerning breast cancer stage and socio-economic status can be found. A study from the south Australian cancer registry compared diameters between 1980-1986 and 1997-2002 and analyzed socioeconomic predictors for large tumors in 1997-2002. [START_REF] Luke | Female breast cancers are getting smaller, but sociodemographic differences remain[END_REF] Their data showed an increase in smaller tumors over time, however indicated that earlier diagnosis is not evenly distributed among socioeconomic classes. [START_REF] Luke | Female breast cancers are getting smaller, but sociodemographic differences remain[END_REF] In the present study, we observed more in situ tumors (mostly screen detected) in patients from the higher SES, indicating that attendance for the screening program is higher in higher SES groups. A study from Stockholm (n=15021) showed significant differences in SES for clinical stage and survival. [START_REF] Rutqvist | Socioeconomic gradients in clinical stage at presentation and survival among breast cancer patients in the Stockholm area 1977-1997[END_REF] However, in this study differences in survival were mostly the consequence of differences in non breast cancer mortality and no differences in stage-specific breast cancer mortality were seen, while in the present study differences in relative survival were seen. [START_REF] Rutqvist | Socioeconomic gradients in clinical stage at presentation and survival among breast cancer patients in the Stockholm area 1977-1997[END_REF] Schrijvers et al reported the results of 29676 women in southeast England. [START_REF] Schrijvers | Deprivation and survival from breast cancer[END_REF] Remarkably, only in elderly women aged >65 years, the severity of deprivation related to the presence of advanced disease at time of diagnosis. [START_REF] Schrijvers | Deprivation and survival from breast cancer[END_REF] On the contrary, two other studies found no association between SES and tumor size. [2;15] Regional small socioeconomic differences in breast cancer survival have been described before in the southeastern part of the Netherlands; Schrijvers et al studied patients diagnosed between 1980 and 1989 (n=3928) in this part of the country. [START_REF] Schrijvers | Socioeconomic status and breast cancer survival in the southeastern Netherlands, 1980-1989[END_REF] However, the differences could mostly be explained by differences in stage at diagnosis, while in the present study differences in Overall and Relative Survival remain significant after adjustment for stage differences. There are a number of individual patient and tumor characteristics that might affect survival and may vary across social groups, such as known risk behaviors as smoking and comorbidity. [START_REF] Jeffreys | Socioeconomic inequalities in cancer survival in New Zealand: the role of extent of disease at diagnosis[END_REF] The use of relative survival reflects disease-specific survival.

Hence, results can not be explained by a higher rate of death due to other causes in the lower SES; although the presence of comorbidity may limit the treatment choices available to the patients. [START_REF] Janssen-Heijnen | Comorbidity in older surgical cancer patients: influence on patient care and outcome[END_REF] Although scant research has been published, evidence shows that physician decisions may contribute to inequalities in cancer survival. [START_REF] Jeffreys | Socioeconomic inequalities in cancer survival in New Zealand: the role of extent of disease at diagnosis[END_REF] A prospective study showed that within breast cancer patients women with a lower level of education were administrated lower doses of first-cycle chemotherapy. [START_REF] Griggs | Effect of patient socioeconomic status and body mass index on the quality of breast cancer adjuvant chemotherapy[END_REF] Other studies have also found evidence for lower rates of breast conserving treatment, radiation therapy and adjuvant chemotherapy among women of lower socioeconomic status. [11;16;26] In the present study an indicator of socio-economic status based on the postal code of a residential area was used. This indicator was based on income, employment and education and collected by information of telephone calls with one person per 6-digit postal code area. This aggregate covers a relatively small geographical area, and thus probably represents a reliable approximation of individual SES status. [START_REF] Louwman | Impact of a programme of mass mammography screening for breast cancer on socio-economic variation in survival: a population-based study[END_REF] Dominguez and colleagues studied the association between health outcomes and several small-area-based socioeconomic measures and also with individual socioeconomic measures. [START_REF] Dominguez-Berjon | The usefulness of area-based socioeconomic measures to monitor social inequalities in health in Southern Europe[END_REF] Results showed that, in general, the inequalities that were measured with the area-based measures were in the same range as those obtained with individual-based measures. [START_REF] Dominguez-Berjon | The usefulness of area-based socioeconomic measures to monitor social inequalities in health in Southern Europe[END_REF] Besides, Hyndman et al studied the possible misclassification of social disadvantage when SES is based on postcode versus collector's district analyses and found that misclassification of individuals to SES groups on the basis of postcode could lead to an underestimation of the true relationship between SES and health-related measures. [START_REF] Hyndman | Misclassification of social disadvantage based on geographical areas: comparison of postcode and collector's district analyses[END_REF] Ideally, individual-based measures of socioeconomic position would be used. Unfortunately, this information was not available as other information concerning several potential confounders such as comorbidity, receptor status and type and dose of the chemotherapy.

Possible explanations for the differences in survival according to SES include differences in access to early diagnosis (possibly trough screening), tumor biology, and access to optimal treatment.[2;3;5;12-15] However, despite persistent publications on social and ethnical disparities in breast cancer outcome, we still do not know to which extent these factors explain social inequalities in breast cancer prognosis. [START_REF] Bouchardy | Social class is an important and independent prognostic factor of breast cancer mortality[END_REF] There is evidence that cancer presents with a more aggressive phenotype among patients from minority racial/ethnic groups in the US. [START_REF] Byers | The impact of socioeconomic status on survival after cancer in the United States : findings from the National Program of Cancer Registries Patterns of Care Study[END_REF] Social factors, however, seem to be more important than biological factors in explaining racial and ethnic cancer disparities, especially in the US where access to medical care is closely tied to economic status. [START_REF] Byers | The impact of socioeconomic status on survival after cancer in the United States : findings from the National Program of Cancer Registries Patterns of Care Study[END_REF] A recent study confirmed this: Yood et al showed ethnic differences in stage at diagnosis among women with similar medical care access. [START_REF] Yood | Race and differences in breast cancer survival in a managed care population[END_REF] That difference in distribution of stage had a major influence on differential African-American/European-American survival but does not fully explain it. However, adjustment for income, age, and marital status resulted in a negligible effect of race on survival.

Data from the Norwegian Women and Cancer Study showed an overall negative socioeconomic gradient in cancer survival. [START_REF] Braaten | Socioeconomic differences in cancer survival: the Norwegian Women and Cancer Study[END_REF] Inclusion of stage modestly changed the risk difference, comorbidity did not alter the estimates, but further adjustment for smoking did change the estimates considerably. Other studies show that both patient and physician barriers to, expectation of, and communications about adjuvant therapy also contribute to the observed differences in survival. [START_REF] Bickell | Missed opportunities: racial disparities in adjuvant breast cancer treatment[END_REF] and Social Exclusion. Given its incidence, the number of cancer deaths in Europe that could potentially be avoided by reducing socioeconomic health inequalities appears particularly important for breast cancer. The present study shows that also in an equal-access health care system socioeconomic differences do exist in survival from breast cancer, even when adjusted for stage at diagnosis. This study suggests that health policies aimed at the reduction of social exclusion might be important to increase breast cancer specific survival.

# 20 Figure 1 :

 201 Figure 1: Distribution of T-stage according to socio-economic status.

Figure 2 :

 2 Figure 2: Relative Survival according to socioeconomic status.

Figure 1 :

 1 Figure 1: Distribution of T-stage according to socio-economic status.

Figure 2 :

 2 Figure 2: Relative Survival according to socioeconomic status.

Table 1 :

 1 Characteristics of study population (n=127599).

Table 2 :

 2 Relative Survival (10-years RS) according to stage and SES.

	Socio-economic status

Table 3 :

 3 Results multivariable analysis Overall Survival and Relative Survival Adjusted for age, year, histology, grade, T-stage, nodal status, distant metastases, surgery and adjuvant treatment

				Multivariable analyses		Multivariable analyses
			10-yr OS	HR (95% CI)	p-value	10-yr RS	RER (95% CI)	p-value
	SES	Continuous		1.04 (1.03-1.05)	<0.001		1.07 (1.05-1.08)	<0.001
	score
	Survival analysis according to SES groups
	SES	Very high	65.3	1	<0.001	79.0	1	<0.001
	groups	High	64.9	1.04 (1.00-1.07)		78.6	1.06 (1.01-1.12)
		Intermediate	62.4	1.06 (1.02-1.10)		76.7	1.11 (1.05-1.17)
		Low	61.5	1.08 (1.04-1.11)		76.4	1.11 (1.06-1.17)
		Very low	58.3	1.10 (1.06-1.13)		74.0	1.19 (1.13-1.25)

Table 4 :

 4 Avoidable deaths within 5 years of diagnosis by deprivation category

  The 10-years Relative Survival was 79.0% for the highest SES and 74.0% for the lowest SES with a corresponding adjusted RER 1.2 (p<0.001).

		100
		90
	(%)
	Survival	80
	100% Relative	3.8 4.9		3.6 5.3			4.0 5.6		3.8 6.0			3.9 6.9
		80% 70
			33.1		33.0		33.1		33.9		35.1
		60%
		60
		0	1	2	3	4	5	6	7	8	9	10	11	12	13
		40%						Years
		49.5 Very high status		49.4 High status		48.5 Average status	47.9 Low status	46.3 Very low status
		20%
			8.9		8.8			8.9		8.4			7.9
		0%
			Very high	High		Intermediate	Low			Very low
			(n=25496)	(n=25538)		(n=25524)	(n=25525)		(n=25516)
					In situ		T1 T2 T3 T4
															21

the authors would like to thank the Dutch Cancer Society (KWF


Ann H Rosendahl

Maria Hietala

Maria Henningson

Håkan Olsson

Dr Helena Jernström
email: helena.jernstrom@med.lu.se

and polymorphisms predict circulating IGFBP-3

Keywords: IGFBP-3, single nucleotide polymorphism, diplotype, breast cancer, BRCA, oral contraceptives

come

Introduction

Breast cancer is the most commonly diagnosed cancer among women in the Western world and the lifetime risk is approximately 12%. Women with one first-degree relative with breast cancer have an approximately 2-fold greater risk compared to women in the general population. Mutations in the high-penetrance BRCA1 or BRCA2 genes confer 60-80% lifetime risk of breast cancer [START_REF] Narod | BRCA1 and BRCA2: 1994 and beyond[END_REF]. Still, >50% of the genetic predisposition to hereditary breast cancer remains unexplained and the influence of other low-penetrance genetic or non-genetic factors is under intense investigation [START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF].

Insulin-like growth factor I (IGF-I) is a polypeptide growth hormone that promotes cell proliferation and inhibits apoptosis of both normal and malignant breast epithelial cells [START_REF] Pollak | Insulin-like growth factors and neoplasia[END_REF].

Given the high circulating IGF-I concentration and wide tissue distribution, tight regulation and control of its actions is required to maintain homeostasis. The vast majority of circulating IGF-I is bound to IGF binding proteins, predominantly IGFBP-3 in complex with an acidlabile subunit, which restrict the bioactive IGFs and limit their interaction with receptors.

Dysregulation of this control resulting in increased IGF-I levels or altered IGF-I/IGFBP-3 ratios may contribute to an increased risk of breast cancer. IGFBP-3 has a well-defined role in sequestering IGF-I and thereby attenuating its mitogenic actions. However, in addition to this regulatory role on IGF-I actions, IGFBP-3 may exert IGF-I independent effects promoting cellular growth [START_REF] Rosendahl | Influence of IGF-IR stimulation or blockade on proliferation of human renal cell carcinoma cell lines[END_REF][START_REF] Martin | Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity[END_REF]. High circulating IGFBP-3 levels have been associated with proliferative benign breast disease and increased breast cancer risk [START_REF] Su | Genetic variation and circulating levels of IGF-I and IGFBP-3 in relation to risk of proliferative benign breast disease[END_REF][START_REF] Key | Insulin-like growth factor 1 (IGF1), IGF binding protein 3 (IGFBP3), and breast cancer risk: pooled individual data analysis of 17 prospective studies[END_REF].

Non-genetic factors such as oral contraceptives (OC) have been shown to increase IGFBP-3 levels in most women [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF][START_REF] Jernstrom | Genetic and nongenetic factors associated with variation of plasma levels of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in healthy premenopausal women[END_REF]. In addition to lifestyle and environmental factors, IGFBP-3 plasma levels are likely to be influenced by genetic variation in the IGFBP1 and IGFBP3 genes [START_REF] D'aloisio | IGF-I and IGFBP-3 polymorphisms in relation to circulating levels among African American and Caucasian women[END_REF]. The gene for IGFBP3 is located at chromosome 7p14-p12, in a tail-to-tail configuration with IGFBP1 at 7p13-p12 [START_REF] Ehrenborg | Contiguous localization of the genes encoding human insulin-like growth factor binding proteins 1 (IGBP1) and 3 (IGBP3) on chromosome 7[END_REF]. Twin studies indicate that genetic variants may account for up to 60 % of the inter-individual variation in circulating IGFBP-3 levels [START_REF] Harrela | Genetic and environmental components of interindividual variation in circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3[END_REF][START_REF] Pantsulaia | Pedigree-based quantitative genetic analysis of interindividual variation in circulating levels of IGFBP-3[END_REF][START_REF] Pantsulaia | Genetic regulation of the variation of circulating insulin-like growth factors and leptin in human pedigrees[END_REF].

Our group has previously reported significant associations between the AA genotype of the IGFBP3 SNP rs2854744 (A-202C) in the promoter region of IGFBP3 and higher circulating IGFBP-3 levels, especially in women from BRCAX families, in the present study population [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF]. This polymorphism may directly influence IGFBP3 gene promoter activity [START_REF] Deal | Novel promoter polymorphism in insulin-like growth factor-binding protein-3: correlation with serum levels and interaction with known regulators[END_REF][START_REF] Paquette | Identification of upstream stimulatory factor binding sites in the human IGFBP3 promoter and potential implication of adjacent single-nucleotide polymorphisms and responsiveness to insulin[END_REF]. To our knowledge, associations between multiple genetic polymorphisms and diplotypes in IGFBP1 and IGFBP3 and circulating IGFBP-3 levels among women from BRCA1, BRCA2 or BRCAX families have not been previously investigated. The aims of present study were to examine the associations between genetic variation in the IGFBP1 and IGFBP3 genes and IGFBP-3 plasma levels, BRCA status, and breast cancer risk among women from high-risk breast cancer families.

Materials and methods

Study Population

The study population included 323 women from 192 families from two inclusion arms, all belonging to high-risk breast cancer families in the South Swedish Health Care Region with DNA available for genotyping. The first inclusion arm has been described in detail elsewhere [START_REF] Jernstrom | Insulin-like growth factor-1 (IGF1) genotype predicts breast volume after pregnancy and hormonal contraception and is associated with circulating IGF-1 levels: implications for risk of earlyonset breast cancer in young women from hereditary breast cancer families[END_REF]. Briefly, 267 young healthy women with no previous cancer history or prophylactic mastectomy or bilateral oophorectomy were enrolled in the study between 1996-2006.

Eligible participants had to belong to high-risk breast cancer families and be either 1) known BRCA1 or BRCA2 mutation carriers or 2) first or second-degree relatives of a breast cancer case or 3) first or second-degree relatives of a known male or female BRCA1 or BRCA2 mutation carrier. In the second inclusion arm an additional 40 BRCA1 and 16 BRCA2 mutation carriers born between 1950-1988 were included irrespective of cancer status, in order to include all BRCA1/2 mutation carriers in the South Swedish Health Care Region with DNA available for SNP testing. Information on BRCA mutation status was obtained through medical records from the Oncogenetic Clinic at the Department of Oncology, Lund. Written informed consent was obtained from all participating women and the study was approved by the local ethics committee at Lund University. Extensive questionnaire data with information on reproductive factors, the use of combined oral contraceptives (OC) and other medications etc. were obtained from the majority of cohort members. Characteristics of the women are presented in Table 1.

IGFBP-3 plasma levels

IGFBP-3 levels were measured in plasma samples obtained between 07:15 am and 12:15 pm 5-10 days prior to the predicted onset of the next menstrual period, i.e. during cycle day 18-23 in most women, using the IMMULITE 2000 IGFBP-3 enzyme-labeled chemiluminiscent immunometric assay (Siemens) in Uppsala University Hospital (Uppsala, Sweden) as previously described [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF]. The assay sensitivity was 0.02 μg/ml. The intra-assay and interassay variation was 4.1% and 7.3%, respectively.

SNP selection and genotyping

Four IGFBP1 (rs1995051, rs3763497, rs1065780, and rs4988515) in one haplotype block and three IGFBP3 haplotype-tagging (ht)SNPs (rs2471551, rs2854744, and rs2132572) in another haplotype block were selected to capture 95% of the genetic diversity of the IGFBP1 and IGFBP3 genes in a Swedish population, based on personal communication with Dr Mattias Johansson (International Agency for Research against Cancer, IARC, Lyon, France) and data from a Swedish cancer cohort [START_REF] Johansson | Genetic and plasma variation of insulin-like growth factor binding proteins in relation to prostate cancer incidence and survival[END_REF]. Two additional IGFBP3 SNPs (rs6670 and rs2453839) between the blocks were also included.

Genomic DNA was extracted from peripheral blood using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Genotyping was performed in the Department of Genotyping and Sequencing at Region Skåne's Competence Center of Clinical Research (RSKC, Malmö, Sweden).

The SNP (rs3763497, rs1065780, rs4988515, rs6670, rs2471551, rs2854744, and rs2132572) analyses were performed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on a Sequenom MassARRAY® platform (Sequenom, San Diego, CA, USA), using iPLEX reagents according to the manufacturers' protocol. The Sequenom MassARRAY® designer software was used for multiplex SNP analysis design. The SNPs (rs1995051 and rs2854744) were genotyped using a Taqman SNP allelic discrimination assay in 384-well format on ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Six out of 323 women were not successfully genotyped for IGFBP3 rs2854744 SNP using TaqMan. For those six women, rs2854744 genotypes were available from iPLEX and DNA sequencing from the previous study [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF] (100% concordance) and used in the study models. For quality control of genotype data, more than 10% of the samples were run in duplicates, with 100% concordance. The genotyping success rate was ≥ 95% for all SNPs.

Diplotype assignment

The htSNPs were used to predict the most likely haplotypes and their corresponding diplotypes, i.e. paired haplotypes. Each of the htSNPs within IGFBP1 or IGFBP3 was crosstabulated against the remaining htSNPs within their respective genes to generate possible linkage combinations and to identify non-existing or unlikely combinations. The most likely haplotypes were created and compared with previously published data [START_REF] Johansson | Genetic and plasma variation of insulin-like growth factor binding proteins in relation to prostate cancer incidence and survival[END_REF]. The women were subsequently assigned to their most likely diplotypes. Diplotypes with an assignment of five women or fewer were combined together into a composite "rare diplotypes" category.

Follow-up

Women were considered at risk for breast cancer from age 18 and were followed until the development of a first breast cancer according to the Regional Cancer Registry, until the date of a self-reported prophylactic mastectomy or oophorectomy, or until May 31 2009, whichever came first. The women in the study who were considered to have a high-risk of developing breast cancer were offered clinical follow-up including annual mammograms, ultrasounds or magnetic resonance imaging and physical examination of the breasts. The report rate of the Swedish cancer registries is close to 100%.

Statistical Analysis

The statistical software SPSS 17.0 was used for most analyses and the multivariate linear regression models were also adjusted for family clustering by using the cluster option of the regress command in STATA. For analyses of IGFBP-3 levels, we excluded women who were breast-feeding at blood draw (n=4), women using hormonal contraceptives other than combined OCs (n=19), or both (n=1), as well as one woman who had not answered the question on hormonal contraceptive use, leaving 87 current OC users and 144 non-users. For SNP and diplotype analyses, frequencies and associations with circulating levels were calculated for all women as well as stratified according to OC status. Multivariate linear regression models were used to estimate the standardized [age(29 years), weight(ln67 kg) and no current OC use] mean IGFBP-3 levels for each SNP. Non-linear additive effects for the minor allele were accounted for in the analyses through the use of dummy variables for each copy of the minor allele and the wild type allele as reference. An interaction term between OC status and cumulative number of SNP alleles were created.

Non-standardized means for circulating IGFBP-3 levels for each diplotype were obtained using one-sample t-tests. Kaplan-Meier survival analyses were used to investigate incident breast cancers after 18 years of age in relation to htSNPs or diplotypes. For healthy women, we censored at the woman's age on May 31 2009, or at the age of a prophylactic mastectomy.

Nominal P-values are presented. All P-values were two-tailed.

Results

Frequencies of htSNPs in IGFBP1 and IGFBP3 and associations with IGFBP-3 levels

Several SNPs in IGFBP1 and IGFBP3 were associated with circulating IGFBP-3 levels (Table 2). The strongest associations with IGFBP-3 levels were observed with IGFBP1 (rs1995051 and rs4988515) and IGFBP3 (rs2471551 and rs2854744) (P<0.05 for all). The minor alleles of IGFBP1 SNPs rs1995051 and rs4988515 were associated with lower IGFBP-3 levels. These minor alleles segregated at a higher frequency among women from BRCA2 families (35.5% and 20.6%, respectively) than among women from BRCA1 (25.4% and 7.5%, respectively) or BRCAX families (27.3% and 10.9%, respectively) (additional data given in Online Resource 1). Similarly, the heterozygous variant of the IGFBP3 rs2471551 was associated with lower IGFBP-3 levels. In contrast, the minor allele of the IGFBP3 rs2854744 was associated with higher IGFBP-3 levels. Women from BRCA2 families were less frequently homozygous for this variant allele (5.9%) than women from BRCA1 or BRCAX families (12.9% or 18.2%, respectively) (additional data given in Online Resource 1). These four SNPs were associated with changes in mean circulating IGFBP-3 levels ranging from 4% (rs2471551) to 12% (rs2854744). However, only rs2854744 remained significantly associated with circulating IGFBP-3 levels (nominal P=0.0002) after adjustment for multiple testing.

The differences in standardized mean IGFBP-3 levels according to rs2854744 genotype were 271 ng/ml (6%, P=0.009) for heterozygotes (CA) and 547 ng/ml (12%, P=0.0002) for homozygotes (AA) compared with the wild type (CC).

OC use influences IGFBP-3 levels and was considered as effect modifier of the SNP and IGFBP-3 level relationship. Hence, analyses were also stratified for OC use (Table 2). The mean IGFBP-3 levels were lower in the 144 non-users 4768 ng/ml (95%CI: 4636-4899) than in the 87 current OC users 5190 ng/ml (95%CI: 5039-5341). An interaction was observed between cumulative number of IGFBP1 SNP (rs3763497) and OC use (P interaction =0.034).

Diplotype associations with circulating IGFBP-3 levels

Eight IGFBP1 and nine IGFBP3 diplotypes were created from four IGFBP1 and three IGFBP3 htSNPs, respectively, based on assignment to >5 women (Figure 1). One additional composite group of each rare IGFBP1 and rare IGFBP3 diplotypes (≤5 women) were included in the analyses. Two IGFBP3 SNPs outside of the haplotype blocks (rs6670 and rs2453839) were excluded from the diplotype analyses.

Four IGFBP1 diplotypes and four IGFBP3 diplotypes were associated with changes in mean IGFBP-3 levels of >200 ng/ml relative to the mean level of 4927 ng/ml for all women (Figure 1). Among the four IGFBP1 diplotypes, GTAC/ACAT and the composite rare IGFBP1 diplotypes group were associated with decreased mean IGFBP-3 levels (-654 and -427 ng/ml, respectively), while two IGFBP1 diplotypes (GCGC/GCGC and GTAC/GTAC) were associated with increased mean IGFBP-3 levels (201 and 227 ng/ml, respectively). Among the IGFBP3 diplotypes, GCG/CCG and GCA/GCG were associated with decreased IGFBP-3 levels (-488 and -301 ng/ml, respectively), while GAG/GAG and the composite rare IGFBP3 diplotypes group were associated with increased IGFBP-3 levels (314 and 673 ng/ml, respectively). Using linear regression models, the adjusted mean IGFBP-3 level associated with the IGFBP1 diplotype GTAC/ACAT was significantly lower compared with the reference diplotype GCGC/GTAC (P=0.004), adjusted for age, weight, OC use and other IGFBP1 diplotypes. Similarly, the adjusted mean IGFBP-3 levels associated with the IGFBP3 diplotypes GCA/GCG and GCG/CCG were significantly lower compared with the reference diplotype GAG/GCA (P=0.004 and P=0.002, respectively). The associations between diplotypes and IGFBP-3 levels differed between OC users and non-users (Figure 1). The IGFBP1 diplotype GCGC/ACGC had opposing associations on IGFBP-3 levels dependent on OC use, while the IGFBP-3 levels in GTAC/ACGC carriers was similar irrespective of OC status.

After adjusting the models for BRCA family status, both the BRCA1 and BRCA2 family status (P≤0.006) and the IGFBP1 diplotype GTAC/ACAT (P=0.004) were associated with lower IGFBP-3 levels, adjusted for age, weight, OC use, and other IGFBP diplotypes. Similarly, both the BRCA1 and BRCA2 family status (P≤0.03) and the IGFBP3 diplotypes GCA/GCG (P=0.007) and GCG/CCG (P=0.002) were significantly associated with lower IGFBP-3 levels, adjusted for age, weight, OC use, and other IGFBP diplotypes. This suggests independent effects from IGFBP1, IGFBP3 diplotypes and BRCA family status on IGFBP-3 levels. After adjustment for family clustering, all statistical associations between IGFBP1 and IGFBP3 diplotype data and IGFBP-3 levels remained.

Diplotype co-segregation with BRCA1/2 mutation status

We examined possible co-segregation of diplotype frequencies and BRCA family status in all women (Figure 2a and3a) and in the first included woman from each family (Figure 2b and3b). GCGC/GTAC was the most common IGFBP1 diplotype across the BRCA1, BRCA2 and BRCAX families (Figure 2a andb). As previously reported IGFBP-3 levels were higher in BRCAX than in BRCA1/2 families [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF]. Based on the corresponding IGFBP-3 levels the diplotypes were sub-grouped into IGFBP1 low , IGFBP1 high , IGFBP3 low and IGFBP3 high compared with the overall mean IGFBP-3 level. No evident co-segregation of IGFBP1 or IGFBP3 diplotypes was observed among the BRCA1 families. Women from BRCA2 families more frequently carried IGFBP1 diplotypes associated with lower IGFBP-3 levels (60.6%) OR 2.05 (95%CI 0.97-4.30) than women from BRCA1 or BRCAX families (40.8% or 45.5%, respectively) (Figure 2a). In contrast, IGFBP3 diplotypes associated with higher IGFBP-3 levels (IGFBP3 high ) co-segregated among BRCAX families (57.3%) OR 1.68 (95%CI 1.04-2.74) compared with women from BRCA1 or BRCA2 families (45.1% or 41.2%, respectively) (Figure 3a).

Diplotypes associated with breast cancer

Against a background of a limited number of breast cancer cases (n=23), there was a nonsignificant tendency towards increased breast cancer incidence among women carrying a combination of IGFBP1 high and IGFBP3 high diplotypes (9/88, 10.2%) compared with IGFBP1 low and IGFBP3 low diplotypes (4/68, 5.9%). The incidences of breast cancer found among women carrying IGFBP1 low /IGFBP3 low diplotypes (4/69, 5.8%) and IGFBP1 high /IGFBP3 low diplotypes (6/91, 6.6%) were similar to the incidence of breast cancer among women with the IGFBP1 low /IGFBP3 low diplotypes. Among the 23 breast cancer cases, nine (39.1%) carried a IGFBP1 high /IGFBP3 high diplotype and only four (17.1%) carried a IGFBP1 low /IGFBP3 low diplotype (Figure 4a). One IGFBP1 diplotype (GTAC/ACGC) showed a tendency towards being associated with decreased risk of breast cancer (log rank P=0.050) (Figure 4b).

Discussion

To our knowledge, this is the first study to examine genetic variants (SNPs) and diplotypes in the IGFBP1 and IGFBP3 genes in relation to both circulating IGFBP-3 levels and risk of breast cancer among women from BRCA1, BRCA2 or BRCAX families. Two SNPs in IGFBP1 and two SNPs in IGFBP-3 were associated with circulating IGFBP-3 levels, but no associations between individual SNPs and breast cancer incidence were found. Several diplotypes in IGFBP1 and IGFBP3 were associated with circulating IGFBP-3 levels. Breast cancer incidence was non-significantly higher among women carrying a combination of IGFBP1 high /IGFBP3 high diplotypes associated with higher circulating IGFBP-3 levels. The frequency of IGFBP1 high and IGFBP3 high diplotypes differed between women from BRCA1, BRCA2 or BRCAX families, which were further reflected in significant differences in mean circulating IGFBP-3 levels.

The IGF axis plays important roles in normal physiology by regulating cell proliferation, differentiation and apoptosis [START_REF] Pollak | Insulin-like growth factors and neoplasia[END_REF]. In addition, considerable evidence from laboratory, clinical and epidemiological research demonstrates important roles of the IGF-I and its major binding protein IGFBP-3 in the development and progression of several tumor types, including breast cancer [START_REF] Pollak | Insulin-like growth factors and neoplasia[END_REF]. Large inter-individual variations in circulating levels of IGF-I and IGFBP-3 exist, which in part may be related to different genotypes. Previous studies have evaluated associations between circulating IGFBP-3 levels and risk of sporadic breast cancer in relation to individual IGFBP1 and IGFBP3 SNPs or haplotypes and reported conflicting results. Although many SNPs have been associated with the corresponding IGFBP-3 plasma levels, their respective associations with breast cancer risk have been inconsistent across studies.

Reports from the Nurses Health Study II and from the NCI Breast and Prostate Cancer Cohort Consortium found associations between IGFBP3 polymorphisms and circulating IGFBP-3 levels, but no substantial influence on breast cancer risk [START_REF] Schernhammer | Insulin-like growth factor-I, its binding proteins (IGFBP-1 and IGFBP-3), and growth hormone and breast cancer risk in The Nurses Health Study II[END_REF][START_REF] Patel | IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3)[END_REF]. In some studies, genetic variants and IGFBP-3 levels were inversely associated with breast cancer risk [START_REF] Al-Zahrani | IGF1 and IGFBP3 tagging polymorphisms are associated with circulating levels of IGF1, IGFBP3 and risk of breast cancer[END_REF]22], while in other studies IGFBP-3 levels were positively associated with breast cancer risk [START_REF] Key | Insulin-like growth factor 1 (IGF1), IGF binding protein 3 (IGFBP3), and breast cancer risk: pooled individual data analysis of 17 prospective studies[END_REF][START_REF] Yu | Insulin-like growth factors and breast cancer risk in Chinese women[END_REF]. In addition, meta-analyses report that high circulating IGFBP-3 levels were associated with increased risk of pre-menopausal breast cancer [START_REF] Renehan | Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk: systematic review and meta-regression analysis[END_REF][START_REF] Shi | IGF-I and breast cancer: a metaanalysis[END_REF].

Our results suggest that women carrying the heterozygous variant of IGFBP1 SNPs rs1995051, rs4988515 or IGFBP3 SNP rs2471551 have lower IGFBP-3 levels, consistent with results observed by others [START_REF] Diorio | Genetic polymorphisms involved in insulin-like growth factor (IGF) pathway in relation to mammographic breast density and IGF levels[END_REF]. Notably, these heterozygous variants all co-segregated among women from BRCA2 families. A recent study reported positive associations for the rare allele of the extensively studied IGFBP3 rs2854744 (A-202C) with both IGFBP-3 levels and proliferative benign breast disease, a marker of increased breast cancer risk [START_REF] Su | Genetic variation and circulating levels of IGF-I and IGFBP-3 in relation to risk of proliferative benign breast disease[END_REF]. The higher circulating IGFBP-3 levels observed with the AA genotype are consistent with the functional differences between the A and C alleles indicated in vitro, describing significantly higher promoter activity of the A allele compared to the C allele [START_REF] Deal | Novel promoter polymorphism in insulin-like growth factor-binding protein-3: correlation with serum levels and interaction with known regulators[END_REF][START_REF] Paquette | Identification of upstream stimulatory factor binding sites in the human IGFBP3 promoter and potential implication of adjacent single-nucleotide polymorphisms and responsiveness to insulin[END_REF]. Consistent with these findings, we have previously reported that women carrying the AA genotype have higher IGFBP-3 levels and that this genetic variant segregated at a higher frequency among women from BRCAX families in the present study population [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF], while the CC genotype segregated among women from BRCA1 families [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF]. However, there were residual effects of BRCA family status on IGFBP-3 levels beyond the effect of the rs2854744 genotype and beyond the effects of diplotypes.

Although mutations in the high-penetrance BRCA1 and BRCA2 genes predispose to earlyonset breast cancer, considerable individual variation in tumor incidence and onset exist. In addition, only one-third of women from Swedish high-risk breast cancer families carry disease-causing mutations in BRCA1 and BRCA2. Considering that differences in individual breast cancer susceptibility probably result from the additive effect of multiple genetic variants, where each variant contribute but a modest risk increase [START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF], we investigated associations between in vivo IGFBP-3 levels and IGFBP1 and IGFBP3 diplotypes. The diplotype analyses revealed significant variation in circulating IGFBP-3 levels between the highest and lowest IGFBP1 diplotypes (range ~900 ng/ml). Interestingly, the homozygous IGFBP1 diplotypes were all associated with the highest IGFBP-3 levels. Similar variation was observed for the IGFBP3 diplotype associations with circulating IGFBP-3 levels (range ~1100 ng/ml). Two out of three homozygous IGFBP3 diplotypes, of which one contained the AA genotype of IGFBP3 SNP rs2854744, were associated with the highest IGFBP-3 levels.

By applying diplotype analyses in preference to individual SNP analyses, we found a tendency towards increased breast cancer incidence among women carrying a combination of IGFBP1 high /IGFBP3 high diplotypes. The higher frequency of IGFBP3 high diplotypes among BRCAX families implies that the subgroup of these women who carry a combination of IGFBP1 high /IGFBP3 high diplotypes may have an increased breast cancer risk related to high IGFBP-3 levels. This is the first study where IGFBP1 and IGFBP3 diplotype analyses have been applied to examine influence on breast cancer risk; previous studies have only examined haplotype data [START_REF] Schernhammer | Insulin-like growth factor-I, its binding proteins (IGFBP-1 and IGFBP-3), and growth hormone and breast cancer risk in The Nurses Health Study II[END_REF][START_REF] Patel | IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3)[END_REF][START_REF] Al-Zahrani | IGF1 and IGFBP3 tagging polymorphisms are associated with circulating levels of IGF1, IGFBP3 and risk of breast cancer[END_REF]. One IGFBP1 diplotype (GTAC/ACGC) was borderline associated with decreased breast cancer incidence after age 18. However, these observations warrant replication in larger study populations.

Current OC use increases the overall mean circulating IGFBP-3 levels [START_REF] Jernstrom | Differences in IGFBP-3 regulation between young healthy women from BRCAX families and those belonging to BRCA1/2 families[END_REF][START_REF] Jernstrom | Genetic and nongenetic factors associated with variation of plasma levels of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in healthy premenopausal women[END_REF]. However, the associations between diplotypes and IGFBP-3 levels differed between OC users and nonusers. The variations present at position 2 and 3 in the IGFBP1 GCGC/ACGC and GTAC/ACGC diplotypes suggest that the CC and GG genotypes of these polymorphisms may causally influence IGFBP-3 levels during OC use, or tag for other unknown functional SNPs. These data suggest that women from high-risk families carrying specific combinations of IGFBP1 and IGFBP3 diplotypes may be more susceptible to IGFBP-3 level modulation by OC use, which could further modify their risk of breast cancer. Further studies and the identification of causal mechanisms are needed to better understand how these genotypes and diplotypes may modify non-genetic factors related to IGFBP-3.

In conclusion, this is the first study to investigate associations between multiple genetic polymorphisms and diplotypes in IGFBP1 and IGFBP3 and circulating IGFBP-3 levels among women from BRCA1, BRCA2 or BRCAX families. Our findings suggest that individual SNPs and diplotypes are associated with circulating IGFBP-3 levels. Theses associations vary according to OC status and may influence the risk of breast cancer among women from highrisk breast cancer families. We observed co-segregation of IGFBP1 low diplotypes among BRCA2 families and of IGFBP3 high diplotypes among BRCAX families. This study highlights the informative advantage of applying diplotype analyses over individual SNPs or haplotypes.

Our findings warrant confirmation in independent study populations.

Figure 1 .

 1 Figure 1. Associations between common diplotypes in the IGFBP1 and IGFBP3 genes with

Figure 2 .

 2 Figure 2. IGFBP1 diplotype distribution among women from BRCA families. Frequency (%)

Figure 3 .

 3 Figure 3. IGFBP3 diplotypes distribution among women from BRCA families. Frequency (%)

Table 1 .

 1 Characteristics of all women and of women with available IGFBP-3 levels

Table 2 .

 2 Associations between IGFBP1 and IGFBP3 SNP genotypes and standardized mean IGFBP-3 levels

Acknowledgments

We thank our research nurses Kerstin Nilsson, Monica Pehrsson, Anita Schmidt-Casslén for assistance with body measurements and blood drawing and Johanna Wagenius, Johanna Frenander, Helen Sundberg, Malin Sternby, and Susanna Holmquist for their assistance with recruitment. We also thank Dr Pär-Ola Bendahl for assistance with the family clustering analyses and Dr. Eric T. Dryver for proofreading the manuscript.

This study was supported by grants from Vetenskapsrådet (the Swedish Research Council, K2001-27GX-14120-01A and K2008-68X20802-01-3), the Medical Faculty, Lund University, the Mrs. Berta Kamprad's Foundation, the Gunnar Nilsson Foundation, the Crafoord Foundation, the South Swedish Health Care Region (Region Skåne), Lund Hospital Fund and the Swedish Cancer Society.

Conflict of interest

Supplementary data: Table 1. Distribution of IGFBP1 and IGFBP3 SNP genotypes among all women and women from BRCA1, BRCA2 or BRCAX families.


Maria A Loizidou

Dr Andreas Hadjisavvas

John P A Ioannidis

Kyriacos Kyriacou

Replication of genome-wide discovered breast cancer risk loci in the Cypriot population

Keywords: breast cancer, Cyprus, GWAS replication, SNP

come

Introduction

Breast cancer is the most common cancer in women in developed countries [START_REF] Parkin | Global cancer statistics, 2002[END_REF].

Genetic factors play an important role in both sporadic and familial breast cancer [START_REF] Balmain | The genetics and genomics of cancer[END_REF][START_REF] Stratton | The emerging landscape of breast cancer susceptibility[END_REF][START_REF] Houlston | The search for low-penetrance cancer susceptibility alleles[END_REF]. In the mid 1990s, the two major breast cancer susceptibility genes, BRCA1 and BRCA2, were identified [START_REF] Miki | A strong candidate for the breast and ovarian cancer susceptibility gene brca1[END_REF][START_REF] Wooster | Identification of the breast cancer susceptibility gene brca2[END_REF]. Inherited mutations in these high-penetrance genes explain only a small fraction of the familial risk for the disease [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with brca1 or brca2 mutations detected in case series unselected for family history: A combined analysis of 22 studies[END_REF] and attempts to localize additional highly penetrant breast cancer susceptibility genes have failed [START_REF] Smith | A genome wide linkage search for breast cancer susceptibility genes[END_REF].

This led to the suggestion that the remaining breast cancer susceptibility is polygenic in nature and that multiple low penetrance alleles, each conferring a small risk, are involved [START_REF] Pharoah | Polygenic susceptibility to breast cancer and implications for prevention[END_REF][START_REF] Galvan | Beyond genome-wide association studies: Genetic heterogeneity and individual predisposition to cancer[END_REF]. In the last few years, a new harvest of breast cancer susceptibility alleles has emerged using genome-wide association studies (GWAS). Scanning common variants in an agnostic fashion, these studies have identified several new associations with robust statistical support for influencing breast cancer susceptibility [START_REF] Hunter | A genome-wide association study identifies alleles in fgfr2 associated with risk of sporadic postmenopausal breast cancer[END_REF][START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF][START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Gold | Genome-wide association study provides evidence for a breast cancer risk locus at 6q22.33[END_REF][START_REF] Stacey | Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Zheng | Genome-wide association study identifies a new breast cancer susceptibility locus at 6q25.1[END_REF][START_REF] Thomas | A multistage genome-wide association study in breast cancer identifies two new risk alleles at 1p11.2 and 14q24.1 (rad51l1)[END_REF][START_REF] Ahmed | Newly discovered breast cancer susceptibility loci on 3p24 and 17q23.2[END_REF]. Between 2007 and 2009, a total of eleven breast cancer susceptibility loci in or near genes such as FGFR2, MAP3K1, LSP1, TOX3 (formerly known as TNRC9), COX11 and at 1p11.2, 2q35, 3p24, 6q25.1, 6q22.23 and 8q24.21, were discovered [START_REF] Ioannidis | A compendium of genome-wide associations for cancer: Critical synopsis and reappraisal[END_REF]. Newer and larger GWAS efforts continue to yield additional risk loci [START_REF] Turnbull | Genome-wide association study identifies five new breast cancer susceptibility loci[END_REF].

The majority of breast cancer GWAS and replications published to-date have been conducted in Northern European populations [START_REF] Hunter | A genome-wide association study identifies alleles in fgfr2 associated with risk of sporadic postmenopausal breast cancer[END_REF][START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF][START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Thomas | A multistage genome-wide association study in breast cancer identifies two new risk alleles at 1p11.2 and 14q24.1 (rad51l1)[END_REF][START_REF] Ahmed | Newly discovered breast cancer susceptibility loci on 3p24 and 17q23.2[END_REF][START_REF] Stacey | Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF] and to a lesser extent in Asians [START_REF] Zheng | Genome-wide association study identifies a new breast cancer susceptibility locus at 6q25.1[END_REF] and Ashkenazi Jews [START_REF] Gold | Genome-wide association study provides evidence for a breast cancer risk locus at 6q22.33[END_REF]. It is important to assess whether these variants confer risk across different populations and also to assess the magnitude of risk conferred [START_REF] Ioannidis | Population-wide generalizability of genome-wide discovered associations[END_REF].

The aim of this study was to evaluate previously GWAS-identified breast cancer risk variants in the Cypriot population using a case-control design. The Cypriot population belongs to the wider Caucasian family, but it is genetically distinct from other Caucasian populations and substantially different from Northern European populations [START_REF] Novembre | Genes mirror geography within europe[END_REF]. In this report, a total of eleven genetic variants were examined for their association with breast cancer risk.

Materials and Methods

Study subjects

The subjects included in the current study are drawn from the MASTOS study, a population-based case-control study of breast cancer in Cyprus. Blood samples were collected between 2004 and 2006 from 1109 female breast cancer patients diagnosed between 40-70 years old and 1177 unrelated healthy controls of the same age range as the cases. No matching was performed. Cases participating in the study were women who were previously diagnosed with breast cancer between January 1999 and December 2006. The majority of cases (n=981) were ascertained from the Bank of Cyprus Oncology Centre that operates as a referral centre and offers treatment and follow-up for 80-90% of all breast cancer cases diagnosed in Cyprus. The rest of the patients (n=128), were recruited at the Oncology Departments of the Nicosia, Limassol, Larnaca and Paphos district hospitals. The control group consisted of healthy women who were participating in the National program for breast cancer screening with the use of mammography. Volunteers were enrolled in the study during the same calendar period as the cases, from the 4-district mammography screening centers that operate in Cyprus. Eligible controls were women with no previous history of breast cancer who had a negative mammography result. All study participants, both cases and controls, were of Greek Cypriot Caucasian origin. The participation rate of cases and controls was very high covering around 98% of eligible cases and controls who were approached to participate and provide DNA. Breast cancer cases were verified by reviewing histological reports. The study protocol was approved by the National Bioethics Committee of Cyprus. All participants provided written informed consent.

SNP Selection and Genotyping

We selected and genotyped 11 SNPs that had been associated with increased risk of breast cancer in at least one publication using the genome-wide significance threshold of p<5x10 -8 as of the end of 2009 [START_REF] Hoggart | Genomewide significance for dense snp and resequencing data[END_REF]. These include rs2981582 in FGFR2, rs889312 in MAP3K1, rs3817198 in LSP1, rs3803662 in TOX3, rs6504950 in COX11, rs11249433 at 1p11.2, rs13387042 at 2q35, rs4973768 at 3p24, rs2180341 at 6q22.23, rs2046210 at 6q25.1 and rs13281615 at 8q24.21.

Genotyping was performed using the Taqman SNP genotyping assays from Applied Biosystems Inc. (ABI). Each assay was carried out using 10 ng genomic DNA in a 5 μl reaction using Taqman Universal PCR Master Mix (ABI), forward and reverse primers, and FAM and VIC-labelled probes purchased from Applied Biosystems (ABI Pre-Designed assays). All assays were carried out in 384-well plates. The fluorescence profile was read on an ABI PRISM 7900HT instrument and the results analyzed with Sequence Detection Software (ABI). For quality control, 96 random samples were genotyped in duplicate and had identical genotyping assignments.

Investigators were blinded as to whether samples were duplicates, cases or controls.

The order of the DNA samples on 384-well plates was randomized in order to ensure the same study conditions for samples from cases and controls. Genotyping call rates ranged from 96 to 99% and duplicate concordance rates were 100%.

Data Analysis

Hardy Weinberg equilibrium (HWE) was assessed in the control samples by applying an exact test. Deviation from HWE was considered nominally statistically significant at the P< 0.05 level. Genotype frequency differences between cases and controls were tested using unconditional logistic regression without any adjustments. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated under the log-additive model, using the major allele in the Cypriot control population as reference. ORs thus represent the risk conferred per copy of the minor allele. Secondary analyses examined also recessive and dominant models of inheritance.

Pearson's correlation coefficient (r) was used to estimate the correlations in minor allele frequencies between our study, HapMap CEU population [START_REF]The international hapmap project[END_REF] and the GWAS population where each SNP was first discovered. The overall correlation between ORs in the Cypriot population and the GWAS population where each SNP was first discovered was also calculated.

The power of the study to detect ORs similar to those previously found in the GWAS, given the allele frequencies observed in the Cypriot population, was estimated at alpha value of 0.05.

Statistical analyses were run in SNPStats [START_REF] Sole | Snpstats: A web tool for the analysis of association studies[END_REF] for HWE and logistic regressions, PS [START_REF] Dupont | Power and sample size calculations. A review and computer program[END_REF] for power calculations, and Stata, version 10.1 (College Station, TX), for correlation analyses. P-values for association are two-tailed and not adjusted for multiple comparisons, since this is a replication effort for associations that already have robust statistical support. Design and reporting follow the STREGA guidance [START_REF] Little | Strengthening the reporting of genetic association studies (strega): An extension of the strobe statement[END_REF].

Results

Table 1 shows the allele frequencies in cases and controls for the 11 SNPs which were genotyped in this study and genotype frequencies appear in Supplementary Table 1.

Genotype distributions of controls at each locus were consistent with HWE (Supplementary Table 1).

Four of the eleven breast cancer-associated SNPs discovered in GWAS (i.e. rs2981582, rs889312, rs13387042 and rs2046210) were statistically significantly associated with breast cancer risk in this study at P less than 0.05 (Table 1). All associations were of very modest magnitude, and the largest OR association observed in the Cypriot population was with the G allele of rs889312, located in the MAP3K1 gene (OR=1.18 per copy of risk allele).

The other seven SNPs were not associated with breast cancer risk in our study. For three of the seven SNPs (rs4973768, rs6504950 and rs2180341) the point estimates of the ORs were in the opposite direction than previously described and one other SNP (rs11249433) had OR=1.00 in the Cyprus population.

Evaluation of the 11 SNPs under recessive and dominant models of inheritance did not show lower p-values for the association for any of the SNPs, with the exception of rs2180341 that was marginally associated with breast cancer risk in a recessive model (OR=1.36; P=0.054). P-values were also lower with the recessive rather than logadditive model for rs889312, and rs2046210 and they were nominally significant with both models.

The correlation coefficients of effect sizes (per-allele OR) and minor allele frequencies between the Cypriot and the GWAS populations are r=0.58 (p=0.064) and 0.88 (p<0.001) respectively (Figure 1). Similarly, the Pearson correlation coefficient in minor allele frequencies between the Cypriot and the CEU Hapmap population is 0.83 (p=0.001).

The power of our study (1,109 case patients and 1,177 control subjects) to detect ORs similar to those previously found, given the allele frequencies observed in the Cypriot population ranges from 8% for rs6504950 to 97% for rs2180341, at alpha value of 0.05 (see Figure 1). By summing the power estimates for all SNPs to detect the respective ORs previously seen in GWAS, it is estimated that if ORs were identical in the Cypriot population, our study would be expected to have found five nominally statistically significant associations among the eleven tested. This is not significantly different from the 4 nominally significant associations that we observed. [START_REF] Hunter | A genome-wide association study identifies alleles in fgfr2 associated with risk of sporadic postmenopausal breast cancer[END_REF][START_REF] Easton | Genome-wide association study identifies novel breast cancer susceptibility loci[END_REF][START_REF] Stacey | Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF][START_REF] Thomas | A multistage genome-wide association study in breast cancer identifies two new risk alleles at 1p11.2 and 14q24.1 (rad51l1)[END_REF][START_REF] Ahmed | Newly discovered breast cancer susceptibility loci on 3p24 and 17q23.2[END_REF][START_REF] Stacey | Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer[END_REF] and other populations [START_REF] Zheng | Genome-wide association study identifies a new breast cancer susceptibility locus at 6q25.1[END_REF][START_REF] Gold | Genome-wide association study provides evidence for a breast cancer risk locus at 6q22.33[END_REF] have recently identified and confirmed new genetic variants associated with breast cancer. However, it is recognized that the prevalence of breast cancer as well as the allele frequencies differ across populations. It is therefore important to understand the effect of the GWAS discovered markers in women of other ethnicities. This is the first study in Cypriot women that has systematically evaluated breast cancer susceptibility loci recently identified through GWAS. Of the eleven SNPs evaluated, four (rs2981582, rs889312, rs13387042, rs2046210) were found to be nominally significantly associated with breast cancer risk in the Cypriot population.

Discussion

GWAS in Europeans

The effect estimates of these four SNPs are in the same direction as previously reported in the original studies. Based on estimated power, five associations would be expected to be nominally significant among the eleven tested , if the effects were of the same magnitude as seen in the GWAS-not very inconsistent with the four statistically significant associations actually observed.

The strongest association was found for rs889312, for which there was an 18% elevated risk of breast cancer with each risk allele. The per-allele OR in the current study for this SNP was slightly higher than that estimated in the original GWAS. This enrichment could be due to modification by other risk factors, such as gene-gene or gene environment interactions or differences in LD pattern. Alternatively, it could be a chance finding, given that the 95% CI for that SNP goes as low as 1.04. The other three SNPs were, in general, weakly associated with the risk of breast cancer, with

The overall correlation between genetic effects (per-allele ORs) in the Cypriot and the GWAS populations is modest and not even nominally significant. Conversely, for the same variants, there is a very strong correlation in allele frequencies (correlation coefficient=0.88, P<0.001). This suggests that genuine differences may exist in the genetic effects. If so, then risk models built with these variants may give different results in different ethnicities [START_REF] Gail | Value of adding single-nucleotide polymorphism genotypes to a breast cancer risk model[END_REF].

As with any unrelated case-control genetic association study, population stratification and "cryptic relatedness" between subjects may be potential sources of bias in our study. Population stratification may occur when study participants are selected from subpopulations with a different prevalence of the phenotypes and genotypes. In order to avoid confounding from population stratification, we only enrolled women of selfverified Greek-Cypriot ancestry into the study. "Cryptic relatedness" between subjects leads to violation of the assumption of independent data points. The effect of "cryptic relatedness" is likely to be more important in inbred population isolates than in large out-bred populations [START_REF] Thomas | Point: Population stratification: A problem for casecontrol studies of candidate-gene associations?[END_REF]. Cyprus is a small island with less than a million population. Despite its small size, inbreeding is practically absent since consanguineous marriages are not permitted by law. Furthermore, in our study, all participants were unrelated minimizing potential bias due to relatedness.

The Cypriot population is a Caucasian-origin population. However, principal component analysis of samples from diverse regions of Europe shows substantial genetic diversity across different ethnicities that largely reflect the geographic distribution of populations [START_REF] Novembre | Genes mirror geography within europe[END_REF]. In this regard, the Cypriot could be distinguished clearly from populations of neighboring countries and were quite distant on both Fig. 1 Correlation of genetic effects and risk allele frequencies in GWAS populations vs. the Cypriot population. A) Genetic effects expressed in the ORs scale B) Risk allele frequencies. The data are shown by colored circles. The boxed number neighbouring each data point shows the estimated power (in %) at alpha value of 0.05 to detect the OR of the GWAS also in the Cypriot population, given the allele frequency observed in the Cypriot control subjects Table 1. Allele frequencies in case patients and control subjects and odds ratio (OR) for previously identified risk SNPs + Tested allele / reference allele * GWAS-described risk effect is for the major allele

Acknowledgements

We thank Drs Yiola Marcou, Eleni Kakouri, Maria Daniel, Panayiotis Papadopoulos and Simon Malas for their assistance in recruiting breast cancer patients. We also thank Rena Papachristoforou and Thalia Michael for their assistance in data collection and Christina Flouri and Ioanna Neophytou for help with genotyping. We are indebted to all study participants. This work was supported by ""Cyprus Research Promotion Foundation"" grant 0308/08 and the Cyprus Institute of Neurology and Genetics.

each allele conferring elevated risks of 15% or lower. These modest effects are consistent with what GWAS have also shown for these SNPs.

Seven previously identified risk SNPs were not associated with breast cancer risk in the Cypriot population (P values ranged from 0.066 to 0.88). For the majority of these SNPs, limited power is the most likely explanation for the null associations observed.

However, this study was well-powered (97% estimated power) to detect the effect of rs2180341, recently identified following a GWAS in the Ashkenazi Jewish genetic isolate [START_REF] Gold | Genome-wide association study provides evidence for a breast cancer risk locus at 6q22.33[END_REF]. This association was validated in a subsequent replication analysis of independent cohorts of cases and controls of both Ashkenazi Jewish as well as non-Jewish, predominantly European American, populations [START_REF] Kirchhoff | The 6q22.33 locus and breast cancer susceptibility[END_REF] . It is noted that the aggregated analysis of European breast cancer cases and controls showed a significant association in all modes of analysis, with the strongest association signal assuming the recessive model [START_REF] Kirchhoff | The 6q22.33 locus and breast cancer susceptibility[END_REF]. Under the recessive model, rs2180341 was marginally associated with breast cancer risk in the Cypriot population. Most GWAS-discovered SNPs fit best to a log-additive model, most likely due to power considerations [START_REF] Salanti | Underlying genetic models of inheritance in established type 2 diabetes associations[END_REF][START_REF] Pereira | Discovery properties of genome-wide association signals from cumulatively combined data sets[END_REF]. However, for some SNPs, alternative models of inheritance may also apply and would be interesting to compare such models in different populations.

For four of the SNPs, the point estimates were not in the same direction in the Cyprus population versus the GWAS populations. Two of these SNPs (rs4973768 and rs11249433) have been previously found to be stronger with estrogen receptor (ER) positive than with ER negative breast cancer [START_REF] Thomas | A multistage genome-wide association study in breast cancer identifies two new risk alleles at 1p11.2 and 14q24.1 (rad51l1)[END_REF][START_REF] Ahmed | Newly discovered breast cancer susceptibility loci on 3p24 and 17q23.2[END_REF]. We have not evaluated tumor pathology in our study. It is possible that the presence of an excess of ER negative cases in our sample, may have masked the effects of these SNPs.

principal component dimensions from all Northern European sets [START_REF] Novembre | Genes mirror geography within europe[END_REF]. The data of our study reinforce the need to replicate the GWAS discovered variants across different populations and ethnicities. The effects sizes of GWAS need to be verified separately in different populations, even when these populations are all under the Caucasian group.

Conclusions

In conclusion, we replicated four of the eleven regions identified as significantly associated with breast cancer from the GWAS, in a sample of Cypriot breast cancer cases and ethnically matched controls. The results of our study provide further support for the need to critically assess the generalizability of previously identified breast cancer associations and show the complexity of uniformly applying GWAS findings across different populations.

Competing interests

The authors declare that they have no competing interests.


W Groothoff

J J L Klink

U Bültmann

Corné A M Roelen
email: corne.roelen@arboned.nl

Petra C Koopmans

MD PhD; Willem Van Rhenen

PhD Johan W Groothoff

MD Jac J L Van Der Klink

U Bültmann Corné

MD PhD; A M Roelen

Trends in return to work of breast cancer survivors

Keywords: sick leave, breast cancer, return to work, epidemiology

Trends in return to work of breast cancer survivors

INTRODUCTION Word count: 2020

Breast cancer is the most common malignancy in women of working ages. The majority of women survive because they are diagnosed with early-stage disease and receive a treatment that is focused on curing breast cancer and preventing relapse of disease. The age-adjusted five-year survival varies between European countries from 70% in the Czech Republic and Poland to more than 80% in the Scandinavian countries and the survival rates are increasing [1]. In the Netherlands, the five-year survival after breast cancer was 77% in 1998, 81% in 2003 and 85% in 2008 [2,3]. Employed women usually interrupt their work for the treatment of breast cancer. Prolonged sickness absence often generates loss of income and social exclusion. Therefore, return to work (RTW) during or after breast cancer treatment is important, all the more because RTW restores social relations and participation, which adds to the patient's quality of life [START_REF] Ganz | Breast cancer survivors: psychosocial concerns and quality of life[END_REF][START_REF] Kennedy | Returning to work following cancer: a qualitative exploratory study into the experience of returning to work following cancer[END_REF][START_REF] Groenvold | Health-related quality of life in early breast cancer[END_REF].

RTW after breast cancer has been studied in different countries with different jurisdictions. In a prospective multicenter study from 1990 to 1994 including research groups from the UK, Sweden and Italy, it was reported that 79% of premenopausal women had returned to work 12 months after the diagnosis of breast cancer and 86% within 36 months of diagnosis [START_REF] Johnsson | Factors associated with return to work after breast cancer treatment[END_REF]. In a Canadian study, 71% of women diagnosed with breast cancer in 1996-1997 resumed work within 12 months of diagnosis and 89% within 36 months [START_REF] Drolet | Work absence after breast cancer diagnosis: a population-based study[END_REF]. In 2001-2002, 82% of American women had returned to work 18 months after the diagnosis of breast cancer [START_REF] Bouknight | Correlates of return to work for breast cancer survivors[END_REF]. Recent European studies reported lower percentages of women resuming work after breast cancer. For example, [START_REF] Johnsson | Predictors of return to work ten months after primary breast cancer surgery[END_REF] found that 59% of 102 Swedish women had returned to work within 10 months of surgery for primary early-stage breast cancer [START_REF] Johnsson | Predictors of return to work ten months after primary breast cancer surgery[END_REF]. [START_REF] Fantoni | Factors related to return to work by women with breast cancer in Northern France[END_REF] found that 54% of 379 women diagnosed with breast cancer in 2004-2005 in France resumed work within 12 months after starting cancer treatment [START_REF] Fantoni | Factors related to return to work by women with breast cancer in Northern France[END_REF]. The different RTW rates may be explained by differences in health care systems and social insurances across countries [START_REF] Snashall | Health of the working age population. New report recommends integration of occupational health into mainstream healthcare[END_REF][START_REF] Bloch | Who returns to work and why ? A six country study on work incapacity and integration[END_REF]. In the Netherlands, for example, sickness absence is usually fully compensated by the employer in the first year and up to 70% in the second year, irrespective of the cause of sickness absence. Thus, not only work-related injuries are compensated, but also non work-related disorders such as breast cancer. breast cancer and women with recurrent breast cancer were excluded from analysis as RTW trends in these cases may differ from the trends in primary breast cancer among women.

Ethical considerations

Ethical approval was not required because the Act on Scientific Medical Research does not apply to the analysis of register data without involving the employees themselves [START_REF]Guidelines for the use of personal data in scientific research[END_REF]. All employees consented to the analysis of their sickness absence data when data were registered.

RESULTS

The incidence of primary breast cancer increased from 101 per 100,000 employed women in 2002 to 124 per 100,000 employed women in 2008, which is in line with the Dutch population incidence of 129 per 100,000 women in 2008. The numbers and age distribution of all employed women and the selected breast cancer cases are presented in Table 1.

[insert Table 1 about here]

Proportion of RTW

In the period from 2002 to 2008, the proportion of partial RTW was 65-76% in women aged <40 years, 66-75% in women aged 40-50 years, and 68-79% in women aged >50 years. Among women aged >50 years at diagnosis there was a declining trend (χ 2 for linear trends P=0.037) in the proportion of partial RTW from 79% in 2003 to 68% in 2008 (Figure 1). [insert Tables 2 and 3 about here]

DISCUSSION

Partial RTW one year after the diagnosis of breast cancer was more or less stable around 70% in the period from 2002 to 2008, whereas full RTW showed a decreasing trend in women of all ages.

The strength of our study is that it covered a large population of employees working in different economic sectors. Studying registered sickness absence eliminates the recall bias that affects selfreported sickness absence [START_REF] Van Poppel | Measuring sick leave: a comparison of self-reported data on sick leave and data from company records[END_REF][START_REF] Voss | Comparisons of self-reported and register data on sickness absence among public employees in Sweden[END_REF]. The sickness absence register study could be performed quickly and inexpensively without the need to involve the employees themselves. The main limitation, however, was that the number of variables available from the sickness absence register was limited.

For example, information about work accommodations or placement in another job was not recorded in the register. Furthermore, we had no access to the medical files of the patients. Hence, information on the stage of breast cancer (local, regional or metastatic), type of treatment, and persisting limitations and symptoms such as fatigue [START_REF] Spelten | Cancer, fatigue and the return of patients to worka prospective cohort study[END_REF][START_REF] Bower | Fatigue in long-term breast cancer survivors[END_REF][START_REF] Hansen | Breast cancer survivors at work[END_REF][START_REF] Ahn | Impact of breast cancer diagnosis and treatment on work-related life and factors affecting them[END_REF], distress [START_REF] Henselmans | Identification and prediction of distress trajectories in the first year after a breast cancer diagnosis[END_REF][START_REF] Calvio | Measures of cognitive function and work in occupationally active breast cancer survivors[END_REF] and depressive symptoms [START_REF] Hansen | Breast cancer survivors at work[END_REF][START_REF] Reich | Depression, quality of life and breast cancer: a review of the literature[END_REF] was not available.

The fact that the sickness absence register contained a limited number of variables impeded the interpretation of the results. Therefore, the decreasing proportions of full RTW can not be explained robustly. It is possible that the decreasing proportion of RTW is the result of a change in Dutch disability policies in 2004. Before 2004, sickness absence was compensated by employers for a period of one year, and since 2004 sickness absence compensation is granted for a period of two years.

However, if policy changes were responsible for the decrease in full RTW, then one would also expect a decline in partial RTW, which was not found.

An alternative explanation for the decreasing proportions of RTW one year after the diagnosis of breast cancer may be the broadened indication for chemotherapy in the Netherlands among young breast cancer survivors [START_REF] Sukel | Substantial increase in the use of adjuvant systemic treatment for early stage breast cancer reflects changes in guidelines in the period 1990-2006 in the southeastern Netherlands[END_REF]. It has been recognized that women who are treated with chemotherapy are less likely to resume their work [7, 9-11, 22, 27, 28]. As chemotherapy delays RTW, one would expect a longer time to partial RTW if the decreasing proportion of full RTW after breast cancer is the result of a more sophisticated adjuvant treatment. The study showed, however, that the time to RTW did not change significantly over the years. Thus, women resume work initially, but it is possible that full RTW is impeded by symptoms persisting after the treatment of breast cancer. [START_REF] Hansen | Breast cancer survivors at work[END_REF] reported that fatigue was most strongly related to work limitations and explained 71% of the symptom burden of employees after the diagnosis of breast cancer [START_REF] Hansen | Breast cancer survivors at work[END_REF]. [START_REF] Calvio | Measures of cognitive function and work in occupationally active breast cancer survivors[END_REF] reported that the symptom burden reduced the performance-based work output of breast cancer survivors. They also showed that breast cancer survivors experienced cognitive difficulties, particularly due to deficits in memory and executive function [START_REF] Calvio | Measures of cognitive function and work in occupationally active breast cancer survivors[END_REF], while work is becoming more and more mentally demanding [START_REF] Van Hooff | The Netherlands Working Conditions Survey[END_REF]. Although the level of job control in the Netherlands is relatively high, work pace and work pressure are increasing and cancer survivors may feel distressed that they are not able to perform at their pre-diagnosis level [START_REF] Kennedy | Returning to work following cancer: a qualitative exploratory study into the experience of returning to work following cancer[END_REF]. Therfore, women experiencing fatigue, distress and cognitive difficulties may be uncertain to fully resume their work.

Another explanation for the decline in full RTW after breast cancer may be that attitudes towards work have changed after cancer. Breast cancer survivors may put less value on work and for half of them financial pressure was the primary reason for resuming work [START_REF] Kennedy | Returning to work following cancer: a qualitative exploratory study into the experience of returning to work following cancer[END_REF]. [START_REF] Tiedtke | Experiences and concerns about 'returning to work' for women breast cancer survivors: a literature review[END_REF] reviewed articles published between 1995 and 2008 on the experiences of breast cancer survivors with RTW [START_REF] Tiedtke | Experiences and concerns about 'returning to work' for women breast cancer survivors: a literature review[END_REF]. The authors found that women altered their work priorities and ambitions, which was reported by [START_REF] Maunsell | Work situation after breast cancer : results from a population-based study[END_REF] as one of the reasons for stopping work after breast cancer [START_REF] Maunsell | Work situation after breast cancer : results from a population-based study[END_REF].

Finally, it is important to notice that breast cancer survivors receive little advice about returning to work [START_REF] Tiedtke | Experiences and concerns about 'returning to work' for women breast cancer survivors: a literature review[END_REF]. Work issues were scarcely discussed with treating physicians and in many cases company physicians or insurance physicians followed the patient's wishes, letting them decide for themselves about resuming work [START_REF] Kennedy | Returning to work following cancer: a qualitative exploratory study into the experience of returning to work following cancer[END_REF]. Uncertainty about work ability after treatment and mixed feelings about the work environment may be barriers in the RTW process. A guideline has been developed for Dutch

OPs to support cancer survivors in RTW [START_REF]TABLE 2 Partial return to work with 50% of the earnings before sickness absence within one year of breast cancer diagnosis[END_REF]. This guideline accentuates the importance of advising cancer patients about their RTW, taking the influence of physical and psychological limitations on the patient's work ability into account. Implementation of this guideline in daily occupational health practice may improve RTW of cancer survivors in the years to come.

In conclusion, the proportion of women who resume working after breast cancer has decreased in the Netherlands over the period from 2002 to 2008. This warrants more epidemiological research on the trends in RTW in breast cancer survivors across countries. In this study, follow-up of breast cancer survivors was restricted to one year and longer follow-up is needed to provide insight in sustained work beyond one year of diagnosis.

  days. Full RTW was defined as RTW with 100% of the earnings before sickness absence for ≥28 consecutive days. Proportions of women with partial RTW and full RTW were analysed one year after reporting sick with breast cancer. Trends in the proportions of partial and full RTW were investigated by Chi-square analysis for linear trends stratified by age (<40 years, 40-50 years and >50 years). The time to partial RTW was examined by Cox proportional hazards regression analysis stratifying for age <40 years, 40-50 years, and >50 years. Likewise, the time to full RTW was investigated in a separate Cox regression model. Hazard ratios (HR) relative to RTW in 2002 and their 95% confidence intervals (CI) were calculated with HR<1.0 reflecting a longer time to RTW relative to 2002 and HR>1.0 a shorter time to RTW relative to 2002. All statistical analyses were performed in SPSS for Windows version 16 and statistical significance was set at the 5% level (α=0.05).

TimeFigure 1

 1 Figure 1 Trends in return to work (RTW) after the diagnosis of breast cancer from 2002 to 2008

  In 2002, 52% of women had full RTW within one year of reporting sick with breast cancer as compared to 43% in 2008. Figure1shows linear declining trends in the proportion of full RTW in The median time to partial RTW was 294 days in 2002 and 282 days in 2008. The time to partial RTW did not change significantly over the years compared with 2002 (Table2). As is shown in Table3, the median time to full RTW did not change over the years either, although the overall median time to full RTW was borderline significantly (p=0.05) longer in 2008 compared with 2002.

	women of all ages. Full RTW in women <40 years decreased from 55% in 2002 to 40% in 2008 (χ 2 for
	linear trends P=0.003). In women aged 40-50 years, full RTW decreased from 46% in 2002 to 41% in
	2008 (P=0.042), and in women >50 years from 59% to 46% (P=0.001).
	[insert figure 1 about here]
	Time to RTW

The proportion of partial RTW increased non-significantly (P=0.074) from 66% in 2002 to 74% in 2008 among women who were diagnosed with breast cancer between 40 and 50 years of age.

TABLE 1 Study population stratified by age

 1

	2002	2003	2004	2005	2006	2007	2008

Data analysis

The sickness absence data were retrieved from the ArboNed sickness absence register. Partial RTW was defined as RTW with 50% of the earnings before sickness absence for at least 28 consecutive

CONFLICT OF INTEREST

All authors state that they have no financial or other conflicts of interest that might bias their work and contribution to this paper.