Adriana J Maaskant-Braat

Lonneke V Poll-Franse

Ph.D Adri C Voogd

Willem W Jan

Rudi M Coebergh

M Roumen

Tutein Cathelijne

Grard A Nolthenius-Puylaert

Nieuwenhuijzen

M.D Adriana J Maaskant-Braat

Ph.D Lonneke V Van De Poll-Franse

Jan Willem

M.D W Coebergh

M.D Rudi M Roumen

M Cathelijne

M.D Tutein Nolthenius-Puylaert

M.D Grard A Nieuwenhuijzen

M.D Ajg Maaskant-Braat

Sentinel node micrometastases in breast cancer do not affect prognosis: a population-based study

Keywords: breast carcinoma, sentinel node biopsy, micrometastasis, prognosis

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

Introduction

During the last decades an increase in breast cancer incidence has occurred [START_REF] Louwman | On the rising trends of incidence and prognosis for breast cancer patients diagnosed 1975-2004: a long-term population-based study in southeastern Netherlands[END_REF].

Combined with improving survival rates this implies that the number of prevalent breast cancer cases will continue to rise as well as the health care burden of breast cancer.

Sentinel node biopsy (SNB) as an axillary staging procedure in primary breast cancer was introduced about ten years ago to avoid axillary lymph node dissection (ALND) in patients with tumor negative lymph nodes. The main advantage of performing SNB is a markedly lower morbidity [START_REF] Schijven | Comparison of morbidity between axillary lymph node dissection and sentinel node biopsy[END_REF][START_REF] Ververs | Risk, severity and predictors of physical and psychological morbidity after axillary lymph node dissection for breast cancer[END_REF] whereas axillary staging capacities are similar to ALND [START_REF] Cox | Lymphatic mapping and sentinel lymph node biopsy in patients with breast cancer[END_REF][START_REF] Veronesi | Sentinel-node biopsy to avoid axillary dissection in breast cancer with clinically negative lymphnodes[END_REF]. A randomized trial showed the 8-year survival of patients with primary breast cancer to be equal in the SNB-group and the ALND-group [START_REF] Veronesi | Sentinel-lymph-node biopsy as a staging procedure in breast cancer: update of a randomised controlled study[END_REF]. Furthermore, in observational research with cancer registry data, survival of patients with negative SNB without completion ALND has been shown to be at least equivalent to the survival of node-negative patients with extensive ALND [START_REF] Kuijt | Survival after negative sentinel lymph node biopsy in breast cancer at least equivalent to after negative extensive axillary dissection[END_REF]. These findings support the validity and safety of SNB as a staging procedure and thus the removal of clinically negative axillary lymph nodes by ALND seems no longer justifiable.

Since only a few nodes are being removed, the introduction of the SNB led to application of more extensive time-consuming and costly pathologic examination techniques like serial sectioning and immunohistochemistry. Occult metastases or micrometastases were detected in 9-23% of originally Hematoxylin & Eosin node-negative cases [START_REF] Tjan-Heijnen | Micro-metastases in axillary lymph nodes: an increasing classification and treatment dilemma in breast cancer due to the introduction of the sentinel lymph node procedure[END_REF]. Recent studies showed a 4.3-10% increase among patients diagnosed with micrometastatic disease in the sentinel node as well [START_REF] Maaskant | Stage migration due to introduction of the sentinel node procedure: a population-based study[END_REF][START_REF] Van Der Heiden-Van Der Loo | Prognostic significance of breast cancer axillary lymph node metastases assessed by two special techniques : reevaluation with longer follow-up[END_REF]. This increase led to stage migration after adjustment for the simultaneous, favorable trend in tumor size [START_REF] Maaskant | Stage migration due to introduction of the sentinel node procedure: a population-based study[END_REF]. A discussion followed on prognostic significance and possible need for additional systemic and regional treatment. Some studies on the prognostic significance of micrometastases in ALND before introduction of the sentinel node biopsy showed that patients with axillary micrometastases had higher recurrence rates and lower overall survival [11][START_REF] Hainsworth | Detection and significance of occult metastases in node-negative breast cancer[END_REF][START_REF] Breast | Prognostic importance of occult axillary lymph node micrometastases from breast cancers[END_REF][START_REF] Kuijt | The prognostic significance of axillary lymph-node micrometastases in breast cancer patients[END_REF], others however demonstrated no such difference [START_REF] Nasser | Occult axillary lymph node metastases in nodenegative breast carcinoma[END_REF][START_REF] Fitzgibbons | Prognostic factors in breast cancer: College of American Pathologists Consensus statement 1999[END_REF]. Although most studies with larger sample sizes and longer follow-up tend to show a negative effect of micrometastatic disease on overall and disease free survival [START_REF] Grabau | Breast cancer patients with micrometastases only: Is a basis provided for tailored treatment?[END_REF][START_REF] Tan | Occult axillary node metastases in breast cancer are prognostically significant: Results in 368 node-negative patients with 20year follow-up[END_REF], comparison between these retrospective studies and extrapolation to patients with SNB is hampered by different inclusion criteria and large technical variances in the assessment of micrometastases.

The purpose of our study was to perform a population based analysis to determine survival of patients with sentinel node micrometastases as compared to patients with sentinel node-negative and sentinel node-positive disease and thus comment on the prognostic value of these micrometastases in the era after introduction of SNB.

Patients and methods

Methodology, results and discussion of this study were reported according to the REMARK criteria on reporting of tumor marker studies [START_REF] Shane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF]. Patient data were retrieved from the population based Eindhoven Cancer Registry, which records data on all patients newly diagnosed with cancer in the Southeast region of The Netherlands, an area with approximately 2.4 million inhabitants. Collected data were derived from 10 hospitals, consisting of large non-university teaching hospitals and community hospitals, and two radiotherapy departments. Data on patient and tumor characteristics and local and systemic treatment were collected by the Cancer Registry based on the pathology reports and medical records. The patients were staged according to the Tumor-Node-Metastasis (TNM) system of the International Union Against Cancer (UICC) [START_REF] Hermanek | UICC TNM classification of malignant tumors[END_REF].

Sentinel node biopsy followed by axillary lymph node dissection was introduced in the Southeast Netherlands in 1995. In 1997, surgeons gradually started to perform SNB procedures as a routine staging procedure and since 2000 indications for SNB were described in national guidelines [START_REF] Roumen | Samenvatting van de richtlijn "Schildwachtklierbiopsie bij mammacarcinoom[END_REF]. We included all women who underwent SNB for primary invasive breast cancer in the Southeast Netherlands in the period 1996-2006 and we used characteristics and data of the entire group in the analyses.

In The Netherlands the pathology protocol advocated by the EORTC Breast Cancer Group has been adopted by the pathologists since 2000 and included in the Dutch evidencebased guideline for the treatment of breast cancer. According to this guideline, sentinel nodes should be investigated at three levels at 0.25mm intervals and from each section at least two slides should be made: one for H&E staining and one for IHC [START_REF] Roumen | Samenvatting van de richtlijn "Schildwachtklierbiopsie bij mammacarcinoom[END_REF]. For this study, data were categorized according to axillary lymph node status. Node-negative patients were categorized as pN0. Patients with metastases smaller than 0.2 mm were categorized as pN0(i+) (isolated tumor cells) and as pN1mi (micrometastases) in case of metastases between 0.2 mm and 2.0. Node-positive patients were categorized as pN1a if 1 to 3 axillary lymph nodes were positive and >pN1a if more than 3 axillary lymph nodes were positive or if metastases were present in supraclavicular or internal mammary lymph nodes. The discrimination between micrometastases and isolated tumor cells in the Cancer Registration data has only been made since 2003, after introduction of the revised TNM system in 2002.

Follow-up was completed until January 2008 and endpoint of the study was question whether or not the patient was still alive. This information was obtained from the municipal registries in the area of the Eindhoven Cancer Registry and the Central Bureau for Genealogy. The latter institution collects data on all deceased Dutch citizens via the municipal registries. In this way, information on patients who moved outside the registry area was also obtained. The few patients (<0.3%) who died outside The Netherlands might be wrongly considered as "being alive".

Statistical analyses were carried out using SAS (version 9.1 for Windows, SAS institute Inc., Cary, NC). Survival analyses were carried out using the Kaplan-Meier life-table analysis. Survival time was defined as the period between the diagnosis of breast cancer and death or date of last available follow-up. Patients were stratified according to sentinel lymph node status and survival comparison between these groups was performed by means of the log-rank test. We censored the data if the effective sample size was smaller than 10 in the overall survival analyses. Multivariate analyses were carried out using Cox proportional hazards analyses. Variables that showed a significant influence on survival in univariate analyses were entered in the multivariate model. We adjusted for the possible confounding influences of age at diagnosis, tumor size (defined as T-stage) and grade. We additionally adjusted for the effects of administrating adjuvant systemic therapy. P-values <0.05 were considered statistically significant.

Results

Between 1996 and 2006 a total of 6803 patients underwent SNB for primary invasive breast cancer in the Southeast region of The Netherlands. Their characteristics stratified according to lymph node status are demonstrated in Table 1. Micrometastatic lymph node involvement was observed in 6.6% of the patients. Age at diagnosis, pT-stage, grade and histology all differed significantly according to nodal status (P<0.0001). Patients without metastases or with micrometastatic disease underwent breast conserving surgery significantly more frequent than those with macrometastases (P<0.0001). Administration of adjuvant systemic therapy significantly increased with lymph node involvement. 28% of the patients with a negative sentinel node received adjuvant systemic therapy (chemo-and/or hormonal therapy) versus 74% in the pN1mi-group and 93% in the pN1a-group.

Median follow-up was 50 months for patients with pN0-and pN1mi-disease, 53 months for patients with pN1a-disease, 47 months for patients with >pN1a-disease and 36 months for patients with pN0(i+)-disease. Overall (unadjusted) survival was significantly worse for patients with pN1a-and >pN1a-disease compared to pN0-disease (P<0.0001). Survival of patients with pN0(i+) and pN1mi did not differ significantly from pN0-disease (P=0.19 and P=0.52) (Figure 1). In multivariate analysis adjusted for age, pT and grade, no significant survival difference was shown between isolated tumor cells and node-negative disease (HR 0.4 (95% CI, 0.14-1.3)) or between micrometastatic disease and node-negative disease (HR 0.9 (95% CI, 0.6-1.3)) (Table 2). Patients with pN1a-disease had an increased risk of overall mortality with a hazard ratio of 1.4 (95% CI, 1.2-1.7) compared to pN0. Patients with >pN1a-disease had a hazard ratio of 2.2 (95% CI, 1.7-3.0) compared to pN0.

After additional adjustment for adjuvant systemic therapy still no significant survival difference was observed for isolated tumor cells (P=0.15) and micrometastatic disease (p=0.97) compared to node-negative patients. Patients with pN1a-disease and >pN1adisease still had an increased risk of overall mortality (Table 2). We performed additional analyses excluding grade as a covariate considering the relatively high percentage of missing values for this variable, but this did not change the result of our analyses in any way. Neither did the additional analyses we performed adjusting for the execution of completion ALND. Separate unadjusted analyses of prognosis by N-stage according to whether patients received no adjuvant systemic therapy, adjuvant chemo-or hormonal therapy or both did also not change the result of our analyses (Figure 2, 3, 4 and 5).

Discussion

Based on studies conducted before the introduction of the sentinel node procedure, administration of adjuvant systemic therapy to patients with micrometastatic disease in the sentinel node seems justifiable under the assumption that these micrometastases are prognostic indicators of worse survival and outcome. Our study however, which is based exclusively on patients who underwent SNB, showed no overall survival difference between patients with micrometastatic disease and those without axillary lymph node metastasis. Even after adjustment for age, pT, grade and administration of adjuvant systemic therapy no significant survival differences could be detected.

By using data of the Eindhoven Cancer Registry, we were able to present results based on a large, unselected population-based patient population. The patients were treated in both teaching and community hospitals and data are thought to reflect the usual care in The Netherlands. This report is one of the first on the prognosis of patients with micrometastases in the sentinel node. Despite a fairly short follow-up time and a small number of events, the 95% confidence intervals of the estimated Hazard Ratio's are small enough to rule out a large difference in survival between node-negative patients and patients with micrometastases.

Apparently, the biological behavior of sentinel node micrometastases is of limited prognostic significance, at least during the first five years after diagnosis. In order for tumor cells to metastasize, a number of sequential processes have to take place, such as tumor cell invasion, adhesion and angiogenesis. This is a rather inefficient process and not all circulating tumor cells are viable and capable of forming regional and distant metastasis [START_REF] Yeatman | Molecular basis of tumor progression: Mechanisms of organ-specific tumor metastasis[END_REF]. Furthermore, expansion of metastasized tumor masses beyond 1-2 mm in diameter depends on the development of a new blood supply by angiogenesis, which again raises the question whether these very small metastases have biologic implications at all [START_REF] Yeatman | The significance of breast cancer lymph node micrometastases[END_REF].

The observation that occult axillary and distant involvement might never become clinically overt [START_REF] Veronesi | Sentinel-lymph-node biopsy as a staging procedure in breast cancer: update of a randomised controlled study[END_REF][START_REF] Mansi | Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study[END_REF] led to the emergence of the stem cell hypothesis, which postulates that a population of cancer cells consists of a limited number of cancer stem cells that cause cancer progression and a larger number of non-stem cancer cells being dormant [START_REF] Al-Hajj | Prospective identification of tumorigenic breast cancer cells[END_REF].

Presence of cancer stem cells in a metastatic focus is hypothesized to be important or possibly crucial for development and growth of these foci. Without these stem cells the metastatic focus would be destined to disappear by apoptosis or have a very long dormancy.

As stated before, studies on the significance of micrometastases that used data from patients before the introduction of the sentinel node biopsy showed contradicting results.

In one of the larger studies with long follow-up, which was also based on data from the Eindhoven Cancer Registry, 10111 patients were included, of whom 179 had micrometastases. They were diagnosed with invasive breast cancer between 1975 and 1997 and had complete follow-up until April 2002. Remarkably, the results of this study showed that patients with axillary nodal micrometastasis in ALND had a significantly worse survival rate than node-negative patients independent of age or tumor size [START_REF] Kuijt | The prognostic significance of axillary lymph-node micrometastases in breast cancer patients[END_REF]. Since these data were derived from the same database, covering the same hospitals and pathology laboratories, we must conclude that ALND nodal micrometastases do not have the same prognostic implication as sentinel node micrometastases. We might have studied two different breast cancer patient populations with different tumor-characteristics and metastatic tumor burden. This seems quite unlikely however, since we adjusted for tumor stage and completion ALND and other studies showed no change in prognosis during this period. Treatment plans have altered and have included the use of systemic adjuvant therapy far more often, but we adjusted for the possible effect of adjuvant systemic therapy in our study. Detection of metastases might be directly dependent on the methods used to investigate them. By using serial sectioning and immunohistochemistry in routine daily practice, more and smaller metastases are being detected which may not be a harbinger of undetected macrometastases as may have been the case in older studies.

In an American publication the SEER database was used to determine prognostic significance of micrometastases in pre-as well as post-SN era [START_REF] Chen | The prognostic significance of micrometastases in breast cancer: A SEER population-based analysis[END_REF]. This relatively large study demonstrated only a minimal detrimental impact in tumors less than 2.0 cm in diameter and a more significant detrimental impact in larger tumors (1 vs. 4-6% decrease in 5-year survival). The authors, however, were not able to track the use of adjuvant therapies and therefore they could not adjust for the possible confounding effects of administration of adjuvant systemic therapy. Another Dutch publication based on a much smaller population sample showed that despite a higher risk of distant metastases in the micrometastatic group there was no significant difference in overall or disease-free survival between pN0-and pN1mi-disease [START_REF] Gobardhan | Prognostic value of micrometastases in sentinel lymph nodes of patients with breast carcinoma: a cohort study[END_REF]. In a recently published study by Hansen et al [START_REF] Hansen | Impact of micrometastases in the sentinel node of patients with invasive breast cancer[END_REF] patients with isolated tumor cells or micrometastases did not have a worse disease-free and overall survival compared to SN-negative patients. Also consistent with our results was their finding that patients with macrometastases have a worse prognosis than the SNnegative patients and patients with isolated tumor cells or micrometastases.

The MIRROR study (Micrometasases and Isolated tumor cells: Relevant and Robust or Rubbish?) showed that patients with isolated tumor cells or micrometastases as final Nstage after SNB had a significantly lower 5-year disease-free survival than patients without nodal involvement [START_REF] De Boer | Micrometastases or isolated tumor cells and the outcome of breast cancer[END_REF]. This study, which is also a retrospective cohort study, only included patients with favorable tumor characteristics for whom adjuvant systemic treatment was not indicated according to the Dutch treatment guidelines. In contrast, we also included patients who had been receiving systemic treatment according to those guidelines, as well as patients with macrometastases to see how their prognosis compares to the prognosis of patients with micrometastastic disease or isolated tumor cells. In the MIRROR study pathology of removed axillary lymph nodes was reviewed. Reviewing of the pathology seems to have led to a detection of more isolated tumor cells as compared to micrometastases and node-negative patients. We chose to base our analyses on the information that was retrieved from the pathology report and thus to present results based on usual care in The Netherlands. Finally, no data on overall survival were available in the MIRROR trial.

In conclusion, our population-based study showed that the presence of sentinel node micrometastases in breast cancer patients did not have significant impact on breast cancer overall survival during the first 5 years after diagnosis. We therefore postulate that micrometastatic disease itself should not be an indication for adjuvant systemic therapy.

Figure 1

 1 Figure 1 Kaplan-Meier life table analysis of overall survival by lymph node status of 6803

Figure 2

 2 Figure 2 Kaplan-Meier life table analysis of survival by lymph node status of patients who

Figure 3

 3 Figure 3 Kaplan-Meier life table analysis of survival by lymph node status of patients who

Figure 4

 4 Figure 4 Kaplan-Meier life table analysis of survival by lymph node status of patients who

Figure 5

 5 Figure 5 Kaplan-Meier life table analysis of survival by lymph node status of patients who

Table 1

 1 Patient characteristics by lymph node status of 6803 patients who underwent SNB for primary invasive breast cancer in the SoutheastNetherlands, 1996Netherlands, -2006. .

	Lymph node status	pN0	pN0(i+)	pN1mi	pN1a	>pN1a	P-value
	Number of patients (%)	4562 (67.1)	126 (1.9)	451 (6.6)	1347 (19.8)	317 (4.7)
	Accrued months of follow-up	228100	4536	22550	71391	14899
	Age at diagnosis
	≤35	107 (2.4)	2 (1.6)	14 (3.1)	47 (3.5)	17 (5.4)	<0.0001
	36-49	993 (21.8)	29 (23.0)	119 (26.4)	409 (30.4)	98 (10.9)
	50-69	2483 (54.4)	67 (53.2)	238 (52.8)	682 (50.6)	155 (48.9)
	≥70	979 (21.5)	28 (22.2)	80 (17.7)	209 (15.5)	47 (14.8)
	pT-stage						<0.0001
	1	3408 (74.7)	84 (66.7)	294 (65.2)	710 (52.7)	114 (36.0)
	2	1037 (22.7)	39 (31.0)	141 (31.3)	564 (41.9)	168 (53.0)
	3	17 (0.4)	0 (0.0)	8 (1.8)	17 (1.3)	15 (4.7)
	4	25 (0.6)	2 (1.6)	3 (0.7)	28 (2.1)	9 (2.8)
	Unknown	75 (1.6)	1 (0.8)	5 (1.1)	28 (2.1)	11 (3.8)
	Grade
	I	1077 (23.6)	29 (23.0)	91 (20.2)	236 (17.5)	40 (12.6)	<0.0001
	II	1590 (43.9)	49 (38.9)	174 (38.6)	488 (36.2)	118 (37.2)
	III	940 (20.6)	30 (23.8)	97 (21.5)	335 (24.9)	91 (28.7)
	Unknown	955 (20.9)	18 (14.3)	89 (19.7)	288 (21.4)	68 (21.5)
	Histology
	Ductal	3618 (79.3)	101 (80.2)	361 (80.0)	1100 (81.7)	241 (76.0)	<0.0001
	Lobular/mixed	635 (13.9)	18 (14.3)	75 (16.6)	216 (16.0)	68 (21.5)
	Mucinous/tubular/medullary	236 (5.2)	3 (2.4)	8 (1.8)	24 (1.8)	5 (1.6)
	Other	73 (1.6)	4 (3.2)	7 (1.6)	7 (0.5)	3 (1.0)
	Type of definitive surgery
	Breast-conserving surgery	3520 (77.2)	84 (66.7)	307 (68.1)	817 (60.7)	149 (47.0)	<0.0001
	Mastectomy	1029 (22.6)	42 (33.3)	144 (31.9)	528 (39.2)	167 (52.7)
	None/unknown	13 (0.3)	0 (0.0)	0 (0.0)	2 (0.2)	1 (0.3)
	Type of axillary surgery
	SNB alone	4097 (89.8)	49 (38.9)	126 (27.9)	133 (9.9)	8 (2.5)	<0.0001
	SNB+ALND	465 (10.2)	77 (61.1)	325 (72.1)	1214 (90.1)	309 (97.5)
	Radiotherapy
	Yes	3460 (75.8)	81 (64.3)	296 (65.6)	875 (65.0)	259 (81.7)	<0.0001
	No	1102 (24.2)	45 (35.7)	155 (34.4)	472 (35.0)	58 (18.3)
	Adjuvant systemic therapy
	Chemotherapy	447 (9.8)	16 (12.7)	71 (15.7)	368 (27.3)	109 (34.4)	<0.0001
	Hormonal therapy	526 (11.5)	35 (27.8)	180 (39.9)	539 (40.0)	81 (25.6)
	Chemo-and hormonal therapy	301 (6.6)	12 (9.5)	81 (18.0)	342 (25.4)	98 (30.9)
	None	3288 (72.1)	63 (50.0)	119 (26.4)	98 (7.3)	29 (9.2)

Table 2

 2 Hazard ratio of overall mortality by lymph node status of 6803 patients who underwent SNB for primary breast cancer in the SoutheastNetherlands, 1996Netherlands, -2006. .

					Univariate model	Multivariate model I *	Multivariate model II **
		No. of	Follow-up	No. of	crude HR	P-value	HR (95% CI)	P-value	HR (95% CI)	P-value
		patients	(months)	events	(95% CI)
	pN0	4562	50 ± 27	347	1.00		1.00		1.00
	pN0(i+)	126	36 ± 14	3	0.46 (0.15-1.45)	0.19	0.42 (0.14-1.32)	0.14	0.44 (0.14-1.36)	0.15
	pN1mi	451	50 ± 25	31	0.89 (0.61-1.28)	0.52	0.88 (0.61-1.27)	0.48	0.99 (0.68-1.45)	0.97
	pN1a	1347	53 ± 29	167	1.50 (1.25-1.81)	<0.0001	1.43 (1.18-1.73)	0.0002	1.62 (1.30-2.01)	<0.0001
	>pN1a	217	47 ± 25	57	2.50 (1.89-3.31)	<0.0001	2.23 (1.67-2.98)	<0.0001	2.51 (1.84-3.42)	<0.0001
	HR: Hazard Ratio
	95%CI: 95% Confidence Interval
	* Model I: Adjusted for age, pT and grade
	** Model II: Additionally adjusted for adjuvant systemic therapy

SNB: Sentinel Node Biopsy; ALND: Axillary Lymph Node Dissection

Acknowledgements: None


Maurice J C Sangen

Fenneke M M Wiel

Philip M P Poortmans

Vivianne C G Tjan-Heijnen

Grard A P Nieuwenhuijzen

Rudi M H Roumen

Miranda F Ernst

M Cathelijne

B J E Tutein Nolthenius-Puylaert

Adri C Voogd
email: adri.voogd@epid.unimaas.nl

Maurice J C Van Der Sangen

M M Fenneke

Van De Wiel

F M M Van De Wiel

Are breast conservation and mastectomy equally effective in the treatment of young women with early breast cancer? Long-term results

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

Introduction

Multiple randomized controlled trials have shown that survival rates for patients with early-stage breast cancer are comparable after breast-conserving therapy (BCT) and mastectomy [START_REF] Veronesi | Twenty-year follow-up of a randomized study comparing breastconservin surgery with radical mastectomy for early breast cancer[END_REF][START_REF] Fisher | Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF][START_REF] Van Dongen | Long-term results of a randomized trial comparing breast-conserving therapy with mastectomy: European organization for research and treatment of cancer 10801 trial[END_REF][START_REF] Jacobson | Ten-year results of a comparison of conservation with mastectomy in the treatment of stage i and ii breast cancer[END_REF][START_REF] Blichert-Toft | Danish randomized trial comparing breast conservation therapy with mastectomy: Six years of life-table analysis. Danish breast cancer cooperative group[END_REF]. Consequently, BCT is now widely accepted as a local treatment for early-stage breast cancer. Some doubts still remain concerning the treatment of young women since young age has been shown to be an independent risk factor for local relapse after BCT, much more than after mastectomy [START_REF] Voogd | Differences in risk factors for local and distant recurrence after breast-conserving therapy or mastectomy for stage i and ii breast cancer: Pooled results for two large european randomized trials[END_REF][START_REF] Bock | Isolated loco-regional recurrence of breast cancer is more common in young patients and following breast conserving therapy: Long-term results of european organisation for research and treatment of cancer studies[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathology-related characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Elkhuizen | Local recurrence after breast-conserving therapy for invasive breast cancer: High incidence in young patients and association with poor survival[END_REF][START_REF] Jones | Impact of pathological characteristics on local relapse after breast-conserving therapy: A subgroup analysis of the eortc boost versus no boost trial[END_REF]. The 10-year local relapse rate for women aged ≤40 years varies between 19% and 35% after BCT and between 7% and 12% after mastectomy [START_REF] Vrieling | Can patient-, treatment-and pathology-related characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Elkhuizen | Local recurrence after breast-conserving therapy for invasive breast cancer: High incidence in young patients and association with poor survival[END_REF][START_REF] Jobsen | Adjuvant systemic therapy for patients with resectable breast cancer: Guideline from the dutch national breast cancer platform and the dutch society for medical oncology[END_REF]12,[START_REF] Voogd | Differences in risk factors for local and distant recurrence after breast-conserving therapy or mastectomy for stage i and ii breast cancer: Pooled results for two large european randomized trials[END_REF]. In two previous reports concerning women ≤40 years we found 5and 10-year actuarial local relapse rates of 9% and 17.9%, respectively, after BCT [13] and of 5.6% and 7.3% after mastectomy [14].

Only 5% to 10% of all women with breast cancer is aged 40 years or younger.

Because of this relatively low incidence, subgroup analyses of the randomized controlled trials could not confirm whether BCT is as safe as mastectomy in the treatment of early-stage breast cancer in young women. Even by pooling data derived from all available trials, the number of young patients would be too small to draw firm conclusions. Therefore, evidence for the safety of BCT was searched in a few non-randomized studies [15][16][17]. Limitations of these studies are the relatively small number of patients, ranging from 540 to 668, insufficient information on the risk factors of local relapse and a follow-up time up limited to only 10 years. Therefore, we evaluated a large cohort of 1451 patients aged ≤40 years with a sufficiently long follow-up to determine treatment results up to 15 years.

Patients and methods

Patients

Patient data were obtained from the population-based Eindhoven Cancer Registry (ECR). This serves a population of approximately 2.4 million inhabitants in the south of the Netherlands and records data on all patients with newly diagnosed cancer since 1955. These data were compared to data derived from the two radiotherapy departments in this region, the Catharina Hospital in Eindhoven and the Dr. Bernard Verbeeten Institute in Tilburg. Records of patients who did not receive radiotherapy after mastectomy were provided by the surgical departments of the 10 regional hospitals.

Only patients with stage I or II breast cancer (pT1-2, N0-2, M0) were considered eligible for the study. The ECR identified 846 patients aged ≤40 years that underwent mastectomy between 1988 and 2005, from which 745 medical records were available. For the other 101 patients records were either missing or incomplete after digitalising paper medical records. Over the same period, 937 patients aged ≤40 years received BCT, for whom all patient records were available.

The medical files were reviewed to extract information with respect to patient-, tumorand treatment characteristics as well as to outcome. After exclusion of 122 patients with stage III or IV breast cancer, 44 patients with neo-adjuvant systemic treatment, 8 patients with synchronous bilateral breast cancer, 19 patients with non-invasive breast cancer, and 8 patients without radiotherapy of the breast following breastconserving surgery, 562 patients remained available for analysis in the mastectomy group and 889 patients in the BCT group.

Treatment

Patients underwent mastectomy or BCT. All patients received either a sentinel lymph node procedure or an axillary lymph node dissection. Indications for locoregional radiotherapy after mastectomy included an involved lymph node in the apex of the axilla, fixed axillary lymph nodes or ≥4 lymph node metastases. This locoregional radiotherapy was given to a dose of 46 to 50 Gy. In case of an incomplete tumor resection of the primary breast tumor, a boost to a dose of 13.8 to 26 Gy was given to part of the chest wall. Until 2002 the guidelines also recommended the use of postmastectomy radiotherapy for patients with extracapsular lymph node invasion. In the framework of BCT, patients received 46 to 50 Gy to the whole breast, in 796 patients (91.8%) followed by a boost of 10 to 26 Gy to the primary tumor bed.

Indications for locoregional radiotherapy after breast conserving surgery were similar to those after mastectomy.

Before 1998, adjuvant systemic treatment was based on regional guidelines and was recommended only for patients with involved axillary lymph nodes. In 1998, a new national guideline was published [18], based on the St. Gallen guidelines [START_REF] Goldhirsch | Meeting highlights: International consensus panel on the treatment of primary breast cancer[END_REF][START_REF] Goldhirsch | Meeting highlights: International consensus panel on the treatment of primary breast cancer. Seventh international conference on adjuvant therapy of primary breast cancer[END_REF], after which lymph node-negative patients with high-risk breast cancer were advised to receive adjuvant systemic treatment, depending on size, grade, and hormone receptor status. In 2001, the guidelines were updated, recommending adjuvant systemic treatment also for most patients aged ≤35 years, irrespective of their lymph node status.

The standard chemotherapy regimen consisted of cyclophosphamide, methotrexate and fluorouracil (CMF) in the early years of the study period and of anthracycline-containing regimens thereafter. From 1988 until 2001 there was no strict guideline for the use of hormonal therapy for pre-menopausal patients who had receptor-positive tumors. After 2001 hormonal therapy was recommended for all receptor positive high-risk patients. In the study period, trastuzumab was not yet implemented in the adjuvant setting.

Follow-up

For patients treated with mastectomy, follow-up was completed until January 1 st , 2007 or death. Median duration of follow-up was 7.4 years. Patients receiving BCT were followed up until death or January 1 st , 2008. Median duration of follow-up was 9.5 years. Seventy-three patients (5.0%) were lost to follow-up, meaning that their last contact with the surgeon, radiation oncologist or general practitioner was before January 1 st , 2007 in case of mastectomy or January 1 st , 2008 in case of BCT. The median follow-up for these patients was 8.9 years and they have been included in the analysis until their last date of contact.

Methods

The endpoints of this study were local relapse, distant recurrence and death.

Local relapse was defined as reappearance of tumor growth in the treated breast or overlying skin or, in case of mastectomy, on the ipsilateral chest wall. The time to an event was estimated using the life-table method and was considered to be the interval between the date of primary treatment and occurrence of the event.

Observation time was censored at January 1 st , 2007 for patients undergoing mastectomy and January 1 st , 2008 for those undergoing BCT, or death or the last date of contact for patients who were lost to follow-up. Local relapses that were diagnosed after distant metastases or with synchronous distant disease were not counted as events in the survival analyses, because these are considered to be of little clinical importance and because the risk of underreporting is high. These patients were censored at the date of diagnosis of distant disease.

A multivariate analysis was carried out using Cox proportional hazard model to determine the impact of the type of surgery on the risk of distant recurrence, adjusting for age at diagnosis, the period of diagnosis, tumor size, axillary nodal status and use of adjuvant systemic treatment.

Results

Patient, tumor and treatment characteristics

Table 1 lists the patient, tumor and treatment characteristics of the study population.

Median age at diagnosis was 37.2 years for patients treated with mastectomy and 37.4 years for those treated with BCT. As expected, despite the restriction to early stage breast cancer, patients treated with mastectomy generally had worse prognostic factors, when compared to those treated with BCT (Table 1). Because of worse prognostic factors, patients treated with mastectomy more often received systemic therapy (P<0.0001). The proportion of young women undergoing BCT decreased from 69% in the period 1988-1993 to 55% in the period 2000-2005 (P<0.0001).

After mastectomy, 37% of the patients received radiotherapy. The overall proportion of patients aged ≤40 years receiving adjuvant systemic treatment increased from 35% in the period 1988-1993 to 77% in the period 2000-2005 (Figure 1).

Local tumor control

Of the 131 local relapses occurring after BCT, 61 (45%) were diagnosed within 5 years, 44 (33%) between 5 and 10 years, and 30 (22%) thereafter. Twenty-three local relapses occurred after mastectomy, of which 19 (83%) within 5 years and 4 (17%) between 5 and 10 years after treatment. No local relapses were diagnosed more than 6 years after mastectomy.

Of the local relapses after BCT, 19 (14%) were in situ and 111 (82%) were invasive tumors. From five patients this information was missing. Of the 23 local relapses after mastectomy, 22 were invasive and one was an in situ lesion. Ninety (67%) of the 135 local relapses following BCT were localized at or near the site of the primary tumor, 15 (11%) were localized elsewhere in the breast, seven (5%) were diffuse and one involved the skin. For 22 patients information on the location of the local relapse was missing.

The actuarial risk of local relapse was significantly worse for patients undergoing BCT compared to patients undergoing mastectomy (Figure 2a). The 5-, 10-and 15-year local relapse rates were 8.3% (95% CI, 6.3-10.5), 18.3% (95% CI, 14.9-21.7) and 27.9% (95% CI, 22.9-32.9), respectively. For the patients who underwent mastectomy, these figures were 4.4% (95% CI, 2.4-6.4) after 5 years and 6.0% (95% CI, 3.5-8.5) after 10 and after 15 years of follow-up. When also taking into account the local relapses that occurred simultaneously with distant metastases (11 after mastectomy and 19 after BCT), the 5-, 10-and 15-year rates were 10.3% (95% CI, 8.1-12.5), 20.4% (95% CI, 17.0-23.8) and 30.1% (95% CI, 25.0-35.2), respectively, after BCT and 6.4% (95% CI, 4.1-8.7), 8.2% (95% CI, 5.4-11.0) and 8.2% (95% CI, 5.4-11.0) after mastectomy (P<0.0001).

A significant improvement was observed in the local control rate after BCT during the study period (Table 2). A stratified analysis according to axillary nodal status showed that this improvement did only occur in patients with negative lymph nodes. This appeared to be related to the increased use of systemic therapy over the years, as the risk for local relapse was significantly lower for patients who received adjuvant systemic treatment (Table 2, Fig 2b). A higher local relapse risk was observed for patients whose excision of the primary tumor was incomplete or doubtful, as compared to those with a complete excision (Table 2). No significant associations were found between the risk of local relapse after BCT and tumor size and between the different age groups (<30, 30-35 and 35-40 years) (Table 2). The local relapse rate was significantly higher after BCT in the patient group without a boost dose of radiotherapy to the tumor bed (Table 2). No dose-effect relationship was observed for the patients receiving a boost dose.

Distant recurrence-free and overall survival

No statistically significant difference in distant recurrence-free survival was observed between the patients undergoing BCT or mastectomy (Figure 3). The 10-year distant recurrence-free survival rates were 71.0% (95% CI, 67.6-74.4) and 67.0% (95% CI, 62.4-71.6), respectively. The 10-year overall survival rate was 74.9% (95% CI, 71.7-78.1) after BCT and 71.2% (95% CI, 66.4-76.0) after mastectomy (P=0.215).

A stratified analysis according to tumor size (pT1 or pT2) and nodal status (pN0 and pN1-2) was performed, in which four strata were created: pT1-pN0, pT2-pN0, pT1-pN1-2 and pT2 and pN1-2. In none of these four strata a statistically significant difference was observed in the distant recurrence-free survival between the patients undergoing BCT versus those with mastectomy, with P-values of 0.52, 0.17, 0.72 and 0.64, respectively.

In the first seven years after treatment, distant recurrence risk was significantly lower for the patients treated with BCT (P=0.009). However, for the patients surviving without distant metastases until the seventh year of follow-up, the risk of developing distant disease is lower for the patients with mastectomy (P=0.044). This reversal of the risk was also expressed by the distant disease hazard ratio for BCT compared to mastectomy, which was 0.75 (95% CI, 0.61-0.93) during the first seven years of follow-up and 1.96 (95% CI, 1.02-3.76) for the patients followed from seven years onward until censoring.

A significant improvement in the distant recurrence-free survival was observed during the study period (Figure 3). The 5-year distant recurrence-free survival improved from 72.2% (95% CI, 67.9-76.5) in the period 1988-1993 to 84.8% (95% CI, 81.0-88.6) in the period 2000-2005 (HR 0.61 (95% CI, 0.45-0.82) (Table 3). Other independent favourable prognostic factors were: a negative axillary nodal status, a tumor size of 2 cm or less and the use of adjuvant systemic treatment. Also the multivariate analysis showed no statistically significant difference in distant recurrence-free survival between patients treated with BCT and mastectomy (HR, 0.97; 95% CI, 0.78-1.20).

Discussion

The current study is one of the few studies focussing on young women with early stage breast cancer, comparing local control and survival after BCT and mastectomy.

Our results indicate a worse local control after BCT with a linearly increasing cumulative risk of developing a local relapse even after more than 15 years of followup, whereas a plateau was reached after 6 years for the patients treated with mastectomy. Despite the more favourable stage distribution of the patients who underwent BCT, disease-free survival at 10 years was similar for both treatment groups. The local relapse rate after BCT showed a decreasing trend during the study period but still remains at 1% annually for the patients treated in the period 2000-2005. We also noted a significant decrease of the distant recurrence rate over time, associated with increased use of systemic therapy, irrespective of the type of surgery.

Many studies on BCT have shown that young women have an increased local relapse risk compared to older women [START_REF] Voogd | Differences in risk factors for local and distant recurrence after breast-conserving therapy or mastectomy for stage i and ii breast cancer: Pooled results for two large european randomized trials[END_REF][START_REF] Bock | Isolated loco-regional recurrence of breast cancer is more common in young patients and following breast conserving therapy: Long-term results of european organisation for research and treatment of cancer studies[END_REF][START_REF] Vrieling | Can patient-, treatment-and pathology-related characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?[END_REF][START_REF] Elkhuizen | Local recurrence after breast-conserving therapy for invasive breast cancer: High incidence in young patients and association with poor survival[END_REF][START_REF] Jones | Impact of pathological characteristics on local relapse after breast-conserving therapy: A subgroup analysis of the eortc boost versus no boost trial[END_REF][START_REF] Zhou | Factors affecting outcome for young women with early stage invasive breast cancer treated with breast-conserving therapy[END_REF]. However, few data are available from studies in young women on treatment outcomes after BCT compared to mastectomy. Our data show that local relapses keep on occurring after BCT, even after prolonged follow up. This continuing risk was previously reported by Kreike et al who report 10-and 15-year local relapse-rates of 9.3% and 13.8%, respectively, in a cohort of 1024 patients with a mean age of 50 years [START_REF] Kreike | Continuing risk of ipsilateral breast relapse after breastconserving therapy at long-term follow-up[END_REF]. A possible explanation for at least part of the late local relapses is the occurrence of new primary tumors.

Minimising the risk of local relapse remains an important clinical issue. The EBCTCG meta-analysis has shown that local control has an influence on long term breast cancer related mortality: for every 4 local relapses prevented at 5 years, 1 breast cancer related death is avoided at 15 years [START_REF] Clarke | Effects of radiotherapy and differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: An overview of the randomised trials[END_REF]. Punglia et al emphasize the importance of a careful selection of patients for BCT as this strategy leads to a decrease in breast-cancer and overall mortality [START_REF] Punglia | Local therapy and survival in breast cancer[END_REF]. Although the proportion of local relapses is higher after BCT, studies among young women show no difference in distant recurrence-free survival [17,15]. Neither did we find a significant difference with a distant recurrence-free survival at 10 year of 67.0% after mastectomy and 71.1% after BCT. The multivariate analysis also showed no difference in distant recurrence-free survival after treatment with BCT and mastectomy. Results from retrospective studies are harder to interpret than results from randomised controlled studies, because of a possible bias due to baseline characteristics and the selection of treatments. However, randomized studies were not able to give a definitive answer and new randomized controlled trials are hard to imagine. Our study is the largest study with long-term follow-up comparing the outcome after BCT and mastectomy in young women with early-stage breast cancer. Moreover, it is population-based and follow up was meticulously done. One could argue that the poor local control after BCT is dominated by a group of patients treated rather shortly after the introduction of BCT in The Netherlands, when less attention was given, for example, to preoperative imaging of the tumor and inking of surgical margins (both invasive and DCIS). Fact is however, that a poor local tumor control was also observed in the patients treated in the second period (1994)(1995)(1996)(1997)(1998)(1999) (Table 2) Fortunately, in the period 2000-2005, we observed a clear trend towards improved local control. There are several factors which could have contributed to this improvement. First of all, the awareness of different risk factors for local relapse after BCT, not only may have led to a more stringent selection of patients for BCT [START_REF] Voogd | Changing attitudes towards breast-conserving treatment of early breast cancer in the south-eastern netherlands: Results of a survey among surgeons and a registry-based analysis of patterns of care[END_REF], but also to a certain reluctance towards treating young women with BCT. Over time, the BCT rate decreased from 69% to 55%. Secondly, improvements in the locoregional treatment of breast cancer have taken place over the past decades. However, the most important reason for the better local control is the increased use of adjuvant systemic treatment. Table 2 shows a significant increase in local tumor control over time for patients treated with BCT. This can be largely explained by the increasing proportion of node negative patients receiving adjuvant systemic therapy. In the period 1994-1999 15% of node negative patients received adjuvant systemic therapy, which increased to 60% in the period 2000-2005. Apart from this, we did not notice an additional influence of second or third generation regimens over first generation regimens on the local relapse rate. Our results, showing the importance of adjuvant systemic treatment and its potential to decrease the risk of local relapse, are in line with other studies [START_REF] Bock | Isolated loco-regional recurrence of breast cancer is more common in young patients and following breast conserving therapy: Long-term results of european organisation for research and treatment of cancer studies[END_REF]13,[START_REF] Clarke | Effects of radiotherapy and differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: An overview of the randomised trials[END_REF][START_REF] Buchholz | Impact of systemic treatment on local control for patients with lymph nodenegative breast cancer treated with breast-conservation therapy[END_REF][START_REF] Kroman | Factors influencing the effect of age on prognosis in breast cancer: Population based study[END_REF][START_REF] Park | Outcome at 8 years after breastconserving surgery and radiation therapy for invasive breast cancer: Influence of margin status and systemic therapy on local recurrence[END_REF].Patient and tumor related risk factors for local relapse besides young age, are high tumor grade, positive excision margin, and lymphovascular invasion (LVI). In our series, 90% of the patients had a reported complete tumor excision and of all local relapses, 92% occurred in patients with a microscopically complete excision. This is in line with the results from Jones et al where only young age, grade 3, the lack of systemic treatment and the omission of a boost dose were found to be independent prognostic factors for local relapses after BCT [START_REF] Jones | Impact of pathological characteristics on local relapse after breast-conserving therapy: A subgroup analysis of the eortc boost versus no boost trial[END_REF]. Data on LVI and tumor grade were not or only partly available in our material.

Local control is a result of the interaction between tumor remnants, treatment and tumor biology. An ipsilateral breast tumor recurrence (IBTR) can be a new primary tumor or the result of outgrowth of a tumor remnant in the treated breast, a so called true recurrence. New primary tumors often appear later in time and there is some proof that they have a more favorable prognosis than true recurrences.

Molecular clonality assays can help to establish the type of IBTR [START_REF] Mcgrath | Long-term patterns of in-breast failure in patients with early stage breast cancer treated with breast-conserving therapy: A molecular based clonality evaluation[END_REF][START_REF] Schlechter | Quantitative DNA fingerprinting may distinguish new primary breast cancer from disease recurrence[END_REF], but larger studies are needed to draw conclusions about their potential prognostic and therapeutic value.

Recently, studies of tumor biology by gene expression micro-array technology have demonstrated patterns correlating with breast cancer in young women [START_REF] Anders | Young age at diagnosis correlates with worse prognosis and defines a subset of breast cancers with shares patterns of gene expression[END_REF] and even local relapses in young women [START_REF] Kreike | Local recurrence after breast-conserving therapy in relation to gene expression patterns in a large series of patients[END_REF]. However, this new technology is not yet robust enough for clinical use with regard to local treatment decisions until results from ongoing prospective clinical trials are available. Genetic alterations in histologically normal tissue adjacent to the breast tumor, such as chromosomal instability en DNA hypermethylation, could also play a role [START_REF] Ellsworth | Genomic patterns of allelic imbalance in disease free tissue adjacent to primary breast carcinomas[END_REF][START_REF] Yan | Mapping geographic zones of cancer risk with epigenetic biomarkers in normal breast tissue[END_REF].

For the patients in our study information about BRCA1/2 status was largely unknown. Population-based data indicate that approximately 10% of the breast cancers in women younger than 40 years are related to a BRCA1 or BRCA2 mutation [START_REF] Sanjosé | Prevalence of brca1 and brca2 germline mutations in young breast cancer patients: A population based study[END_REF].Although women who carry BRCA mutations have a very high risk of developing breast cancer, there is no convincing evidence that their risk of developing local relapse after BCT is significantly different from the risk in non-carriers [START_REF] Pierce | Ten-year multi-institutional results of breast-conserving surgery and radiotherapy in brca1/2-associated stage i/ii breast cancer[END_REF][START_REF] Haffty | Outcome of conservatively managed early-onset breast cancer by brca1/2 status[END_REF][START_REF] Alpert | Conservative management of breast cancer in brca1/2 mutation carriers[END_REF]. The risk of developing a contralateral breast cancer is, on the other hand, much higher than in non-carriers [START_REF] Pierce | Ten-year multi-institutional results of breast-conserving surgery and radiotherapy in brca1/2-associated stage i/ii breast cancer[END_REF][START_REF] Alpert | Conservative management of breast cancer in brca1/2 mutation carriers[END_REF][START_REF] Haffty | Outcome of conservatively managed early-onset breast cancer by brca1/2 status[END_REF].

The categorisation into breast cancer subtypes, based on the combination of hormonal receptor status and HER-2 status, may be predictive for recurrence risks and survival. Kyndi et al found a significantly lower locoregional recurrence rate and a significant survival improvement in hormone receptor positive patients treated with mastectomy and radiation therapy whereas in triple negative and hormone receptor negative patients the benefit of radiotherapy was much less pronounced [START_REF] Kyndi | Estrogen receptor, progesterone receptor, her-2, and response to mastectomy radiotherapy in high-risk breast cancer: The danish breast cancer cooperative group[END_REF]. In HER-2 positive patients, trastuzumab reduces the risk of locoregional recurrence by 57% [START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable her2-positive breast cancer[END_REF].

In summary, local control after BCT in patients ≤40 years is significantly worse than after mastectomy with local relapses continuing to occur during long-term followup. Local relapses might negatively influence the long term survival of patients with breast cancer, although we were unable to demonstrate this. Adjuvant systemic therapy has been the main contributor to the observed improvement of local control over the past decades. Nevertheless, the local relapse rate remains approximately 1% per year, which underlines the necessity of improvement and continuation of follow-up. Whether the marked reluctance against BCT in young women is less appropriate with current, more potent systemic treatment has to be confirmed. The

  Fig. 1a, 1b Proportion of patients receiving adjuvant systemic treatment, according to

Fig. 2a Fig. 3

 2a3 Fig. 2a Actuarial local tumor control in patients aged ≤40 years, according to local

Table 1

 1 General characteristics of patients aged 40 years or younger undergoing breast-conserving therapy (BCT) or mastectomy in the period 1988-2005.

	Characteristic	BCT (n=889)	Mastectomy	P-value
				(n=562)
		N	(%)	N	(%)
	Age (yrs)					0.048
	≤30	69	(8)	65	(12)
	31-35	253	(28)	149	(27)
	36-40	567	(64)	348	(62)
	Period					<.0001
	1988-1993	309	(35)	136	(24)
	1994-1999	306	(34)	198	(35)
	2000-2005	274	(31)	228	(51)
	Tumor size (pT)					<.0001
	T1	610	(70)	227	(42)
	T2	266	(30)	313	(58)
	Unknown	13		22
	Nodal status (pN)					<.0001
	pN-	581	(66)	271	(48)
	pN+	304	(34)	288	(52)
	Unknown	4		3
	Tumor type					<.0001
	Ductal	802	(90)	478	(85)
	Lobular/mixed	60	(7)	81	(14)
	Other	27	(3)	3	(1)
	Grade					0.016
	Good	52	(12)	30	(8)
	Intermediate	151	(34)	109	(29)
	Poor	239	(54)	242	(64)
	Unknown	447		181
	Estrogen receptor					0.389
	Negative	255	(36)	158	(32)
	Positive	455	(64)	314	(68)
	Unknown	179		90
	Microscopic completeness of tumor excision					<.0001
	Complete	836	(94)	477	(85)
	Incomplete	39	(4)	10	(2)
	Doubtful or unknown	14	(2)	75	(12)
	Radiotherapy					<.0001
	Yes	889	(100)	206	(37)
	No	0	(0)	355	(63)
	Unknown			1

Table 3

 3 Multivariable analysis for time until distant metastases for patients aged 40 years or younger

	(n=1408)
	Characteristic	HR	(95% CI)	P


Dr Anthony Gonçalves
email: goncalvesa@marseille.fnclcc.fr

Pascal Finetti

Renaud Sabatier

Marine Gilabert

José Adelaïde

Jean-Paul Borg

Max Chaffanet

Patrice Viens

Daniel Birnbaum

François Bertucci

José Adelaide

-mRNA expression in human breast cancer: a meta-analysis

Keywords: PARP1, breast cancer, triple-negative, basal, microarrays PARP1, Poly(ADP-ribose) polymerase-1, SSBs, single-strand breaks, DSBs, double-strand breaks, TN, triple-negative, NB, normal breast, SSP, single sample predictor, DWD, distance weighted discrimination, aCGH, arraycomparative genomic hybridization, MFS, metastasis-free survival, IHC, immunohistochemical, ER, estrogen receptor, PR, progesterone receptor

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

Introduction

Poly(ADP-ribose) polymerase-1 (PARP1), the most abundant member of the PARP superfamily, is a nuclear chromatin-associated protein involved in a wide range of biological processes including cell proliferation, apoptosis, malignant transformation, transcriptional regulation and DNA repair. PARP1 is essential to the base excision repair of DNA single-strand breaks (SSBs) [START_REF] Ame | The PARP superfamily[END_REF]. In response to DNA damage, PARP1 senses and binds to DNA nicks and breaks, resulting in activation of catalytic activity, causing poly(ADP)ribosylation of PARP1 itself, as well as other acceptor proteins, such as histones and topoisomerases. This modification potentially stimulates the recruitment and activity of other components of DNA repair pathways [START_REF] Ame | The PARP superfamily[END_REF]. In its absence, DNA SSBs accumulate and degenerate to DNA double-strand breaks (DSBs), which are not appropriately repaired if the BRCA pathway is deficient or dysfunctional. This is thought to explain the exquisite sensitivity to PARP inhibitors of tumors with BRCA inactivation, a concept called "synthetic lethality" [START_REF] Farmer | Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy[END_REF][START_REF] Bryant | Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase[END_REF]. Recent clinical evidence confirmed that PARP inhibition is a promising therapeutic strategy either as single-agent in BRCA1 or BRCA2-mutated breast cancers [START_REF] Fong | Inhibition of Poly(ADP-Ribose) Polymerase in Tumors from BRCA Mutation Carriers[END_REF], or in combination with cytotoxic chemotherapy in triple-negative (TN) breast cancers, a subgroup that shares many features with BRCA1-mutated tumors [START_REF] O'shaughnessy | Efficacy of BSI-201, a poly (ADP-ribose) polymerase-1 (PARP1) inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): Results of a randomized phase II trial[END_REF]. Several PARP inhibitors are currently in clinical development. However, until now, PARP1 mRNA expression has not been described in breast cancer. A few and relatively small (less than 95 samples) studies have been reported in melanoma [START_REF] Csete | Poly(adenosine diphosphateribose) polymerase-1 expression in cutaneous malignant melanomas as a new molecular marker of aggressive tumor[END_REF][START_REF] Staibano | Poly(adenosine diphosphate-ribose) polymerase 1 expression in malignant melanomas from photoexposed areas of the head and neck region[END_REF], ovarian cancer [START_REF] Brustmann | Poly(adenosine diphosphate-ribose) polymerase expression in serous ovarian carcinoma: correlation with p53, MIB-1, and outcome[END_REF] and colon cancer [START_REF] Nosho | Overexpression of poly(ADP-ribose) polymerase-1 (PARP-1) in the early stage of colorectal carcinogenesis[END_REF], and showed frequent PARP1 overexpression, associated with poor-prognosis histo-clinical features.

Here, we explored our gene expression database of 326 breast cancer samples profiled using oligonucleotide microarrays. Data were then combined with 12 publicly available expression datasets, resulting in a total of 2,485 invasive breast cancers informative for meta-analysis. Our primary objective was to describe the molecular epidemiology of this novel therapeutic target in a large population of early breast cancer patients. Secondary objectives included correlation between PARP1 expression and other clinical, pathological and molecular features, including survival.

Patients and Methods

Gene expression data of breast cancer

To determine PARP1 mRNA expression in breast cancer, we analyzed gene expression data of 326 breast tumor samples that we had profiled using oligonucleotide microarrays. Tumor tissues had been collected from 326 patients with invasive adenocarcinoma who underwent initial surgery at the Institut Paoli-Calmettes (Marseilles, France) between 1987 and 2007. Each patient gave written informed consent and the study was approved by our institutional review board.

Samples were macrodissected and frozen in liquid nitrogen within 30 min of removal.

All specimens contained >60% of tumor cells (as assessed before RNA extraction using frozen sections adjacent to the profiled samples). After surgery, patients were treated using a multimodal approach according to standard guidelines. DNA and RNA were extracted from frozen samples by using guanidium isothiocynanate and cesium chloride gradient as described previously [START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF]. RNA integrity was controlled on Agilent Bioanalyzer (Agilent Technologies). We had also profiled 11 normal breast (NB) tissue samples pooled in 4 RNA samples. Expression profiles had been established for these 326 cancers and 4 NB pools with Affymetrix U133 Plus 2.0 human oligonucleotide microarrays as previously described [START_REF] Bertucci | Gene expression profiling shows medullary breast cancer is a subgroup of basal breast cancers[END_REF]. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GeneChip Operating Software. Data were analyzed by the Robust Multichip Average method [START_REF] Irizarry | Exploration, normalization, and summaries of high density oligonucleotide array probe level data[END_REF] in R using Bioconductor and associated packages. PARP1 expression was measured by analyzing the sole probe set present on our Affymetrix microarrays, ID 208644_at. Before analysis, gene expression levels for each tumor sample were centered by the average expression levels of the four NB samples. All data were then log2 transformed for display and analysis.

To examine the correlation between PARP1 mRNA expression and histoclinical features of tumors in a large series of samples, we analyzed 12 publicly available datasets collected from the National Center for Biotechnology Information (NCBI)/ Genbank GEO database (series entry GSE2034, GSE2990, GSE4922, GSE1456, GSE7390, GSE2741, GSE1992, GSE2740, GSE2607, GSE6130, GSE3165, GSE6128, GSE10886) or at the following web addresses http://bioinformatics.mdanderson.org/pubdata.html;http://microarraypubs.stanford.edu/wound_NKI/ ; http://www.rii.com/publications/2002/vantveer.html.

To be comparable across datasets and to exclude bias from population heterogeneity, PARP1 expression levels were standardized within datasets using the luminal A population as reference. Combined with our IPC series, this resulted in a total of 2,485 invasive breast cancers with PARP1 expression and histo-clinical data available for meta-analysis (Table 1).

The molecular subtypes related to the Stanford intrinsic breast cancer classification were determined using Single Sample Predictor (SSP) classifier based on a list of 306 intrinsic genes [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF]. Briefly, after having identified the genes common between the intrinsic gene set and each expression dataset, we then used Distance Weighted Discrimination (DWD) [START_REF] Benito | Adjustment of systematic microarray data biases[END_REF] to normalize each dataset in order to be comparable to the 315 samples of the Hu"s combined test sample set. Next, we defined the expression centroid of each subtype for the common probe sets in this combined test sample set [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF]. Finally, we measured the correlation of each sample with each centroid. The sample was attributed the subtype corresponding to the most correlated centroid.

Array-comparative genomic hybridization data of breast cancer

Our analysis included data of genomic imbalances of 260 out of the 326 breast tumors. Data had been generated by array-comparative genomic hybridization (aCGH) using 244K CGH Microarrays (Hu-244A, Agilent Technologies) following a described protocol [START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF]. A pool of 13 normal male DNA had been used as reference.

Scanning was done with Agilent Autofocus Dynamic Scanner (G2565BA, Agilent Technologies). Data analysis was done as described [START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF]. Extraction of data (log2 ratio) was done from CGH Analytics, whereas normalized and filtered log2 ratio was obtained from ""Feature Extraction"" software (Agilent Technologies). The PARP1 locus at 1q41 was analyzed and copy number changes were characterized as reported previously [START_REF] Adelaide | Integrated profiling of basal and luminal breast cancers[END_REF]. Eight probes matched the PARP1 gene on our Agilent chips. A tumor was considered as harboring a gain for PARP1 if at least 5 (out of 8) consecutive probes displayed a log2 ratio tumor/normal >|0.5|.

Statistical analyses

Study is retrospective and multicentric based on a large public data set.

Comparisons of mean PARP1 expression level according to classical histo-clinical factors were done using Student t-test (2 variables) or one-way analysis of variance (ANOVA; more than 2 variables). Metastasis-free survival (MFS) was calculated from the date of diagnosis until date of distant relapse and follow-up was measured from the date of diagnosis to the date of last news for patients without relapse. Overall survival (OS) was calculated from the date of diagnosis until date of death and followup was measured from the date of diagnosis to the date of last news for patients without death. Survival curves were generated using Kaplan-Meier method and compared with the log-rank test. Univariate and multivariate analyses were done using Cox regression analysis. Variables tested in univariate analysis included patients"age at time of diagnosis (≤50 years vs >50), pathological axillary lymph node status (pN: negative vs positive), pathological tumor size (pT: pT1 vs pT2-4), pathological grade (I vs 2-3), immunohistochemical (IHC) estrogen receptor (ER), progesterone receptor (PR), and ERBB2 status (negative vs positive), and PARP1 mRNA expression (continuous value). Variables with a p-value <0.10 in univariate analysis were tested in multivariate analysis. All statistical tests were two sided at the 5% level of significance. Statistical analysis was done using the survival package (version 2.30) in the R software (version 2.4.1; http://www.cran.r-project.org/). We followed the reporting recommendations for tumour marker prognostic studies (REMARK) [START_REF] Mcshane | REporting recommendations for tumor MARKer prognostic studies (REMARK)[END_REF].

Results

PARP1 mRNA expression and DNA copy number: IPC series

We analyzed expression data generated using Affymetrix microarrays from 330 samples including 326 pre-treatment primary breast cancers and 4 NB samples collected at our institution. First, we observed that PARP1 mRNA expression was highly heterogeneous, with expression level varying across a large dynamic range and encompassing almost four base 2 logs. To address the impact of potential intratumor heterogeneity in this pattern, we took advantage of recently published microarray data generated from a series of 18 breast cancer samples [START_REF] Barry | Intratumor heterogeneity and precision of microarray-based predictors of breast cancer biology and clinical outcome[END_REF]. In this study, gene expression profiles obtained from 2 to 3 biopsy replicates of the same tumor sample were available for comparison; we observed an intra-patient PARP1 mRNA expression variance significantly lower than inter-patient variance (p=9.19E-29, ANOVA), suggesting that intra-tumor heterogeneity has a minor influence on the heterogeneous expression of PARP1 we have observed.

Whole-genome hierarchical clustering showed that PARP1 was located within a proliferation gene cluster, including for example MKI67 and PCNA, along with other genes involved in DNA damage repair such as RAD51 and ERCC4. Of note, PARP1 expression did not correlate with BRCA1 and BRCA2 expression (data not shown).

Compared to NB, PARP1 was overexpressed (≥2-fold increase) in 58% of cancer samples (Figure 1a). Array-comparative genomic hybridization (aCGH) data, simultaneously available for 260 of the 326 samples, allowed us to analyze the PARP1 locus at 1q41. Amplification of the 1q41-q44 region is a frequent genetic alteration in breast cancer [START_REF] Bieche | Loss and gain of distinct regions of chromosome 1q in primary breast cancer[END_REF][START_REF] Knuutila | Online access to CGH data of DNA sequence copy number changes[END_REF][START_REF] Larramendy | Comparative genomic hybridization reveals complex genetic changes in primary breast cancer tumors and their cell lines[END_REF]. A significant genomic gain (log2 ratio>|0.5|) or amplification (log2 ratio>|1|) was observed in 91 out of 260 (35%) samples (the mean percent of probes displaying this copy number alteration was 99.2% [95CI, 97.8 -100] within the 91 altered tumors). Moreover, a significant association between PARP1 mRNA expression and gene copies was observed: the mean expression level of PARP1 mRNA was more than twice higher in tumors with PARP1 gain or amplification compared with tumors displaying a normal gene copy number (p<1.E-8, t-test; Figure 1b). However, gain/amplification was not the sole mechanism of overexpression, which was also found in tumors without any gene copy number gain.

PARP1 mRNA expression and histo-clinical correlations: meta-analysis

Meta-analysis examined correlations between PARP1 mRNA expression and histo-clinical features of tumors in a dataset of 2,485 invasive breast cancer samples, including our series and 12 public microarray datasets. As shown in Table 2, PARP1 expression was significantly (t-test) associated with immunohistochemical (IHC) negativity for estrogen receptor (ER), progesterone receptor (PR) and ERBB2, with high grade, histological type (being the highest in medullary cancers and the lowest in lobular tumors), high pathological tumor size, with a trend for axillary lymph node involvement (p=0.067), but not with age. For 430 cases with available IHC information, PARP1 expression was higher in TN samples (negative for ER, PR and ERBB2), compared to ERBB2+ (positive for ERBB2, whatever the ER and PR status) and ERBB2-/HR+ (positive for ER and/or PR, negative for ERBB2) samples (p=0.05, one-way ANOVA, data not shown).

We looked for PARP1 expression in the transcriptional molecular subtypes defined by the intrinsic gene set [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF]. PARP1 was overexpressed in basal samples compared to other subtypes (Figure 2). Interestingly, PARP1 expression was much more tightly associated with the basal subtype than with the TN subgroup. Indeed, among the 430 above-described cases with available IHC classification, the correlation of PARP1 mRNA with the molecular subtypes (basal, ERBB2, and others) was high (p=4.6E-6, one-way ANOVA) and greatly superior to that observed with the IHC classification (data not shown). Examining the sole IPC dataset (where simultaneous mRNA and genomic data were available), it was possible to address within each molecular subtype the above-described association between PARP1 expression and gene copy gain. The association was significant (t-test) in basal (p=5.06E-4), ERBB2 (p=7.2E-3) and luminal B (p=6.28E-3) samples, but not in luminal A and normal samples. PARP1 was overexpressed in 84, 100 and 79% of PARP1-amplified/gained basal, ERBB2 and luminal B samples, respectively, versus 61 and 50% of luminal A and normal samples, respectively. Finally, we examined the prognostic impact of PARP1 expression, along with other known clinical or pathological factors. We first examined MFS. In this series, follow-up was available for 1,637 patients: 494 women developed metastatic relapse with a median time to relapse of 32 months, and 1,143 remained relapse-free with a median follow-up of 96 months. In univariate analysis, PARP1 expression (together with pN, pT, grade, and ER, PR, and ERBB2 IHC status) was associated with a worse MFS (HR=1.12 [1.04-1.22]; p=0.004; Figure 3a). However, this was not maintained in multivariate analysis (data not shown). Since potential prognostic factors for MFS may interact with and thus be confounded by systemic adjuvant therapies, we re-analyzed data according to the treatment received, adjuvant chemotherapy and/or hormonal therapy, no patient being exposed to adjuvant trastuzumab. There were 261 patients who had not received any adjuvant systemic therapy (subgroup CT0HT0). Among them, 91 developed metastatic relapse with a median time to relapse of 27 months, and 170 remained relapse-free with a median follow-up of 99 months. There were 602 patients who had not received any adjuvant chemotherapy, with or without hormonal therapy (subgroup CT0HT+/-), including 210 who experienced metastatic relapse with a median time of 28 months, and 392 who did not relapse with a median follow-up of 100 months. As shown in Table 3 and Regarding OS, follow-up was available for 1,115 patients: 285 women died with a median time of 49 months and 830 were alive with a median follow-up of 100 months. In univariate analysis, PARP1 expression was associated with a worse OS (HR=1.16 [1.04-1.29]; p=0.006; Figure 3b). However, this was not maintained in multivariate analysis (data not shown). In the CT0HT0 subgroup (163 patients with available follow-up), 43 patients died with a median time of 46 months and 120 were alive with a median follow-up of 108 months. In the CT0HT+/-subgroup, 213 patients had follow-up data available, including 53 who died with a median time of 47 months and 160 alive with a median follow-up of 101 months. In the CT1 subgroup (326 patients with available follow-up), 93 patients died with a median time to death of 41 months and 233 were alive with a median follow-up of 74 months. No significant association was found between PARP1 expression and OS in any subgroup (HR=1.23 [0.93-1.62], p=0.15 in CT0HT0; HR=1.18 [0.92-1.52], p=0.2 in CT0HT+/-; HR=1.13 [0.94-1.36], p=0.18 in CT1), even though trends similar to MFS (lower survival in high PARP1-expressing tumors in untreated patients only) were observed (Figure 3b).

Discussion

To date PARP1 mRNA expression and DNA copy number alterations have not been described in breast cancer. In this large series, we show that PARP1 expression is heterogeneous, with overexpression found in a significant number of breast cancers, in part due to genomic gain/amplification. This is particularly true in TN and even more in basal samples, and to a lesser degree in ERBB2+ and luminal samples. However, the role of such an overexpression in tumor initiation or progression, if any, remains to be elucidated. In addition, and for the first time to our knowledge, we have shown a significant and independent association between PARP1 expression and metastasis-free survival in breast cancer. Importantly, this association was restricted to patients not treated with any adjuvant chemotherapy.

Exploiting DNA repair alterations using PARP inhibition [START_REF] Fong | Inhibition of Poly(ADP-Ribose) Polymerase in Tumors from BRCA Mutation Carriers[END_REF][START_REF] O'shaughnessy | Efficacy of BSI-201, a poly (ADP-ribose) polymerase-1 (PARP1) inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): Results of a randomized phase II trial[END_REF] was recently shown as a promising approach in BRCA-mutated or TN breast cancers, which are classically associated with the basal subtype [START_REF] Cleator | Triple-negative breast cancer: therapeutic options[END_REF]. Thus, in a phase I dose-escalation study, Fong and colleagues selected a population enriched in BRCA-associated cancers [START_REF] Fong | Inhibition of Poly(ADP-Ribose) Polymerase in Tumors from BRCA Mutation Carriers[END_REF]. In the overall population, there was no objective response. In the group of 19 patients with a documented BRCA mutation, including breast, ovarian, and prostate malignancies, there was a 47% response rate and a 63% clinical benefit rate. In TN breast cancer, a randomized phase II study compared addition of a PARP1 inhibitor, BSI-201, to chemotherapy with gemcitabine and carboplatin versus chemotherapy alone [START_REF] O'shaughnessy | Efficacy of BSI-201, a poly (ADP-ribose) polymerase-1 (PARP1) inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): Results of a randomized phase II trial[END_REF]. Preliminary data demonstrated that the addition of BSI-201 was associated with highly significant improvements in response (from 16% to 48%), in progression-free survival (median from 3.3 to 6.9 months), as well as overall survival. A randomized phase III study is currently underway to confirm these results with gemcitabine, carboplatin, and BSI-201. Moreover, cisplatin-based neoadjuvant treatment [START_REF] Byrski | Pathologic complete response rates in young women with BRCA1-positive breast cancers after neoadjuvant chemotherapy[END_REF] provided dramatic pathological response rate (nearly 75%) in a smallsized population of tumors occurring in BRCA-mutated patients. In addition, cisplatin as single-agent induced a promising response rate of 22% in TN breast cancer, which was correlated with low BRCA expression [START_REF] Silver | Efficacy of Neoadjuvant Cisplatin in Triple-Negative Breast Cancer[END_REF]. These convergent clinical data suggest that tumors with deficient DNA-repair phenotypes, thought to be enriched in BRCA-mutated and TN subtypes, could be very sensitive to PARP inhibitors and DNA-damaging agents, alone or in combination. However, reliable and robust biomarkers that allow the accurate identification of alterations in functional DNA repair pathways (the so-called "BRCAness" phenotype, with the highest probability to benefit from these therapies) are still lacking. It still remains to be demonstrated whether the major increase in PARP1 expression observed in our study in some breast cancers might be such a marker. Speculatively, it could reflect a regulatory response to genetic instability, aiming to compensate for an abnormal rate of DNA damages, explaining why this event is more pregnant in the most instable subtypes, such as TN and basal. In this regard, our observation that the prognostic value of PARP1 expression was lost in patients receiving adjuvant chemotherapy could suggest that overexpression of PARP1 may be associated with tumor sensitivity to cytotoxic treatment, essentially cyclophosphamide and/or anthracycline-based in our retrospective series. Such an hypothesis is consistent with the recently reported positive correlation between PARP1 protein expression and response to neoadjuvant chemotherapy [START_REF] Von Minckwitz | Denkert C 443 PARP is expressed in all subtypes of early breast cancer and is a predictive factor for response to neoadjuvant chemotherapy[END_REF][START_REF] Loibl | Denkert C PARP expression in early breast cancer and its predictive value for response to neoadjuvant chemotherapy[END_REF]. In this study, 646 breast cancer samples from the GeparTrio neoadjuvant phase III trial were centrally stained by IHC for PARP protein. Similarly to what we observed at the mRNA level, PARP protein was found in all molecular subtypes, more frequently in ERBB2-positive and TN tumors, and correlated with most known prognostic factors, including poor differentiation, non-lobular histological type and negative hormonal receptivity. Notably, PARP expression independently predicted for the pathological complete response (pCR) to neoadjuvant taxaneanthracycline-based chemotherapy: patients with high expression showed a pCR rate of 25.7% compared to 18.8% and 6.1% in patients with medium or low expression (p<0.001). Interestingly, it is worth emphasizing that the deficient DNA-repair phenotype may be observed beyond these subtypes. For example, recent data have suggested that PTEN deficiency may associate with reduction in RAD51 expression and perturbation of homologous recombination in BRCA-intact human cancer cells, leading to high sensitivity to PARP inhibition [START_REF] Mendes-Pereira | Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors[END_REF]. Moreover, a similar deficiency in DSBs repair may be functionally induced in various non-genetic conditions, including hypoxia or even PARP inhibition itself, both setting being associated with a repression in BRCA1 and RAD51 transcription and therefore an increased efficiency of anti-PARP treatment [START_REF] Hegan | Inhibition of poly(ADP-ribose) polymerase down-regulates BRCA1 and RAD51 in a pathway mediated by E2F4 and p130[END_REF]. Altogether, these data support the hypothesis that PARP inhibition may have broader applications in breast cancer treatment than anticipated based on initial preclinical and clinical results.

In conclusion, we report the first large-scale integrated analysis of PARP1 expression and genomic status in breast cancer. Whether PARP1 overexpression may identify breast cancers with higher probability of response to PARP inhibitors or DNA-damaging agents needs to be determined. But, if this hypothesis were confirmed, our data should not only strengthen the interest for these compounds in basal breast cancers, but also suggest promising applications in a larger fraction of patients, beyond the basal subtype. b/ Kaplan-Meier OS curves (the legend is similar to a/).

All BC samples

Figure 3a ,

 3a Figure 3a, PARP1 mRNA expression was associated with a worse MFS in univariate

Figure 2 :

 2 Figure 2: mRNA expression of PARP1 according to breast cancer molecular

Figure 3 :

 3 Figure 3: Metastasis-free and overall survivals according to PARP1 mRNA

Figure1

  Figure1Click here to download Figure: Figure_1_may2010.ppt

Figure2

  Figure 2

  Figure 3 A All patients CT0HT0

Figure 3

 3 Figure 3 Click here to download Figure: Figure 3 Revised.ppt

Table 1 :

 1 Histo-clinical characteristics of the 2,485 breast cancer patients.

	Variables and categories	N (%)
	Sex
	Female	2485 (100%)
	Age (years)
	≤50	897 (51%)
	>50	856 (49%)
	Histological type
	DUC	495 (79%)
	LOB	35 (6%)
	MIX	30 (5%)
	MED	24 (4%)
	TUB	6 (1%)
	MUC	5 (1%)
	Other	32 (5%)
	Pathological axillary lymph node invasion
	(pN)
	No	1280 (66%)
	Yes	671 (34%)
	Pathological tumour size (pT)
	pT1	753 (40%)
	pT2	877 (47%)
	pT3	169 (9%)
	pT4	65 (3%)
	Grade
	1	337 (16%)
	2	820 (39%)
	3	932 (45%)
	ER (IHC)
	Negative	653 (32%)
	Positive	1362 (68%)
	PR (IHC)
	Negative	306 (42%)
	Positive	418 (58%)
	ERBB2 (IHC)
	Negative	334 (78%)
	Positive	96 (22%)
	Adjuvant chemotherapy
	No	625 (57%)
	Yes	480 (43%)
	Chemotherapy regimen
	CMF	10 (2%)
	AC	195 (40%)
	AC-T	165 (34%)
	Unknown	110 (24%)
	Adjuvant hormonal therapy
	No	815 (76%)
	Yes	254 (24%)
	Follow-up (months)
	Median	96
	Metastatic relapse
	No	1143 (70%)
	Yes	494 (30%)

Table 2 :

 2 PARP1 mRNA expression according to histo-clinical features in 2,485 breast cancer samples.

	Variables and categories (N)	Mean PARP1 mRNA expression (log2)	p-value
	Age (years)		0.44
	< 50 (897)	1.08
	> 50 (856)	1.12
	Histological type		4.49E-9
	DUC (495)	1.15
	LOB (35)	0.85
	MED (24)	2.61
	MIX (30)	0.77
	MUC (5)	0.96
	TUB (6)	0.95
	Other (32)	1.37
	Pathological axillary lymph node invasion		0.067
	(pN)	1.08
	no (1280)	1.18
	yes (671)
	Pathological tumour size (pT)		0.0007
	pT1 (753)	0.98
	pT2 (877	1.16
	pT3 (169)	1.30
	pT4 (65)	1.29
	Grade		4.64E-20
	1 ( 337)	0.67
	2 ( 820)	0.96
	3 (932)	1.32
	ER (IHC)		7.27E-6
	positive (653	1.02
	negative (1362)	1.28
	PR (IHC)		0.018
	positive (306)	1.04
	negative (418)	1.24
	ERBB2 (IHC)		0.05
	positive (96)	0.96
	negative (334)	1.21

Table 3 :

 3 Univariate and multivariate analyses in MFS in patients untreated with

	systemic adjuvant chemotherapy
	A/ Subgroup CT0HT0
			Univariate			Multivariate
	Variables	N	HR [CI95%]	p-value	N	HR [CI95%]	p-value
	Age >50	261	0.81 [0.49-1.31]	0.38
	ER (IHC) pos	259	0.58 [0.38-0.89]	0,012	133	0.69 [0.4-1.19]	0,18
	PR (IHC) pos	38	0.77 [0.21-2.87]	0,695
	ERBB2 (IHC) pos	34	NA	NA
	pN pos	134	2.88 [1.66-5]	0,0001	133	2.12 [1.22-3.7] 0,0078
	pT T2-4	260	2.07 [1.37-3.13] 0,0005	133	1.83 [1.07-3.12] 0,027
	Grade 2-3	259	4.15 [2.01-8.59] 0,0001	133	2.22 [0.76-6.46]	0,14
	mRNA PARP1	261	1.25 [1.04-1.51]	0,017	133	1.24 [1-1.53]	0,054
	B/ Subgroup CT0HT+/-
			Univariate			Multivariate
	Variables	N	HR [CI95%]	p-value	N	HR [CI95%]	p-value
	Age >50	316	0.76 [0.50-1.17]	0.21
	ER (IHC) pos	600	0.72 [0.54-0.97]	0,029	166	0.6 [0.36-0.98]	0,042
	PR (IHC) pos	73	0.85 [0.3-2.47]	0,772
	ERBB2 (IHC) pos	65	0.00 [0.00-Inf]	1
	pN pos	456	2.5 [1.66-3.78]	1,35E-05	166	2.34 [1.42-3.84] 0,0008
	pT T2-4	314	2.18 [1.47-3.24] 0,0001	166	1.59 [0.98-2.58] 0,059
	Grade 2-3	312	4.24 [2.14-8.4]	3,54E-05	166	1.99 [0.83-4.76]	0,12
	mRNA PARP1	602	1.12 [0.99-1.27]	0,063	166	1.24 [1.02-1.51] 0,035
	NA, not applicable (no ERBB2-positive patient)

b/ Expression across 260 BC samples profiled also by aCGH using 244K CGH microarrays (Hu-244A, Agilent Technologies). Box plots of PARP1 expression in BC are shown according to PARP1 genomic status, with (left) and without (right) gain defined as a DNA copy number ratio tumor/NB ≥2 (horizontal dashed line).

Differences in PARP1 expression levels between both groups were tested for significance using Student t-test. For each box plot, median and ranges are indicated.

Conflict of interest

All authors declare that they have no conflict of interest to disclose

Aknowledgments