MD Florian Fitzal
email: florian.fitzal@meduniwien.ac.at

Riedl

Oncologic safety of breast conserving surgery after tumor downsizing by neoadjuvant therapy: A retrospective single centre cohort study

Keywords: breast conservation, local recurrence, prospective trials, retrospective analyses, preoperative treatment, local treatment, local failure, preoperative therapy, breast preservation, downstaging, ABCSG 7, ABCSG 14, ABCSG24 4

Objective:

The objective of this study is to analyse local recurrence rates in patients receiving neoadjuvant Chemotherapy (nCT) comparing mastecomized (MX) patients with those undergoing breast conserving therapy (BCT). Method: Patients undergoing breast cancer surgery after nCT (3xCMF or 3-6xED) between 1995 and 2007 at our department were retrospectively analysed. Results: The median follow up was 60 months for 308 patients. Patients who were downsized from MX to BCT with partial or complete response (n=104) had similar local recurrence free survival (LRFS) compared to patients who did not experience successful downsizing (n=67) and finally undergoing MX (LRFS MX-BCT 81% versus MX-MX 91%: p=0.79). Uni-and multivariate analyses demonstrated that BCT itself was not an independent prognostic factor for a worse LRFS (p=0.07 and 0.14). After pathologic no change or progressive disease the risk of local recurrence was increased in patients undergoing BCT (MX-BCT; n=6 LRFS 66%) compared with MX (n=44; LRFS 90%; p=0.04). Overall survival in general was better for the BCT group (n=197) compared with MX group (n=111) regardless of clinical response (92% versus 72% p<0.0001). Breast conservation, nodal negativity and low or medium grade histology were prognostic factors for an improved OS (p = 0.02; 0.01; 0.004). Conclusion: Our study suggests that BCT is oncologically safe after tumor downsizing by nCT in patients primarily scheduled for mastectomy. These patients, however, should not be treated with breast conservation in the absence of any proven response after nCT.

Aknowledgement

Florian Fitzal is independent of any commercial funder and he had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Introduction

The meta-analysis by Mauri et al [START_REF] Mauri | Neoadjuvant versus adjuvant systemic treatment in breast cancer: a meta-analysis[END_REF] with nine randomized prospective trials of 3946 patients comparing neoadjuvant chemotherapy (nCT) with adjuvant therapy demonstrated a significant increased risk for local relapse in the nCT group (RR: 1.22). The inclusion of patients without optimal local treatment after nCT in this meta-analysis, however, was a major bias. A recent Cochrane database review by Mieog et al [START_REF] Mieog | Neoadjuvant chemotherapy for operable breast cancer[END_REF] clearly showed that in prospective randomized trials with adequate local therapy after nCT there is no significant increase in local relapse as compared to primary operation and adjuvant treatment. Patients with adequate local therapy had a significant reduction in local recurrence free survival (LRFS; p=0.02) compared with inadequate local therapy suggesting that breast conserving therapy (BCT) may be safe after nCT.

Prospective studies about BCT after nCT demonstrated a reduced LRFS in patients scheduled for mastectomy before and finally undergoing BCT (MX-BCT) after nCT compared with patients scheduled for BCT before and undergoing BCT (BCT-BCT) after nCT [START_REF] Fisher | Effect of preoperative chemotherapy on local-regional disease in women with operable breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-18[END_REF][START_REF] Fisher | Effect of preoperative chemotherapy on the outcome of women with operable breast cancer[END_REF][START_REF] Van Der Hage | Preoperative chemotherapy in primary operable breast cancer: results from the European Organization for Research and Treatment of Cancer trial 10902[END_REF]. These retrospective subgroup analyses, however, had a major bias as primary cancer biology differs within these two groups. Multivariat analyses have also not been reported. Moreover, patients who are eligible for BCT should not proceed to nCT outside clinical trials as they have no evident benefit so far but might acquire serious side effects from chemotherapy. In this regard, comparison of oncologic outcome of patients scheduled for mastectomy (MX) but undergoing BCT after nCT (MX-BCT) with those patients scheduled for and undergoing mastectomy after nCT (MX-MX) may be clinically more relevant.

In addition there is no guideline or statement so far regarding the use of BCT after nCT in patients without response to nCT. We hypothesize that those patients should not be treated with BCT as this may in fact increase local relapse.

Thus, the aim of our study in breast cancer patients undergoing nCT due to primarily scheduled mastectomy at a single cancer centre was to compare the oncologic outcome between final mastectomized (MX-MX) and breast conserved patients (MX-BCT) after nCT. Furthermore, we wanted to compare oncologic outcome between clinical responders and non-responders in this respect.

Patients and Methods

Design

We retrospectively analysed our prospectively build internal patient data base. Data are prospectively entered from a study nurse during each outpatient ward contact into a pre-existing EXCEL work sheet.

Inclusion criteria

All patients who completed nCT and local therapy at the Medical University Vienna from January 1995 up until May 2007 were included in the analyses (n=400). Eligibility for BCT or mastectomy before nCT has been re-evaluated by searching the patients' reports (radiographs, outpatient ward report, operation report). Patients without any clear pre-therapeutic decision for either mastectomy or BCT have been excluded from further analyses (n=75). Patients who were scheduled for BCT before and received MX after neoadjuvant therapy have been eliminated from the final analyses as it was of no interest for our research question (n=17). Thus, data from 308 patients are finally presented.

Definition of scheduling patients for mastectomy

Over the mentioned time period patients were seen by 6 different special breast surgeons dedicating more than 50% of their clinical activity to breast cancer surgery. If the size relation between breast and tumor exceeded 1:4 (more than one breast lump has to be excised) patients were scheduled for mastectomy. Accordingly, multicentricity seen in pretherapeutic radiologic examinations was another factor for scheduling patients for mastectomy before but also after nCT. For primary staging evaluation, clinical assessment as well as mammography and ultrasound or MRI-mammography was mandatory. Restaging was conducted routinely every two to three cycles of therapy.

Exclusion criteria

Metastatic breast cancer

Inflammatory breast cancer

Infiltration of the thoracic wall ECOG >2

Bilateral breast cancer

Any previous malignancy treated with curative intent and the patient has not been disease-free for 5 yearsexceptions are: carcinoma in situ of the cervix squamous carcinoma of the skin basal cell carcinoma of the skin Any recurrent cancer disease Pregnant or lactating women For detailed inclusion and exclusion criteria please refer to [START_REF] Steger | 6 vs. 3 Cycles of epirubicin/docetaxel + G-CSF in operable breast cancer: Results of ABCSG-14[END_REF][START_REF] Steger | A randomized phase III study comparing epirubicin, doxetacel, and capecitabine (EDC) to epirubicin and docetaxel (ED) as neoadjuvant treatment for early breast cancer -first results of the ABCSG Trial 24[END_REF][START_REF] Taucher | The potential risk of neoadjuvant chemotherapy in breast cancer patients--results from a prospective randomized trial of the Austrian Breast and Colorectal Cancer Study Group (ABCSG-07)[END_REF].

Cohort groups

Patients (n=308) were splitted into three groups.

Group 1 BCT-BCT (n=87)

Patients who were scheduled for BCT before and received BCT after neoadjuvant therapy.

Group 2 MX-BCT (n=110)

Patients who were scheduled for MX before but received BCT after neoadjuvant therapy.

Group 3 MX-MX (n=111)

Patients who were scheduled for MX before and received MX after neoadjuvant therapy . We were interested in differences between the three groups with respect to local recurrence free survival (LRFS), overall and distant recurrence free survival (OS, DRFS). Moreover the influence of pathologic response to nCT within the three groups regarding LRFS, OS and DRFS was investigated.

Neoadjuvant chemotherapy

Most patients received neoadjuvant chemotherapy within three prospective randomized trials conducted by the Austrian Breast and Colorectal Cancer Study Group (ABCSG; trials ABCSG-7, ABCSG-14, and ABCSG-24) [START_REF] Steger | 6 vs. 3 Cycles of epirubicin/docetaxel + G-CSF in operable breast cancer: Results of ABCSG-14[END_REF][START_REF] Steger | A randomized phase III study comparing epirubicin, doxetacel, and capecitabine (EDC) to epirubicin and docetaxel (ED) as neoadjuvant treatment for early breast cancer -first results of the ABCSG Trial 24[END_REF][START_REF] Taucher | The potential risk of neoadjuvant chemotherapy in breast cancer patients--results from a prospective randomized trial of the Austrian Breast and Colorectal Cancer Study Group (ABCSG-07)[END_REF].

ABCSG 7

In ABCSG-7, 423 patients with hormone-receptor negative or high-risk endocrine responsive disease were randomized to three cycles of CMF (cyclophosphamide 600 mg/m², methotrexate 40 mg/m² and 5-fluorouracel 600 mg/m² on days 1 and 8, every four weeks) either as pre-or postoperative treatment. In node-negative patients, another three cycles of adjuvant CMF were administered, whereas node-positive patients received three further cycles of EC (epirubicin 70 mg/m² and cyclophosphamide 600 mg/m² on day 1, every three weeks). Overall response rate to neoadjuvant CMF was 56.2%, with 12 patients (5.9%) achieving pathological complete response (pCR). While no difference in terms of overall survival was observed between the two groups, recurrence-free survival was significantly better in patients receiving chemotherapy postoperatively, leading to the conclusion that three cycles of CMF was insufficient as neoadjuvant chemotherapy [START_REF] Taucher | The potential risk of neoadjuvant chemotherapy in breast cancer patients--results from a prospective randomized trial of the Austrian Breast and Colorectal Cancer Study Group (ABCSG-07)[END_REF].

ABCSG 14

ABCSG-14 compared three cycles of ED (epirubicin 75 mg/m² and docetaxel 75mg/m² on day 1, every three weeks, with granulocyte colony-stimulating factor on days 3 to 10 of each cycle) to six cycles of the same regimen as neoadjuvant treatment for breast cancer.

A total of 292 patients were accrued; six cycles of ED yielded a significantly higher pCR rate (18.6% versus 7.7%; p=0.0045), a significantly higher percentage of patients with negative axillary status (56.6% versus 42.8%; p=0.02), and a trend towards higher rate of breast conserving surgery (75.9% versus 66.9%; n.s.) [START_REF] Steger | 6 vs. 3 Cycles of epirubicin/docetaxel + G-CSF in operable breast cancer: Results of ABCSG-14[END_REF].

ABCSG 24

Based upon a proposed synergistic effect of docetaxel and capecitabine, ABCSG-24 compared six cycles of ED plus capecitabine (EDC; epirubicin 75 mg/m² and docetaxel 75mg/m² on day 1, capecitabine 1000 mg/m² BID days1 to 14, every three weeks, plus pegfilgrastim 6 mg on day 2 of each cycle) with standard six cycles of ED as established in ABCSG-14. Patients with Her2-positive disease were also randomized to neoadjuvant trastuzumab every three weeks or control. No results from that second randomization are available yet. A total of 512 patients were accrued to ABCSG-24. Significantly more patients reached pCR with ECD (23.8% versus15.2%; p=0.036), although less patients on ECD completed all six treatment cycles as scheduled, mainly due to capecitabineassociated toxicity [START_REF] Steger | A randomized phase III study comparing epirubicin, doxetacel, and capecitabine (EDC) to epirubicin and docetaxel (ED) as neoadjuvant treatment for early breast cancer -first results of the ABCSG Trial 24[END_REF].

Good cosmetic outcome after BCT questionable

Non palpable tumors were localized with a hook wire preoperatively. Intra-operative frozen section was done in all cases to determine the resection margins as this reduces the re-operation rate [START_REF] Loibl | Surgical procedures after neoadjuvant chemotherapy in operable breast cancer: results of the GEPARDUO trial[END_REF][START_REF] Riedl | Intraoperative frozen section analysis for breast-conserving therapy in 1016 patients with breast cancer[END_REF]. The resection was done within new resection boundaries after response to nCT in unifocal disease while in multifocal disease resection boundaries were only smaller if all tumors responded to nCT and the total diameter was reduced. Patients with multicentric disease were mastectomized. All patients underwent axillary level I and II dissection except in some selected postmenopausal clinical complete responders with no axillary involvement before nCT who underwent sentinel node biopsy only. However, axillary dissection followed sentinel node biopsy in case of a positive sentinel lymph node.

16

Response evaluation

Clinical

Statistical analyses

Categorical data is described with absolute and relative frequencies. Chi-square tests are used to test categorical data between groups. In case of sparse data Fisher's exact test was used. Time to event data with respect to LRFS, OS and DRFS are described graphically by the method of Kaplan-Meier and tested between groups by the log-rank test. The proportional hazards regression model of Cox was used to model the prognostic value of covariates in a uni-and multivariate manner.

All p-values are two-sided and p≤0.05 were considered significant. All calculations are performed with the statistical software SAS® (Version 9.2, SAS Institute Inc., Cary, NC, USA).

Results

Demographic data

From the 308 patients 221 were scheduled for mastectomy before nCT. 111 patients had to be mastectomized and 110 underwent BCT after nCT while 87 patients were scheduled for and received BCT after nCT. Primary level I axillary dissection was conducted in 273 patients, Sentinel only in 7 patients (clinical negative lymph nodes and good response, pathologic negative sentinel node) and sentinel and axillary dissection in 28 patients. All patients had at least 1mm free margin and underwent postoperative radiotherapy with a boost.

Table 1 shows the demographic data of the groups and compares between all patients finally mastectomized (MX) with those finally undergoing BCT. Table 1 also compares between MX-MX and MX-BCT and between BCT-BCT and MX-BCT. Preoperative clinical and postoperative pathologic tumor size were significantly different between patients undergoing MX and BCT while there was no difference in tumor size between BCT-BCT and MX-BCT patients after nCT. Lymph node status differed significantly between the groups while menopause, endocrine responsiveness (any receptor positive) and grading did not differ between the groups.

Oncologic outcome

The median follow up period was 60 months.

Local recurrence free survival

Comparing all patients finally mastectomized (n=111) with patients undergoing breast conservation after nCT (n=197) there was no significant difference between the two groups regarding local recurrence free survival (5years: 91% versus 89%; p=0.92). Within all patients who were initially scheduled for mastectomy an showing pathologic response (partial or complete remission), there was no increase of local relapse in those finally undergoing BCT after nCT compared with those finally undergoing mastectomy after nCT as shown in figure 1. Analyzing local relapse within pathological non responders, patients finally undergoing BCT, however, showed a significant increased risk in local recurrence as shown in figure 2. Comparing patients with pathologic response who were scheduled for mastectomy and finally receiving BCT (n=104) with patients who were primarily scheduled for and finally received BCT (n=69) the local recurrence free survival was only marginal significant different in favour of the BCT-BCT group (5-years: 84% versus 97% p=0.046).

Overall and distant free survival

Patients finally undergoing BCT (n=197) had an improved overall and distant recurrence free survival compared with patients finally mastectomized (n=111) regardless of response (5 years-OS: 92% versus 74%; 5 years-LRFS: 81% versus 58% p<0.0001). Patients who showed a pathologic response to nCT and were scheduled for mastectomy had an increased overall survival if breast conservation was performed at last as shown in figure 3. This was similar for LRFS (78% versus 61% p=0.052). There was no difference in 5 years-OS or LRFS comparing patients with pathologic response who were scheduled for mastectomy and finally receiving BCT (n=104) with patients who were primarily scheduled for and finally received BCT (n=69; OS: 89% versus 96% p=0.27; LRFS: 78% versus 89% p=0.10)

Prognostic factors for OS, LRFS and DRFS

Univariate and multivariate analyses as shown in table 2 revealed that BCT (both MX-BCT and BCT-BCT versus MX-MX), nodal negativity and grade 1 or 2 were prognostic for an improved OS (table 2). There were no prognostic factors for LRFS within these patients (table 3) while ductal type, smaller pathologic tumor size, grade 1 and 2 as well as nodal negativity were prognostic factors for a better DRFS (not shown).

Discussion

Our retrospective analysis shows that BCT after nCT does not increase the risk for LRFS compared with MX while OS as well as DRFS were significantly improved in BCT patients independent of their response to nCT. Patients who were scheduled for MX but finally underwent BCT due to clinical response showed no significant difference in LRFS compared with patients finally undergoing MX. Subgroup analyses with non-responders (pathologic no change or progressive disease), however, revealed a significant increased risk of local relapse after BCT compared with MX in this specific group. Multivariate analyses show that BCT is not a significant prognostic factor for reduced LRFS or DRFS but predicted for superior OS.

The first publications of prospective nCT trials suggested an increased risk for death and local relapse for patients undergoing BCT after nCT. Published data from the EORTC 10920 trial suggested that there may be a reduction of OS in patients scheduled for mastectomy comparing with patients scheduled for BCT finally undergoing BCT after nCT (HR=2.53) [START_REF] Van Der Hage | Preoperative chemotherapy in primary operable breast cancer: results from the European Organization for Research and Treatment of Cancer trial 10902[END_REF]. However, pre-therapeutic lymph node status differed between the groups (BCT-BCT 54% cN1 versus MX-BCT 64% cN1) possible influencing the final result.

Similar data from the NSABP-B18 trial demonstrated a reduced LRFS in MX-BCT patients compared with BCT-BCT patients (15.9% versus 9.9%) [START_REF] Wolmark | Preoperative chemotherapy in patients with operable breast cancer: nine-year results from National Surgical Adjuvant Breast and Bowel Project B-18[END_REF]. However, after controlling for patient age and clinical tumor size before treatment that difference was not significant any more [START_REF] Wolmark | Preoperative chemotherapy in patients with operable breast cancer: nine-year results from National Surgical Adjuvant Breast and Bowel Project B-18[END_REF]. Moreover tamoxifen has been shown to reduce LRFS [START_REF] Ebctcg | Effects of radiotherapy and of 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] and all patients regardless of receptor status received tamoxifen in NSABP-B18.

Our analyses support the hypothesis that LRFS within the group of patients finally receiving BCT after nCT is worse in patients scheduled for mastectomy compared with patients scheduled for BCT before nCT. The difference was still significant after excluding non-responders (all patients after nCT with no change or progressive disease). Clinical and pathological tumor size as well as grading, however, differed significantly while nodal status, menopause and endocrine responsiveness did not differ between these two groups.

Thus, we suggest that the difference in LRFS seen in our study as well as in other prospective trials is biased due to different tumor stage and should not lead to any contraindication for BCT after nCT.

Moreover patients scheduled for BCT should only be treated with nCT in prospective randomized trials as there is no proven benefit for this group [START_REF] Loibl | Surgical procedures after neoadjuvant chemotherapy in operable breast cancer: results of the GEPARDUO trial[END_REF][START_REF] Steger | A randomized phase III study comparing epirubicin, doxetacel, and capecitabine (EDC) to epirubicin and docetaxel (ED) as neoadjuvant treatment for early breast cancer -first results of the ABCSG Trial 24[END_REF]. The advantage of achieving pCR after nCT and thus, of knowing the response of the tumor to a certain kind of drug, is certainly a promising treatment guidance. However, pCR should only be used as surrogate marker in clinical trials. In addition, selecting patients with smaller sized breast cancer who are eligible for BCT in neoadjuvant trials may increase the number of patients receiving unnecessary chemotherapy as the use of nCT never improved oncologic outcome [START_REF] Mieog | Neoadjuvant chemotherapy for operable breast cancer[END_REF]. Thus, the surgical question regarding standard nCT outside clinical trials is, whether BCT after downsizing breast cancer in patients scheduled for mastectomy is oncologic safe. This group (MX-BCT) has never been compared with patients needing MX after planned MX (MX-MX).

Our retrospective analyses may in part answer this issue showing that patients after tumor downsizing primarily scheduled for MX but finally undergoing BCT after nCT had no increase in local relapse compared with patients scheduled for and finally undergoing MX.

Only patients who showed no response to nCT undergoing BCT had a significant increased local relapse rate compared with MX patients. Due to a significant difference in nodal status, grading and tumor size within these two groups the results have to be discussed with caution. However, multivariate analyses for OS, LRFS and DRFS show that BCT is no independent predictive factor for LRFS and DRFS supporting the hypothesis that breast conservation is safe after tumor downsizing by nCT. A prospective evaluation is needed to further elucidate this issue.

A meta-analyses published in 2005 [START_REF] Mauri | Neoadjuvant versus adjuvant systemic treatment in breast cancer: a meta-analysis[END_REF] including 9 randomized studies comparing pre-with postoperative chemotherapy in 3946 patients demonstrated a reduced LRFS in the BCT group after nCT compared with patients undergoing BCT and adjuvant therapy.

This difference was largely influenced by the trials in which surgery was omitted and local treatment was achieved with the use of radiotherapy only in several patients [START_REF] Broet | Short and longterm effects on survival in breast cancer patients treated by primary chemotherapy: an updated analysis of a randomized trial[END_REF][START_REF] Mauriac | Neoadjuvant chemotherapy for operable breast carcinoma larger than 3 cm: a unicentre randomized trial with a 124-month median follow-up. Institut Bergonie Bordeaux Groupe Sein (IBBGS)[END_REF]. A Cochrane analysis of 14 prospective studies with 5500 patients comparing pre-with postoperative chemotherapy demonstrated similar LRFS within the BCT group in trials with adequate local therapy (including surgery in all patients) while inadequate local therapy (radiotherapy only in several patients) resulted in a significant worse LRFS [START_REF] Mieog | Neoadjuvant chemotherapy for operable breast cancer[END_REF] supporting our results.

Published guidelines [START_REF] Buchholz | Statement of the science concerning locoregional treatments after preoperative chemotherapy for breast cancer: a National Cancer Institute conference[END_REF][START_REF] Kaufmann | Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: new perspectives 2006[END_REF][START_REF] Von Minckwitz | Preoperative therapy: what, when and for whom?[END_REF] suggested to omit BCT in patients with a high probability of local recurrence (N2 or N3, T >2cm, lymphovascular invasion, multifocal disease, young age). While our study showed that these parameters were no independent predictors for LRFS except clinical response, we suggest including the latter into surgical decision making rather than other factors.

Major drawback of the study is that patients who were scheduled for mastectomy but finally underwent BCT showed significant smaller tumors compared with mastectomized patients. This may bias our finding that local recurrence is not increased in patients undergoing BCT after nCT and tumor downsizing as size increase the risk for local relapse. This heterogeneity is a result of subjective pretherapeutic allocation to primary mastectomy before nCT by the treating physician. However, so far this is the only retrospective analyses of more than 300 patients analysing true cancer downsizing and local recurrence rate comparing mastectomy and final BCT.

Prospective studies have to further investigate this hypothesis. It is also interesting to see that 56 patients underwent mastectomy though a clinical T1/2 staging. The reason is a high rate of additional extensive intraductal components (n=48) necessitating mastectomy and the fact that 8 patients had a very small breast and BCT would have resulted in an inferior cosmetic result.

In conclusion our study suggests that BCT is safe after tumor downsizing and pathologic response by nCT independent of the initial nodal stage and tumor size. Patients scheduled for mastectomy should not be treated with breast conservation in the absence of any proven response to nCT.

Surgery 4 -

 4 6 weeks after nCT patients proceeded to surgery. Patients underwent BCT except: R1 resection after BCT or initially questionable Inflammatory breast cancer Multicentric disease (MRI in unclear cases) Unwillingness to perform radiotherapy postoperatively

  Clinical response has been evaluated clinically by palpation and radiologically by mammography and ultrasound or MRI-imaging according to the following criteria: Clinical complete response (cCR): no radiological and clinical signs of residual disease within the breast and the axilla Clinical partial response (cPR): radiological and/or clinical signs of residual disease with a diameter <50% of the tumor size before nCT within the breast and/or a positive axilla (palpation) Clinical no change (cNC): radiological and/or clinical signs of residual disease with a tumor diameter within the range of 25% of the tumor size before nCT Clinical progressive disease (cPD): radiologic and/or clinical signs of tumor size increase of more than 25% compared to before nCT. From 2006 onward, MRI-mammography was standard of care for assessment of initial tumour size and treatment response. Pathological Pathologic response has been evaluated from the surgical specimen according to the following criteria: Pathologic complete response (pCRTx ±is N0): no invasive cancer within the breast lymph node negative Pathologic partial response (pPR): no invasive cancer within the breast lymph node positive Invasive cancer within the breast >50% size reduction compared with size before nCT as assessed by mammography and ultrasound or MRI-mammography Pathologic no change (pNC) invasive cancer within the breast and tumor size increase <25% or decrease <50% compared with clinical size before nCT Pathologic progressive disease (pPD) invasive cancer within the breast and >25% increase in size or inflammatory breast cancer.

  MX-MX: patients scheduled for mastectomy and receiving mastectomy after neoadjuvant therapy MX-BCT: patients scheduled for mastectomy but receiving breast conserving therapy after neoadjuvant therapy BCT-BCT: patients scheduled for breast conservation and receiving breast conservation after neoadjuvant therapy CR: complete response; PR: partial response; NC: no change; PD: progressive disease

	cT3/4 (after neoadjuvant) tumor type	2 ductal versus lobular 44	2	44		1	0.04	2	0.10
	pathologic response pCR Tx±isN0 (no invasive but in situ Ca) pathological tumorsize pCR Tx±isN1 (no Ca in breast/axillla positive) menopausal status	11 pT0/1/2 versus pT3/4 3 3 5 praemenopausal vs. postmenopausal 10 3 3 5	11 2	0.04 0.90	10 3	0.84 0.67
	pPR pNC/PD lymph node status	<0.001 neg versus pos 75 52 12 40	82 5	52 40	<0.001	66 21 0.0006 82 5	0.008 0.01	N0
	TNM endocrine responsive	Er/Pr neg versus any other				0.03	0.09
	pT0is/1/2 her2neu	96 pos versus neg 48					0.61	0.68
	grading	<0.001 G3 versus G1/2					0.002	0.004	G1/2
		percentage	p	percentage	p		percentage	p
	Group	BCT	MX	X 2	MX-BCT	MX-MX	X 2	BCT-BCT	MX-BCT	X 2
	n = 308	197 (64%) 111 (36%)	110 (36%) 111 (36%)		87 (28%) 110 (36%)
	clinical tumor size before surgery
	cT1/2 (before neoadjuvant)	73	24		54	24		97			54
	cT3/4 (before neoadjuvant)	27	76	<0.001	46	76	<0.001	3			46	<0.001
	cT1/2 (after neoadjuvant)	98	56	<0.001	98	56	<0.001	99			98	0.837

Table 3 prognostic factors for local recurrence univariate multivariate

 3 -MX: patients scheduled for mastectomy and receiving mastectomy after neoadjuvant therapy MX-BCT: patients scheduled for mastectomy but receiving breast conserving therapy after neoadjuvant therapy BCT-BCT: patients scheduled for breast conservation and receiving breast conservation after neoadjuvant therapy CR: complete response; PR: partial response; NC: no change; PD: progressive disease

	Surgical group	MX-MX vs. MX-BCT vs BCT-BCT	0.07	0.11
	clinical response	CR/PR versus NC/PD	0.86	0.89
	pathological response	CR versus PR/NC/PD	0.51	0.51
	tumor type	ductal versus lobular	0.08	0.09
	pathological tumorsize	pT0/1/2 versus pT3/4	0.96	0.76
	menopausal status	praemenopausal vs. postmenopausal	0.64	0.64

MX


Thomas Dünnebier

Klaus Schlaefer

Michael Gilbert

Christian Baisch

Christina Justenhoven

Hiltrud Brauch

Volker Harth

Anne Spickenheuer

Sylvia Rabstein

Beate Pesch

Thomas Brüning

Yon-Dschun Ko

PhD Ute Hamann
email: u.hamann@dkfz-heidelberg.de

No association of polymorphisms in the cell polarity gene with breast cancer risk

Keywords: SCRIB, polymorphisms, epithelial cell polarity, breast cancer risk

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Introduction

Breast cancer originates from epithelial cells of the terminal ductal lobular units (TDLU) in the breast [START_REF] Allred | Biological features of premalignant disease in the human breast[END_REF][START_REF] Allred | Histological and biological evolution of human premalignant breast disease[END_REF]. Each TDLU consists of several small units, referred to as acini, which consist of a single polarized layer of luminal epithelial cells surrounding a hollow lumen [START_REF] Allred | Histological and biological evolution of human premalignant breast disease[END_REF][START_REF] Bissell | The organizing principle: microenvironmental influences in the normal and malignant breast[END_REF]. The establishment and maintenance of polarized architecture is critical for normal function of epithelial cells in vivo. During the development of carcinoma, epithelial cells loose their ability to maintain polarized organization, suggesting an important role for molecules that regulate cell polarity in breast cancer.

One of the proteins regulating cell polarity is the tumor suppressor Scribble, recently identified in Drosophila [START_REF] Bilder | Localization of apical epithelial determinants by the basolateral PDZ protein Scribble[END_REF]. Loss of Scribble is sufficient to induce tumorigenic growth and promote spontaneous invasion and metastasis in combination with activated oncogenes Ras, Raf or Notch [START_REF] Brumby | scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila[END_REF][START_REF] Pagliarini | A genetic screen in Drosophila for metastatic behavior[END_REF]. Moreover, loss of Scribble results in loss of polarity and neoplastic tissue overgrowth, demonstrating a direct link between cell polarity regulators and control of proliferation [START_REF] Bilder | Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors[END_REF].

Recent studies suggest that the human homolog of Drosophila Scribble, SCRIB also plays a role in the development of cancer. One study demonstrated that deregulation of SCRIB promotes transformation of mammary epithelial cells in vitro and in vivo by disrupting cell polarity, three-dimensional morphogenesis and by inhibiting cell death [START_REF] Zhan | Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma[END_REF]. Moreover, SCRIB is a target protein for the ubiquitin-mediated degradation by human papillomavirus (HPV) E6 oncoprotein, which is considered to have a causal role in the development of cervical cancer [START_REF] Nakagawa | Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix[END_REF]. This is consistent with data showing that SCRIB expression is decreased in tumors associated with HPV infection [START_REF] Nakagawa | Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix[END_REF][START_REF] Massimi | HPV E6 specifically targets different cellular pools of its PDZ domain-containing tumour suppressor substrates for proteasome-mediated degradation[END_REF]. Additionally, loss of SCRIB expression is frequently observed in colon and lobular breast cancers [START_REF] Gardiol | Human discs large and scrib are localized at the same regions in colon mucosa and changes in their expression patterns are correlated with loss of tissue architecture during malignant progression[END_REF][START_REF] Navarro | Junctional recruitment of mammalian Scribble relies on E-cadherin engagement[END_REF]. Further strong evidence for a role of SCRIB in breast cancer comes from a recent study showing that SCRIB was downregulated in more than 50% of breast cancers and mislocalized from cell-cell junctions to the cytoplasm in numerous breast cancer cell lines and ductal carcinomas in situ [START_REF] Zhan | Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma[END_REF].

Due to the strong relationship between SCRIB and breast tumorigenesis, we investigated whether genetic variation in SCRIB is associated with breast cancer risk and histopathological tumor characteristics using a SNP tagging approach. The dataset includes 1,021 patients from the GENICA population-based collection of incident primary breast cancer cases.

Material and Methods

Study population

The GENICA study participants of the population-based breast cancer case-control study from the Greater Bonn Region, Germany, were recruited between 08/2000 and 9/2004 as previously described [START_REF] Pesch | Factors modifying the association between hormone-replacement therapy and breast cancer risk[END_REF][START_REF] Justenhoven | The CYP1B1_1358_GG genotype is associated with estrogen receptor-negative breast cancer[END_REF]. In brief, 1,143 incident breast cancer cases and 1,155 population controls, matched in 5-year classes, participated in the study. Cases and controls were eligible if they were of Caucasian ethnicity, current residents of the study region and below 80 years of age. Among the recruited individuals, DNA samples were available for 1,021 (89%) breast cancer cases and 1,015 (88%) controls. Information on known and potential risk factors was collected for all participants via in-person interviews. The response rate was 88% for cases and 67% for controls. Selected epidemiological characteristics of the patients are shown in Table 1.

Information on clinical and histopathological tumor characteristics was collected and included histology (ductal, lobular, ductolobular), histological grade (G1, G2, G3), tumor size (T1, T2, T3, T4), lymph node status (N0, N≥1), estrogen receptor (ER) status (positive, negative), progesterone receptor (PR) status (positive, negative), and HER2 status (positive, negative). Tumor grade was determined according to the Nottingham Criteria, which comprises formation of tubuli, nuclear pleomorphism and mitotic rate.

The GENICA study was approved by the Ethic´s Committee of the University of Bonn.

All study participants gave written informed consent.

DNA isolation and genotyping

Genomic DNA was extracted from heparinized blood samples (PuregeneTM, Gentra Systems, Inc., Mineapolis, USA) as previously described [START_REF] Justenhoven | ERCC2 genotypes and a corresponding haplotype are linked with breast cancer risk in a German population[END_REF]. Genotyping of SCRIB polymorphisms was performed by TaqMan® allelic discrimination and PCR-based restriction-fragmentlength-polymorphism analysis. SNP genotyping assays were purchased from Applied Biosystems (Foster City, CA, USA). The pre-designed SNP genotyping assay IDs are C_26008847_10 for rs6558394 and C_30687359_20 for rs11786637 and C_401143_20 for rs7822430; custom assay for rs7844493. PCRs were run in a 5 µl reaction containing 6.25 ng DNA, TaqMan® Genotyping Master Mix (Applied Biosystems) and Assay-Mix. PCR was performed at 50°C for 2 min, 95°C for 10 min and 50 cycles at 92°C for 15 s and 60°C for 1 min. The samples were amplified, read and analyzed using the ABI Prism 7900HT Sequence Detection System and ABI Prism SDS 2.2 software. SNP rs4875054 was amplified using newly designed primers, a forward primer (5'-AGG AGA GCA GGA TCA GGG GT-3') and a reverse primer (5'-GGC TGA CAT CTT GCC TGT GAG-3'). The reaction was set up in 10 µl containing 12.5 ng genomic DNA, 1x PCR buffer (Qiagen, Hilden, Germany), 2.0 mM MgCl2, 0.1 µM of each primer, 250 µM of each dNTP (Promega, Mannheim, Germany), and 0.4 U HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany). After an initial 15 min step at 95°C, DNA was amplified by 35 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C and followed by a final extension of 10 min at 72°C. PCR products were digested with 2.5 U MnlI (New England Bioloabs, Frankfurt, Germany).

Sizes of the ethidium-bromide stained fragments were 37 bp and 75 bp for the G allele and 112 bp for the A allele.

Selection of SNPs in SCRIB for association studies

To define the set of tag SNPs we used the software Tagger and CEU genotype data from the International HapMap Project [16] spanning the region from position 144945078 to 144972537 of chromosome 8. Common variants with a minor allele frequency (MAF) of ≥0.05 were captured using a correlation coefficient (r²) between tag and untyped SNPs greater than 0.8. Coding polymorphisms were analyzed for their potential effect on protein function using the web-tools Polyphen [START_REF] Ramensky | Human non-synonymous SNPs: server and survey[END_REF] and Panther [START_REF] Thomas | PANTHER: a library of protein families and subfamilies indexed by function[END_REF][START_REF] Thomas | Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools[END_REF].

Haplotype analysis

The linkage disequilibrium (LD) between polymorphisms was determined in the GENICA control group using the software Haploview [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF]. The haplotype block structure was defined according to the method of Gabriel et al. [START_REF] Gabriel | The structure of haplotype blocks in the human genome[END_REF]. Haplotype analysis was performed within the identified haplotype block using the SAS genetics package. Haplotypes were estimated using an expectation-maximization algorithm (proc haplotype).

Statistical analyses

The power calculation was performed using the PGA software [START_REF] Menashe | PGA: power calculator for case-control genetic association analyses[END_REF]. SCRIB genotype frequencies were tested for Hardy-Weinberg equilibrium. Associations between genetic variables and breast cancer risk were analyzed by conditional logistic regression adjusted for six potential epidemiological breast cancer risk factors (menopausal status, family history of breast or ovarian cancer, use of oral contraceptives, use of hormone therapy, body mass index and smoking) and stratified for age. Subsequently, subgroup analysis was performed for these six epidemiological variables. Additionally, the associations between SCRIB genotypes and seven clinical and histopathological tumor characteristics (histology, histological grade, tumor size, nodal status, ER status, PR status and HER2 status) of breast cancer cases were analyzed by conditional logistic regression and chi square test. All tests were two-sided. To correct for multiple testing we divided the significance level of 0.05 by the number of tested variables. In case of epidemiological variables 0.05 was divided by six and thus P-values < 0.008 were considered significant. Accordingly for the seven tumor characteristics P-values < 0.007 were considered significant. Risk estimates were given as odds ratios (OR) and 95% confidence interval (CI). Statistical analyses were done using SAS version 9.1.3 (SAS Institute Inc., Cary, NC, USA).

Results

Selection of SNPs in SCRIB for association studies

Based on HapMap data, eight common SNPs (MAF ≥0.05) located in exons and introns of SCRIB were tagged by five genotyped variants, rs6558394, rs7844493, rs11786637, rs7822430 and rs4875054 (Figure 1). Functional implications of SCRIB polymorphisms have not been reported so far. However, the tag SNP rs6558394 resulting in an amino acid substitution (L422P) may impact the function of the protein as suggested by in silico analyses.

Associations of SNPs in SCRIB with overall breast cancer risk and histopathological tumor parameters

We analyzed the five tag SNPs in SCRIB within the GENICA study population. Call rates were greater than 99.3% and repeated analysis of 5% of samples showed 99% concordance.

Genotype frequencies of cases and controls met Hardy-Weinberg equilibrium. No association was observed with breast cancer risk in general (Table 2) or in subgroup analysis with respect to menopausal status, family history of breast or ovarian cancer, use of oral contraceptives, use of hormone therapy, body mass index and smoking (data not shown). Frequencies of SCRIB haplotypes encompassing SNPs in strong linkage disequilibrium (rs7822430, rs11786637) did not differ significantly between cases and controls (data not shown).

Further, none of the SNPs was associated with histopathological tumor parameters considering histological grade, tumor histology, tumor size, lymph node status, ER, PR and HER2 status (data not shown).

Discussion

In the present study we tested the hypothesis whether genetic variability of SCRIB impacts breast cancer risk and/or tumor pathology. Such a relationship seemed plausible because it recently has been shown that SCRIB inhibits breast cancer formation and that deregulation of SCRIB by down regulation or mislocalization promotes dysplastic and neoplastic growth by disrupting morphogenesis and cell polarity and by inhibiting cell death [START_REF] Zhan | Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma[END_REF]. To the best of our knowledge, the present investigation of five SCRIB SNPs in the GENICA population-based case-control study is the first one to address this possible relationship.

None of the five tag SNPs in the SCRIB gene was associated with breast cancer risk in general or any of the subgroup risks addressed by menopausal status, family history of breast or ovarian cancer, use of oral contraceptives, use of hormone therapy, body mass index and smoking. There was also no association with histopathological tumor parameters including histology, histological grade, tumor size, lymph node status, ER, PR and HER2 status.

Our study had a 90% power to detect minimum ORs of 1.34-1.40 (p=0.05, two sided test, dominant model). The range reflects the variation of minor allele frequencies of the five polymorphisms in cases, which were at 10-38%.

The results of this study of no link of SCRIB genetic variation with breast cancer risk and tumor parameters are relevant due to the important role of this polarity gene in breast cancer suppression. However, due to the lack of functional studies it is unknown if the polymorphisms analyzed do influence functional aspects of SCRIB such as protein activity, localization or stability.

At least three evolutionary conserved groups of proteins play a central role in the establishment and maintenance of apical-basolateral polarisation of epithelial cells: the Crumbs-Pals1(Stardust)-Patj and the Par3(Bazooka)-Par6-aPKC protein complexes that localize to the apical membrane domain and promote apical-membrane-domain identity [START_REF] Margolis | Apicobasal polarity complexes[END_REF][START_REF] Suzuki | The PAR-aPKC system: lessons in polarity[END_REF], the function of which are antagonized by the basolaterally localized Lethal giant larvae (Lgl), Scribble (Scrib) and Discs large (Dlg) proteins, which together promote basolateral membrane identity [START_REF] Bilder | Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors[END_REF]. Thus, it may be possible that genetic variability in other genes linked with basolateral polarisation or in genes promoting apical polarisation are associated with breast cancer risk and/or tumor parameters.

Figure legend Figure 1 -

 legend1 Figure legend

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Table 1 -

 1 Characteristics of the GENICA study population

	Characteristic		Cases n (%) Controls n (%)
	Age (years)	20-44	124 (12.1) 129 (12.7)
		45-49	99 (9.7)	97 (9.6)
		50-54	155 (15.2) 149 (14.7)
		55-59	140 (13.7) 141 (13.9)
		60-64	202 (19.8) 193 (19.0)
		65-69	140 (13.7) 138 (13.6)
		≥70	161 (15.8) 168 (16.6)
	Menopausal status	Premenopausal 249 (24.7) 235 (23.5)
		Postmenopausal 758 (75.3) 764 (76.5)
	Breast or ovarian cancer in No	860 (84.2) 927 (91.3)
	first-degree relatives	Yes	161 (15.8)	88 (8.7)
	OC use (years)	Never	372 (36.5) 368 (36.3)
		>0 to <5	180 (17.7) 185 (18.3)
		5 to <10	134 (13.2) 120 (11.8)
		≥10	333 (32.7) 340 (33.6)
	HT use (years)	Never	506 (49.8) 509 (50.3)
		>0 to <10	245 (24.1) 290 (28.6)
		≥10	266 (26.2) 214 (21.1)
	BMI (kg/m 2 )	<20	90 (8.8)	72 (7.1)
		20 to <25	469 (45.9) 471 (46.4)
		25 to <30	305 (29.9) 324 (31.9)
		≥30	157 (15.4) 148 (14.6)
	Smoking	Never	586 (57.5) 555 (54.7)
		Former	192 (18.8) 215 (21.2)
		Current	242 (23.7) 245 (24.1)
	OC: oral contraceptive, HT: hormone therapy, BMI: body mass index

Table 2 -

 2 SCRIB polymorphisms and breast cancer risk Odds ratio conditional on age in 5-year groups adjusted for menopausal status, family history of breast or ovarian cancer, use of oral contraceptives, use of hormone therapy, body mass index and smoking b Global P value of testing the null hypothesis of no association between SNP and breast cancer risk based on conditional logistic regression

	SNP	Genotype [Allele]	Cases n (%) Controls n (%) OR adj	a (95% CI)	Global P value b
	rs6558394 A>G	AA	396 (39.8)	387 (38.9)	1.00 (reference)	0.79
		AG	464 (46.7)	474 (47.7)	0.94 (0.77-1.14)
		GG	134 (13.5)	133 (13.4)	0.98 (0.74-1.30)
		[G]	732 (36.8)	740 (37.2)
	rs7844493 A>G	AA	757 (76.0)	764 (76.9)	1.00 (reference)	0.91
		AG	225 (22.6)	217 (21.8)	1.05 (0.85-1.30)
		GG	14 (1.4)	13 (1.3)	1.03 (0.48-2.24)
		[G]	253 (12.7)	243 (12.2)
	rs11786637 T>C	TT	805 (80.7)	806 (81.3)	1.00 (reference)	0.94
		TC	187 (18.7)	179 (18.1)	1.04 (0.83-1.31)
		CC	6 (0.6)	6 (0.6)	0.97 (0.31-3.06)
		[C]	199 (10.0)	191 (9.6)
	rs7822430 C>A	CC	365 (36.8)	401 (40.4)	1.00 (reference)	0.08
		CA	493 (49.8)	452 (45.5)	1.24 (1.02-1.50)
		AA	133 (13.4)	140 (14.1)	1.03 (0.78-1.36)
		[A]	759 (38.3)	732 (36.9)
	rs4875054 G>A	GG	458 (45.9)	465 (46.7)	1.00 (reference)	0.95
		GA	443 (44.3)	431 (43.3)	1.03 (0.85-1.24)
		AA	98 (9.8)	100 (10.0)	1.00 (0.73-1.36)
		[A]	639 (32.0)	631 (31.7)

a

Acknowledgements

We are indebted to all women participating in the GENICA study. We gratefully acknowledge support by interviewers as well as physicians and pathologists of the study region. High-throughput genotyping analyses were supported by Antje Seidel-Renkert. This work was supported by the Federal Ministry of Education and Research (BMBF) Germany grants 01KW9976/8, 01KW9975/5, 01KW9977/0, and 01KW0114, the Deutsches Krebsforschungszentrum, Heidelberg, the Department of Internal Medicine, Evangelische Kliniken Bonn gGmbH, Johanniter Krankenhaus, Bonn, the Robert Bosch Foundation of Medical Research, Stuttgart and the Institute for Prevention and Occupational Medicine of the German Social Accident Insurance (IPA), Bochum, Germany.

Competing interests

The authors declare that they have no competing interests.


Hatef Darabi
email: hatef.darabi@ki.se

Kamila Czene

Sara Wedrén

Yuqing Li

Jianjun Liu

Keith Humphreys

Genetic Variation in the Androgen Estrogen Conversion Pathway in Relation to Breast Cancer Prognosticators

Purpose:

Genetic variation in the androgen-to-estrogen conversion pathway has been shown to be associated with risk of breast cancer, and in particular with Estrogen Receptor (ER) positive tumours. We aimed at studying how the genetic alterations, which have been identified for risk, are associated with breast cancer prognosticators, with a prior hypothesis that, in general, hormone related breast cancers have a better prognosis than non-hormone related breast cancers.

Methods:

Association between tagging SNPs in genes involved in estrogen metabolism and patient's lymph node status, tumour size and histological grade, were estimated in a sample of 1569 Swedish breast cancer patients.

Results:

Polymorphisms in CYP19A1, which have previously been linked to breast cancer risk, are shown to be associated with breast cancer prognosticators. The strongest association was observed for rs4646, with histological grade. The common allele of rs4646, which has been associated with increased breast cancer risk, was associated with low histological grade and small tumour size (p=0.001 and 0.015; 1-sided, respectively). We also found evidence that SNP rs7167936 is associated with histological grade and tumour size (p=0.010 and 0.005; 1-sided, respectively). We show that rs4646 and rs7167936 are associated with histological grade even amongst only ER-positive tumours (p=0.008 and 0.011; 1-sided, respectively).

Conclusions:

Our results provide new evidence that CYP19A1 is involved in both breast cancer risk and prognosis.

Introduction

Exposure to estrogen is an important determinant of the risk of breast cancer. Breast cancer is believed to be induced by excessive estrogen exposure and genetic variation in genes involved in the estrogen metabolism pathway is therefore likely to be important for the disease. Numerous genetic association studies addressing association with cancer risk and genes in the estrogen metabolism pathway have been reported with conflicting results, probably reflecting the low penetrance of mutations in genes within the estrogen metabolism pathway, suboptimal study designs, underpowered studies or a combination of these factors [START_REF] Justenhoven | Breast cancer: a candidate gene approach across the estrogen metabolic pathway[END_REF][START_REF] Torresan | Genetic polymorphisms in oestrogen metabolic pathway and breast cancer: a positive association with combined CYP/GST genotypes[END_REF][START_REF] Haiman | Genetic variation at the CYP19A1 locus predicts circulating estrogen levels but not breast cancer risk in postmenopausal women[END_REF]. We have recently been able to show, using a pathway approach, that there is an association between breast cancer risk and some of the genes involved in the androgen-to-estrogen conversion pathway (a sub-pathway of the complete estrogen metabolism pathway), particularly for the risk of estrogen receptor(ER)-positive tumours [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF]. Gene-based analysis indicated that CYP19A1 was the major contributor to the observed association.

CYP19A1 is located on chromosome 15q21.1 in humans and encodes the aromatase enzyme, which catalyzes the last step of estrogen biosynthesis from androgens. Mutations in CYP19A1 can result in increased or decreased aromatase activity, and it has been suggested that estrogen functions not only as a sex steroid hormone but also plays a part in cell growth or differentiation [START_REF] Simpson | Aromatase--a brief overview[END_REF][6].

In the current study we investigated whether the top risk SNPs within the androgen-to-estrogen conversion sub-pathway, according to [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF], are associated with breast cancer prognosticators; tumour grade, size and lymph node status. Prior to analysis we hypothesised that alleles associated with an increased risk of breast cancer are associated with good prognosis (and vice-versa) since hormone related breast cancers, identified in terms of menopausal hormone treatment have also been reported to be associated with favourable prognosis [START_REF] Rosenberg | Menopausal hormone therapy in relation to breast cancer characteristics and prognosis: a cohort study[END_REF]. We assessed association overall and within subgroups of patients stratified by ER-status.

Material and Methods

Study Subjects

A Swedish case-control study was initiated in the early 90's to examine the effect of menopausal hormone use on breast cancer risk. The nation-wide case-control study encompassed all Swedish born women between 50 and 74 years of age and resident in Sweden between October 1993 and March 1995 and diagnosed with breast cancer. The cancer patients were identified through the regional cancer registries in Sweden. Women with previous cancers were excluded in order to minimize the risk of including patients with a metastasis in the study. Controls were randomly chosen from the Swedish Registry of Total Population and frequency matched to cases, on age. Characteristics of the participants in the breast cancer case-control study have been described elsewhere [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF][START_REF] Rosenberg | Menopausal hormone therapy in relation to breast cancer characteristics and prognosis: a cohort study[END_REF][START_REF] Wedrén | Genetic Susceptibility to Breast and Endometrial Cancer[END_REF]. For cancer patients, information on tumour characteristics was collected from patient records from surgical and oncological units throughout Sweden. This included estrogen and progesterone receptor expression, histopathological subtype, disease stage according to tumour size, and spread to regional lymph nodes. Tumour samples were not assessed for HER2 status or S-phase. The Bloom-Richardson scale or Nottingham histological grade were used as grading systems for classifying Grade [START_REF] Rosenberg | Menopausal hormone therapy in relation to breast cancer characteristics and prognosis: a cohort study[END_REF]. From the original case-control study, consisting of 3345 cases and 3454 controls, 1569 breast cancer cases and 1730 healthy controls were included in a genetic study.

Written informed consent was obtained from all the participating subjects, and the study was approved by the Institutional Review Boards in Sweden and the National University of Singapore [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF].

Gene and haplotype tagging SNP selection

The SNPs/genes within the androgen-to-estrogen conversion pathway, which are included in the current study, represent a subset of SNPs/genes from a parent study of the entire estrogen metabolism pathway [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF].

Genetic variation in the androgen-to-estrogen conversion (sub-) pathway was shown to be associated with breast cancer risk. In the parent study [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF] gene selections were based on the criteria that the gene should code for an enzyme that contributes to estradiol and estrone metabolism. In total, 35 genes were selected and a two-stage genotyping design was used. In the first stage 1007 single nucleotide polymorphisms (SNPs) were selected within the 35 genes and their 30Kb flanking sequences, aiming for a marker density of at least one SNP per 5Kb. Linkage disequilibrium (LD) patterns in these genes were determined using DNA samples from 92 Swedish control women. Haplotypes were reconstructed using the PLEM algorithm implemented in the tagSNP program [START_REF] Qin | Partition-ligation-expectation-maximization algorithm for haplotype inference with single-nucleotide polymorphisms[END_REF][START_REF] Stram | Choosing haplotype-tagging SNPS based on unphased genotype data using a preliminary sample of unrelated subjects with an example from the Multiethnic Cohort Study[END_REF]. TagSNP selection was based on the R 2 coefficient. We chose tagSNPs so that common SNP genotypes (minor allele frequency ≥ 0.03) and common haplotypes (frequency ≥ 0.03) were predicted with a good coverage of R 2 values ≥ 0.8. Overall 302 tagSNPs were selected across 35 genes, and these tagSNPs, in stage 2 of the procedure, were genotyped in all available DNA samples from cases and controls. Of these, 252 tagSNPs were successfully genotyped, from which a further thirteen SNPs were excluded on the basis of failing a Hardy-Weinberg Equilibrium test, or having a call rate < 0.85, or a minor allele frequency < 0.01. This left a final set of 239 SNPs distributed among 34 genes, all of which were genotyped. Of these 34 genes, 15 genes (including 120 tagSNPs) are involved in androgen-to-estrogen conversion. We recently showed that there is an association between breast cancer risk and some of the tagSNPs in these 15 genes [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF].

Benjamini and Hochberg [START_REF] Benjamini | Controlling the false discovery rate a practical and powerful approach to multiple testing[END_REF] introduced an important concept for multiple comparisons that they called the false discovery rate, or FDR. The FDR is the expected proportion of false positives among all tests declared significant. In the current study, the FDR q-value [START_REF] Benjamini | Controlling the false discovery rate a practical and powerful approach to multiple testing[END_REF][START_REF] Storey | A direct approach to false discovery rates[END_REF][START_REF] Storey | The positive false discovery rate: A Bayesian interpretation and the q-value[END_REF] was estimated for each of the 120 tagSNPs in [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF]. The q-value is the FDR equivalent of the p-value; the q-value of an individual test measures the minimum false discovery rate that is incurred when calling that test significant. Out of the 120 tagSNPs, 13 had a false discovery rate q-value less than 0.20 (with q-values ranging from 0.04 to 0.19); see Table 1. These 13 tagSNPs were included in the present study and examined for association with breast cancer prognosticators. For an overview of the design of the current study see Figure 1. We note that 6 of the 13 tagSNPs are in CYP19A1.

Prognosticators for breast cancer

The most commonly used prognosticators for breast cancer are TNM stage, histological grade, estrogen receptor (ER) status and S-phase [START_REF] Rakha | Histologic grading is an independent prognostic factor in invasive lobular carcinoma of the breast[END_REF]. In the current study we decided to include the component variables of TNM stage, lymph node status and tumour size, along with histological grade. We did not include ER status since we have already established, in our study material, that the top risk SNPs, in genes involved in androgen-to-estrogen conversion, in particular increase the risk of ER-positive breast cancer [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF]. S-phase could not be included because it was not measured for patients in our study. The lymph node status is either 1 (if there are no nodes affected), 2 (if up to 3 glands are affected) or 3 (if more than 3 glands are affected).

Similarly the tumour grade is scored as either 1 (for a grade I, less aggressive appearance), 2 (for a grade II, intermediate appearance) or 3 (for a grade III, more aggressive appearance).

Statistical Analysis

For assessing association between the prognosticators, lymph node status, tumour size and histological grade, and the (13) tagSNPs selected in the present study, we based analysis on all cases with available data (case only analysis). Ordinal (proportional odds) regression was used to model the relationship between ordinal outcome variables (grade and lymph node status) with SNP covariates. Linear regression was used to model the relationship between continuous variables (tumour size) with covariates of interest. Tumour size (in cm) was logarithm transformed in order to obtain an approximately normally distributed dependent variable. Because of our hypothesis that alleles associated with increased risk of breast cancer are associated with good prognosis (and vice-versa), we performed 1-sided hypothesis testsfor each SNP, the alternative hypothesis being that the high risk allele (from [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF]) is associated with a favourable prognosticator value (low grade/low lymph node status/small tumour). We, however, also present p-values for 2-sided tests since these are more conservative/standard. We applied Bonferroni corrections to address multiple testing (39 tests of association).

Overall (global) evidence of association between the (13) genetic variants and each of the prognosticators was evaluated using the Admixture Maximum Likelihood (AML) method, which is described in detail in Tyrer et al [START_REF] Tyrer | The admixture maximum likelihood test: a novel experimentwise test of association between disease and multiple SNPs[END_REF]. This test assesses the experiment-wise significance by examining the empirical distribution of single marker test statistics, in order to determine whether there exists a cumulative effect from multiple variants. For these analyses we dichotomised the prognosticators (grades 1 & 2 vs. 3, lymph node status 1 vs. 2 & 3, tumour size ≤20mm vs. >20mm), since the AML test is for dichotomised outcomes. The AML analysis was performed using software obtained from the authors of [START_REF] Tyrer | The admixture maximum likelihood test: a novel experimentwise test of association between disease and multiple SNPs[END_REF]. All other statistical analyses were performed using the free statistical software R [START_REF]R Project[END_REF].

Results

In Table 2 we present summary statistics for the prognosticators, for the entire case-series, as well as for the subset of cases know to be ER positive. Only just over one-half of the cases have tumour ER status recorded. Of these, approximately three-quarters are ER-postive.

In order to summarise the genetic association with tumour phenotypes in cases overall, we used the AML approach [START_REF] Tyrer | The admixture maximum likelihood test: a novel experimentwise test of association between disease and multiple SNPs[END_REF] with categorised prognosticators as outcome. We carried out three AML tests of association, in order to assess the cumulative association between the 13 tagSNPs and each of the prognosticators, using all cases. The (2-sided) p-values were 0.038, 0.549 and 0.691, for histological grade, tumour size and lymph node status respectively. Indicating there is a cumulative effect from (some of) the 13 markers included in this study with tumour grade.

To investigate our hypothesis, that the most important genetic variants for breast cancer risk, in genes involved in androgen-to-estrogen conversion, are associated with breast cancer prognosticators, we fitted regression models to grade (ordinal), lymph node status (ordinal) and tumour size (linear). Results are displayed in Table 3. The lowest p-values for association were obtained between histological grade and rs7167936 (p=0.010 and p=0.020 for 1-sided and 2-sided tests, respectively) and rs4646 (p=0.001 and p=0.002 for 1-sided and 2-sided tests, respectively). Markers rs7167936 (p=0.005, 1 sided) and rs4646 (p=0.015, 1 sided) also showed association with tumour size (Table 3). None of the selected markers showed association with lymph node status, coded either as an ordinal variable or as a dichotomized variable. All regression coefficients with a corresponding 2-sided p-value < 0.05, for tagSNPs in CYP19A1, were such that the high risk allele is associated with less aggressive tumours (the 1-sided pvalues were less than the 2-sided p-values). The 1-sided p-value for the association between rs4646 and histological grade remains significant (at =0.05) after a (conservative) Bonferroni correction for multiple testing (p=0.039), although the 2-sided p-value does not.

We found that the association between the "top" SNPs in CYP19A1 and histological grade remained even when including only cases which are ER-positive (data not shown). For SNPs rs7167936 and rs4646 1sided p-values of 0.011 and 0.008, respectively, were obtained, with regression coefficients 0.27 and 0.31, corresponding to odds ratios of 1.31 and 1.36. Neither marker remained associated for cases that are ERnegative.

Discussion

We have recently shown an association between breast cancer risk and some of the genes involved in the androgen-to-estrogen conversion pathway [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF]. Gene based analysis indicated that the aromatase gene CYP19A1 was a major contributor to the observed genetic association. In the current study we hypothesized that genetic variation in the top risk genes, within the androgen-to-estrogen conversion pathway, is also associated with breast cancer prognosis. In our study material, cancers occurring among women treated with menopausal hormones on average have a more favorable prognosis [START_REF] Rosenberg | Menopausal hormone therapy in relation to breast cancer characteristics and prognosis: a cohort study[END_REF]. Following this line of reasoning we hypothesised that high risk alleles of polymorphisms of genes in the androgen-toestrogen conversion pathway, are, more specifically, associated with breast cancer prognosticators.

The prognosticator with the strongest association was histological grade. Of the 13 studied markers, in the androgen-to-estrogen conversion pathway, the lowest p-values for association with histological grade were obtained for rs7167936 and rs4646 (1-sided p-values of 0.010 and 0.001), both in CYP19A1.

If genetic variation in the androgen-to-estrogen conversion pathway is primarily associated with ERpositive breast cancer, as we have earlier suggested [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF], this will partially explain the association between risk polymorphisms and the prognosticators. This claim, made in [START_REF] Low | Multi-variant Pathway Association Analysis Reveals the Importance of Genetic Determinants of Estrogen Metabolism in Breast and Endometrial Cancer Susceptibility[END_REF], was made on the basis that MAFs of "risk associated" SNPs were similar in controls and ER-negative breast cancers, but differed in ER-positive cancers. Only in a few cases were there statistically significant differences between MAFs in ER-negative and ER-positive cases (e.g. for rs7167936, p=0.041). It appears that the level of association we observed between grade and SNPs in CYP19A1 exceeds that which we would expect due solely to MAF differences in ER-positive and ER-negative cancers.

Multiple studies have shown independent prognostic significance of histological grade in breast cancer. To examine the prognostic value of histological grade Rakha et al. [START_REF] Rakha | Prognostic significance of Nottingham histologic grade in invasive breast carcinoma[END_REF] investigated the association of grade in patients with operable breast cancer with different tumour size and lymph node subgroups. Multivariate analysis revealed that tumour grade was a significant independent predictor of breast cancer specific survival, with a hazard ratio of 3.9 for grade 3 relative to grade 1 after a median of 9 years, with significant difference in survival for higher histological grade and poorer patient's outcome. Histological grade together with lymph node status and tumour size are the three strongest prognostic determinants for breast cancer. Assessment of degree of biological aggressiveness of a tumour is crucial as biologically more aggressive tumours are likely to increase in size in a short time, in contrast to less aggressive tumours which exhibit more restricted growth rate. Many breast tumours are estrogen sensitive; estrogen helps them to grow. Aromatase is involved in the production of estrogens, and altered expression of it may be associated with prognosis. The single-nucleotide polymorphism rs4646, of CYP19A1, has been reported to be associated with HER2 status of the tumour [START_REF] Fasching | Single nucleotide[END_REF] and circulating steroid hormones [START_REF] Haiman | Genetic variation at the CYP19A1 locus predicts circulating estrogen levels but not breast cancer risk in postmenopausal women[END_REF], making it biologically possible that polymorphisms in CYP19A1 may be associated with response to aromatase inhibitors. To examine this hypothesis Garcia-Casado et al. [19] analysed polymorphisms in CYP19A1 in a series of postmenopausal woman treated with neoadjuvant letrozole and described their association with response to treatment and with progression free survival. The minor genetic variant of rs4646 was shown to be associated with poor response to letrozole, and presented a lower progression-free survival, concluding that analysis of rs4646 could improve the clinical management of breast cancer patients. Our result is in agreement with the result presented by Garcia-Casado et al. [19], since we found the minor variant of this SNP to be associated with high tumour grade. polymorphisms of the aromatase gene (CYP19A1), HER2/neu status, and prognosis in breast cancer patients. Breast Cancer Res Treat. 112:89-98.

[19] Garcia-Casado Z, Guerrero-Zotano A, Llombart-Cussac A, Calatrava A, Fernandez-Serra A, Ruiz-Simon A, Gavila J, Climent MA, Almenar S, Cervera-Deval J, Campos J, Albaladejo CV, Llombart-Bosch A, Guillem V, Lopez-Guerrero JA. (2010) A polymorphism at the 3'-UTR region of the aromatase gene defines a subgroup of postmenopausal breast cancer patients with poor response to neoadjuvant letrozole.

  BMC Cancer. 10:36.

Figure1:

  Figure1 : Breast Cancer prognosticators and polymorphisms in genes involved in estrogen metabolism; study design.

Table 1 :

 1 Top 13 SNPs in the androgen-to-estrogen conversion pathway for association with breast cancer risk, ordered by p-value.

	Rs	Gene	MAF a p-value b OR (95% CI) b q-value c	N
	rs7167936	CYP19A1	0.49	0.0003	0.83 (0.75,0.92)	0.0405	2997
	rs12050767	CYP19A1	0.45	0.0013	1.18 (1.07,1.31)	0.0790	2911
	rs4646	CYP19A1	0.30	0.0077	0.86 (0.77,0.96)	0.1915	2994
	rs2736520	UGT2B4	0.18	0.0139	0.84 (0.73,0.97)	0.1915	2925
	rs7085249	AKR1C4	0.25	0.0144	1.15 (1.03,1.29)	0.1915	2981
	rs12762017	AKR1C4	0.15	0.0147	0.83 (0.71,0.96)	0.1915	2791
	rs4371687	UGT2B11	0.48	0.0148	1.13 (1.02,1.25)	0.1915	2993
	hCV3060064	CYP19A1	0.46	0.0158	0.88 (0.79,0.98)	0.1915	2984

Table 2 .

 2 Numbers of cases overall and selected subsets of cases selected for available data on histological grade, lymph node status and tumour size. Out of the 1569 cases, 1031 had ER status recorded, of which 811 were ER-positive.

	Data Total		Grade	Lymph Node Status	Tumour Size
			1	2	3 NA a	1	2	3 NA a ≤20mm >20mm NA a
	All	1569 144 445 424 556 1004 313 131 121 1081	389	99
	ER+ b 811	72 291 224 224 525 190 76 20	560	246	5
	a: Not Available
	b:

Table 3 -

 3 Single Marker Association with different tumour phenotypes for top 13 risk associated markers.

				Grade			Lymph node status			Tumour Size
	Rs	Gene	β a	OR (95%CI)	P-value 1sided	P-value 2sided	β a	OR (95%CI)	P-value 1sided	P-value 2sided	β b	P-value 1sided	P-value 2sided
	rs7167936	CYP19A1 0.20	1.23 (1.03,1.45)	0.010 0.020	-0.04	0.96 (0.82,1.13)	0.698 0.605 0.06	0.005 0.010
	rs12050767	CYP19A1	-0.12	0.89 (0.75,1.06)	0.093 0.186 0.05	1.05 (0.89,1.24)	0.726 0.547	-0.04	0.050 0.100
	rs4646	CYP19A1 0.31	1.37 (1.12,1.66)	0.001 0.002 0.02	1.02 (0.85,1.22)	0.422 0.844 0.06	0.015 0.030
	rs2736520	UGT2B4	-0.03	0.97 (0.77,1.24)	0.587 0.826	-0.01	0.99 (0.79,1.24)	0.542 0.916 0.01	0.332 0.664
	rs7085249	AKR1C4	0.12	1.13 (0.94,1.35)	0.899 0.202	-0.05	0.95 (0.80,1.13)	0.268 0.536	-0.01	0.372 0.745
	rs12762017	AKR1C4	0.15	1.16 (0.88,1.52)	0.145 0.290 0.03	1.04 (0.80,1.34)	0.398 0.795 0.01	0.345 0.691
	rs4371687	UGT2B11	-0.10	0.90 (0.76,1.07)	0.114 0.228	-0.02	0.98 (0.84,1.15)	0.407 0.814 0.00	0.451 0.902

Acknowledgements

This research was funded by The Swedish Research council (grant numbers 521-2008-2335 for HD and 523-2006-972 for KH), Cancerfonden (contract no. 090420 for HD and 5128-B07-01PAF for KC) and Märit and Hans Rausing's Initiative against Breast Cancer. We are grateful to all the women who took the time and effort to participate in this study.

Strengths of the present study include a large sample size, extensive coverage of SNPs in genes in the androgen-to-estrogen conversion sub-pathway and a well defined hypothesisby building on a comprehensive study of breast cancer risk we were able to focus our analysis of breast cancer prognosticators on a small number of polymorphisms (see Figure 1). A limitation of the present work is that several women included in the study had incomplete data on tumour characteristics.

In summary, we found that the polymorphism rs4646 in the 3'-untranslated region of CYP19A1 is associated with histological grade and tumour size in postmenopausal women. This supports the hypothesis that analysis of rs4646 could improve the clinical management of postmenopausal breast cancer patients.

Further studies based on larger data series are needed to confirm our findings.