Oreste Gentilini
email: oreste.gentilini@ieo.it

Edoardo Botteri

Nicole Rotmensz

Luciana Lima

Mujgan Caliskan

Carlos A Garcia-Etienne

Irina Sosnovskikh

Mattia Intra

Giovanni Mazzarol

Simona Musmeci

A E Edoardo

Botteri Ae

A E Luciana

Da Lima

A E Mujgan

Caliskan Ae

Garcia-Etienne Ae

A E Mattia

Intra Ae

Giovanni Mazzarol Ae

A E Paolo

Veronesi Ae

Viviana Galimberti Ae

Alberto Luini

A E Giuseppe

Viale Ae

Aron Goldhirsch

Umberto Veronesi

L Da Lima

Á M Caliskan

Á M Intra

Á S Musmeci

Á P Veronesi

Á V Galimberti

Á A Luini

Á G Viale

Á U Veronesi

Á N Rotmensz

P Veronesi

Conservative surgery in patients with multifocal/multicentric breast cancer

Keywords: Conservative surgery, Breast cancer, Quadrantectomy, Multifocal, Multicentric

come

Introduction

With widespread use of mammographic screening and increased accuracy of diagnostic imaging, the surgical treatment of multifocal (MF) and multicentric (MC) breast cancer has come to represent a new challenge for the conservative approach [START_REF] Coombs | Multifocal and multicentric breast cancer: does each focus matter?[END_REF]. The presence of multiple foci of cancer within the same breast has traditionally been considered a contraindication for conservative surgery. In fact, many surgeons continue to propose mastectomy for MF/ MC because earlier series have demonstrated high rates of local recurrence following breast conserving surgery [START_REF] Kurtz | Breast conserving therapy for macroscopically multiple cancer[END_REF][START_REF] Wilson | Conservative surgery and radiation in treatment of synchronous ipsilateral breast cancers[END_REF]. Moreover, physicians often recommend mastectomy in order to complete local treatment when unexpected multifocality is documented at the final pathologic report, even when surgical margins are disease-free. When two separate tumours are located in different quadrants, breast conservation is not usually considered even in patients with large breasts who might be suitable for a double wide excision. However, the difficulty of endorsing a conservative approach in these patients could be explained by limited available data.

The aim of this study was to retrospectively evaluate the outcome of patients with multiple invasive breast cancer who received conservative surgery with special attention being paid to local control, and to identify predictive factors for recurrence in these patients.

Patients and methods

Between March 1997 and December 2002, 8100 patients were operated on for invasive breast cancer at the European Institute of Oncology of Milan, Italy and were prospectively entered into our data base. We retrieved 476 patients who underwent conservative surgery for MF or MC breast cancer (at least two different pathologically confirmed invasive foci within the same breast). Diagnosis of multiple invasive breast cancer was performed either clinically by palpation, radiologically or at pathological examination. Multifocality was defined as the presence of different tumours within the same quadrant, and multicentricity as the presence of tumours in different quadrants of the breast, which were managed by a double incision.

All patients' cases were discussed after surgery at a multidisciplinary meeting attended by breast surgery, medical oncology, radiotherapy and pathology specialists. Sixty patients (12.6%) received adjuvant chemotherapy alone, 191 patients (40.1%) received hormonal therapy alone, and 217 (45.6%) patients received both chemotherapy and endocrine treatment. Eight patients (1.7%) did not receive any medical adjuvant treatment.

Four-hundred and fifty-four patients received standard postoperative external whole breast radiotherapy, 13 received intra-operative radiotherapy and nine did not receive any radiation treatment. The median age was 53 years (range 23-86). The median follow up was 73 months (range 11-118).

Two-hundred and seventy eight patients underwent upfront axillary dissection either due to the presence of clinically pathological nodes or because they had been treated before the sentinel node era. Eighty-eight underwent sentinel lymph node biopsy (SLNB) and axillary dissection because of the presence of metastases in the sentinel node, 106 received SLNB alone, and four did not have any axillary staging.

All those patients who were not seen in the last 6 months were contacted by telephone in order to update the follow up. No patients were lost to follow up.

Pathology

The distinction between the tumors was assessed both grossly, recording the distance between the foci and microscopically, routinely sampling the interposed tissue between the neoplastic foci. Multifocality and multicentricity were prospectively diagnosed on the pathology report only if the interposed tissue was free of neoplasia, both in situ or infiltrating.

HER-2/neu over-expression was investigated immunohistochemically, using a specific polyclonal antiserum (Dako A/S, Carpinteria, CA). The staining results were recorded in a four-tier scale, from 0 to 3+, according to the percentage of immunoreactive cells, and to the intensity and completeness of membrane staining as recommendend by the Food and Drug Administration, USA [START_REF] Jacobs | Specificity of Herceptest in determining HER2/neu status of breast cancer using the United States Food and Drug Administration-approved scoring system[END_REF]. Only an intense and complete membrane staining of the tumour cells (3+) was taken as evidence of Her2/neu over-expression.

Patients with distant metastases, bilateral cancer, clinical or pathological T4 or those who received primary medical treatment were excluded from the analysis. Table 1 summarizes the patients' and tumours' characteristics. The reported tumor size represents the largest tumor focus. Ten patients had focally positive margins at the final pathologic report and, after careful discussion of the available options, were not submitted to re-excision according to patient' preference.

Statistical considerations

Primary endpoints were cumulative incidence of local events, cumulative incidence of first events and cumulative mortality. Recurrences within the same breast, with or without involvement of regional nodes, were considered as local events. First events were any type of recurrence, second primary cancer, contralateral breast cancer recurrence or death, whichever occurred first. If patients had simultaneous local and distant recurrence they were considered as having a distant recurrence in the analysis. Cumulative mortality was calculated as the cumulative incidence of deaths from any cause.

Crude cumulative incidence of local events was computed in a competing risk framework as described by Marubini and Valsecchi [START_REF] Marubini | Analysing survival data from clinical trials and observational studies[END_REF], and compared across different subgroups by means of the Gray test [START_REF] Gray | A class of k-sample tests for comparing the cumulative incidence of a competing risk[END_REF]. The Log-rank test was used to assess survival differences between groups for cumulative incidence of first events and cumulative mortality. Multivariate Cox proportional hazards regression models were used to identify the prognostic independent clinico-pathological features associated with survival. For local events, Cox Proportional Hazard model was computed in a competing risk framework [START_REF] Putter | Tutorial in biostatistics: competing risks and multi-state models[END_REF]. All analyses were performed with the SAS software (SAS Institute, Cary, NC) and the R software (The R Development Core Team 2004; Free Software Foundation, Boston, MA). All tests were two-sided.

Results

Median age was 53 years (range 23-86). Table 1 shows patients' and tumor characteristics with stratification for multifocal (MF) and multicentric (MC) tumours. Four hundred and twenty-one patients (88.4%) had MF and 55 (11.6%) had MC disease. The only difference between patients with MF and MC tumours was that the number of foci was significantly higher in patients with MC disease.

Invasive lobular carcinoma was diagnosed in 88 patients (18.5%) and invasive mixed ductal-lobular cancer in 27 (5.7%). Two hundred and sixty-seven patients (76.7%) had 2, 55 patients (15.3%) had three and 29 patients (8.0%) had 4 or more tumor foci. Two hundred and sixty-one patients (55.3%) had nodal involvement. Table 2 describes the events. Briefly, 24 patients (5.0%) developed ipsilateral breast recurrence, 2 (0.4%) had locoregional recurrence, and 51 (10.7%) had distant metastases as first event.

Figures 1 and2 represent cumulative incidence of local recurrence (5.1% at 5 years) and mortality (6.2% at 5 years), respectively.

Factors related to local and distant relapses, and to overall survival at the univariate and multivariate analyses are listed in Tables 3 and4, respectively.

At the multivariate analysis, over-expression of HER2/ neu and lack of both estrogen and progesterone receptors (HR: 3.

Discussion

To our knowledge, few papers [START_REF] Kurtz | Breast conserving therapy for macroscopically multiple cancer[END_REF][START_REF] Wilson | Conservative surgery and radiation in treatment of synchronous ipsilateral breast cancers[END_REF][START_REF] Nos | Conservative treatment of multifocal breast cancer: a comparative study[END_REF][START_REF] Leopold | Results of conservative surgery and radiation therapy for multiple synchronous cancers of one breast[END_REF][10][START_REF] Cho | Conservative surgery and radiation therapy for macroscopically multiple ipsilateral invasive breast cancers[END_REF][START_REF] Kaplan | Breast conservation in patients with multiple ipsilateral synchronous cancers[END_REF][START_REF] Okumura | Feasibility of breast-conserving therapy for macroscopically multiple ipsilateral breast cancer[END_REF] have been published concerning conservative surgery in patients with MF/MC, and all have dealt with a limited number of patients. Former series reported a high risk of local recurrence in patients treated with conservative surgery. In fact, Kurtz et al. [START_REF] Kurtz | Breast conserving therapy for macroscopically multiple cancer[END_REF] in their experience with 61 patients with two or more macroscopic tumor nodules, concluded that patients with macroscopically multiple invasive breast cancer were at a higher risk of local failure if tumors were clinically or radiologically apparent (36% rate of local recurrence). Wilson and colleagues [START_REF] Wilson | Conservative surgery and radiation in treatment of synchronous ipsilateral breast cancers[END_REF] reported that the local recurrence rate was greater in patients with synchronous ipsilateral breast cancer as compared to patients with single lesions (72-month actuarial breast recurrence rate of 25% and 12%, respectively). More recently, Cho et al. [START_REF] Cho | Conservative surgery and radiation therapy for macroscopically multiple ipsilateral invasive breast cancers[END_REF] reported that in selected cases the combination of conservative surgery and radiation therapy resulted in acceptable local-regional control, based on their experience with 15 patients affected by multiple ipsilateral invasive breast cancer. Kaplan et al. [START_REF] Kaplan | Breast conservation in patients with multiple ipsilateral synchronous cancers[END_REF], after evaluating their experience with 36 patients, concluded that breast conservation is an effective treatment for patients with synchronous ipsilateral breast carcinoma. Okumura and colleagues [START_REF] Okumura | Feasibility of breast-conserving therapy for macroscopically multiple ipsilateral breast cancer[END_REF] compared 34 patients with multiple invasive breast cancer to 594 patients with unifocal disease and did not find any difference in terms of local control, disease-free survival or cosmetic result.

Our attitude is strongly in favour of breast conservation, and we elect this surgical approach as the treatment of choice, even for patients with multiple invasive cancer as long as it is technically and cosmetically feasible. Here, we re-evaluate the largest series of patients with pathologically confirmed multiple foci of breast cancer and treated with breast conservation. Our cohort comprises patients with multiple palpable tumours located within the same quadrant or in different quadrants, or patients with an unexpected MF invasive disease found at the final pathological report. Multifocalilty and multicentricity in breast cancer are historically defined as the presence of two or more tumor foci within a single quadrant of the breast or within different quadrants of the same breast, respectively [START_REF] Lagios | The concept and implications of multicentricity in breast carcinoma[END_REF]. The only significant difference between patients with MF and MC tumours was the number of foci, which was higher in patients with MC disease. In our opinion, these definitions help to simply illustrate two different scenarios of multiple invasive breast cancer. As two separate tumors within the same quadrant could in fact be more distant from each other than two tumors located in different quadrants, it is important to point out that strict definitions run the risk of falling into discussions which are merely semantic. The distance between tumors should also be regarded with caution, as overall proportion with breast size should be also taken into account.

It is interesting to note that despite the high rate of node metastases (55% of the patients), as expected in MF/MC breast cancer [START_REF] Andea | Pathologic Analysis of tumor size and lymph node status in multifocal/multicentric breast carcinoma[END_REF][START_REF] Gentilini | Sentinel lymph node biopsy in multicentric breast cancer. The experience of the European Institute of Oncology[END_REF], distant metastases occurred in only 10.7% of the patients. One might speculate that the high rate of endocrine-responsive tumours in this series (87.6%), which is probably related to the high prevalence of invasive lobular carcinomas, might lead to a better outcome following adequate adjuvant treatment at least during the first 5 years. Furthermore, patients with lobular or ductal carcinoma had a similar outcome in terms of local recurrence (4.4 and 6.1%, respectively). This confirms that, despite the common multifocality and bilaterality of invasive lobular carcinoma, its occurrence does not by itself preclude breast conservation, as long as it is adequately excised.

The main finding of our study is that the 5 years cumulative incidence of local relapse was 5.1%, which is in our opinion quite acceptable and similar to that reported in patients with unifocal disease [START_REF] Veronesi | Twenty years follow up of randomized study comparing breast conserving surgery with radical mastectomy for early breast cancer[END_REF][START_REF] Fisher | Twenty year follow up of a randomised study comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF]. Therefore, these data, derived from a large series of patients, emphasize that breast conserving surgery is safe for patients with MF/MC breast cancer, as it provides acceptable local control when the disease is adequately excised. Furthermore, breast conservation achieved a good outcome even in patients with MC disease who were treated with separate wide local excisions (8% 5 years cumulative incidence of local relapse).

The multivariate analysis which showed that over-expression of HER2/neu and lack of estrogen and progesterone receptors worsened local disease free survival. Due to the availability of an effective therapy against cells over-expressing HER2/neu it is likely that outcome will be improved with the adjuvant administration of trastuzumab [START_REF] Piccart-Gebhart | Trastuzumab after adjuvant chemotherapy for HER2-positive breast cancer[END_REF][START_REF] Romond | Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer[END_REF]. This is a retrospective analysis and therefore some limitations have to be underlined. First, we were not able to collect reliable data on clinical and radiological presentation. It would be interesting to have this information in order to better characterize the patient cohort. Obviously, all patients with multicentric cancer had preoperative diagnosis since they underwent double incision but for the majority of the population we cannot say how many patients had either clinically palpable lumps or radiologically detected tumours or incidental finding at surgery or at pathologic evaluation. Secondly, this is a selected population of patients. In fact, all these women were considered suitable for breast conservation from both the oncologic and cosmetic point of view after careful discussion.

Despite the above mentioned limitations and critiques which might be raised, we think that these data are of important clinical interest since only a few papers with a limited number of patients have been reported so far on this topic. In specialized centers with high patient volume conservative surgery is usually performed in presence of multifocal cancer, but many surgeons or medical oncologists acting in community hospitals continue to recommend mastectomy for the presence of multifocality itself.

As stated earlier, patients were selected for the expected acceptable cosmetic outcome as well. In most cases of multicentric cancer women had large breasts with tumors far each other and located at the periphery of the breast. In this clinical scenario it is not hard to imagine that satisfactory outcome could be achieved. We believe that the data collected within this study are relevant to the discussion with motivated patients in order to decide upon the type of surgery to be conducted. Given the low rate of local recurrence, in selected patients breast conservation can be considered for patients with MF/MC breast cancer, whenever adequate surgery and acceptable cosmetic results can be achieved.

  2, 95% C.I. 1.01-10.0, and HR: 2.7, 95% C.I. 1.06-7.7, respectively) were associated with higher ipsilateral breast cancer reappearance rate. Involvement of 4 or more lymph nodes and lack of estrogen and progesterone receptors (HR: 2.7, 95% C.I. 1.06-6.7, and HR: 4.7, 95% C.I. 2.1-10.4, respectively) were associated with poorer overall survival.

  events. Dotted lines: 95% confidence interval bands. Cumulative incidence and 95% confidence interval bands were computed in a competing risk framework.

Fig. 1

 1 Fig. 1 Cumulative incidence of local events

Fig. 2

 2 Fig. 2 Cumulative mortality

Table 1

 1 Patients' characteristics and stratification for multifocal/multicentric tumours Variable Classification Total No. (%) Multifocal No. (%) Multicentric No. (%) Chi-square P-value

	Breast Cancer Res Treat (2009) 113:577-583
	All patients		476	421 (88.4)	55 (11.6)
	Age	\35 years	25 (5.3)	23 (5.5)	2 (3.6)	0.492
		35-49 years	178 (37.4)	159 (37.8)	19 (34.6)
		50-64 years	200 (42.0)	172 (40.9)	28 (50.9)
		C65 years	73 (15.3)	67 (15.9)	6 (10.9)
	Menopause	Pre	193 (40.6)	171 (40.6)	22 (40.0)	0.930
		Peri/post	283 (59.4)	250 (59.4)	33 (60.0)
	Number of foci a	2	276 (76.7)	246 (79.4)	30 (60.0)	0.011
		3	55 (15.3)	42 (13.6)	13 (26.0)
		C4	29 (8.0)	22 (7.1)	7 (14.0)
	pT	pT1a-pT1b	84 (17.7)	77 (18.3)	7 (12.7)	0.529
		pT1c	257 (54.0)	227 (53.9)	30 (54.6)
		pT2-pT3	135 (28.4)	117 (27.8)	18 (32.7)
	Extensive in situ component	Absent	297 (62.4)	262 (62.2)	35 (63.6)	0.840
		Present	179 (37.6)	159 (37.8)	20 (36.4)
	Margins	Negative ([1 mm) 421 (88.5)	372 (88.4)	49 (89.1)	0.983
		Close (B1 mm)	45 (9.4)	40 (9.5)	5 (9.1)
		Positive	10 (2.1)	9 (2.1)	1 (1.8)
	Number of positive lymph nodes a 0	211 (44.7)	186 (44.5)	25 (46.3)	0.795
		1-3	167 (35.4)	150 (35.9)	17 (31.5)
		C4	94 (19.9)	82 (19.6)	12 (22.2)
	Histotype	Ductal	333 (70.0)	294 (69.8)	39 (70.9)	0.928 b
		Lobular	88 (18.5)	78 (18.5)	10 (18.2)
		Mixed Duct.-Lob.	27 (5.7)	22 (5.2)	5 (9.1)
		Other	28 (5.9)	27 (6.4)	1 (1.8)
	Estrogen receptors	Positive	417 (87.6)	369 (87.6)	48 (87.3)	0.937
		Negative	59 (12.4)	52 (12.4)	7 (12.7)
	Progesterone receptors	Positive	372 (78.2)	329 (78.2)	43 (78.2)	0.995
		Negative	104 (21.8)	92 (21.8)	12 (21.8)
	Grading a	G1	65 (14.1)	59 (14.5)	6 (11.1)	0.777
		G2	250 (54.1)	219 (53.7)	31 (57.4)
		G3	147 (31.8)	130 (31.9)	17 (31.5)
	HER2/neu a	Over-expressed	40 (13.3)	32 (12.5)	8 (18.6)	0.272
		Not over-expressed 260 (86.7)	225 (87.5)	35 (81.4)
	Ki-67 a	\20%	235 (49.7)	209 (50.0)	26 (47.3)	0.704
		C20%	238 (50.3)	209 (50.0)	29 (52.7)
	Vascular invasion	Absent	327 (68.7)	292 (69.4)	35 (63.6)	0.584
		Present	81 (17.0)	69 (16.4)	12 (21.8)
		Extensive	68 (14.3)	60 (14.3)	8 (14.3)
	a Information is not available for all the patients
	b Lobular vs. ductal. The reported tumor size represents the largest tumor focus
	Bolded entries indicate statistical significance (i.e. P-value \ 0.05)

Table 3

 3 Univariate analysis of prognostic factors

	Variable	Classification	Local events		All first events		Deaths
			Events (5 years	P-value	Events (5 years	P-value	Events (5 years	P-value
			Cum Inc)		Cum Inc)		Cum Inc)
	All patients		26 (5.1)		111 (23.5)		36 (6.2)
	Age	\35 years	2 (8.5)	0.130	8 (25.9)	0.131	2 (8.0)	0.008
		35-49 years	11 (6.0)		38 (22.6)		6 (3.6)
		50-64 years	6 (2.4)		42 (19.5)		17 (6.4)
		C65 years	7 (9.7)		23 (35.4)		11 (11.2)
	Multifocal/multicentric	Multifocal	23 (4.9)	0.829	97 (23.2)	0.393	31 (6.0)	0.515
		Multicentric	3 (8.0)		14 (26.2)		5 (7.6)
	Number of foci a	2	7 (2.4)	0.192	56 (22.5)	0.832	17 (5.0)	0.802
		3+	5 (5.7)		19 (19.8)		6 (5.4)
	pT	pT1a-pT1b	7 (10.6)	0.347	15 (22.5)	0.014	5 (4.9)	0.210
		pT1c	12 (4.9)		50 (20.6)		16 (5.8)
		pT2-pT3	7 (5.1)		46 (29.1)		15 (7.7)
	Extensive in situ component	Absent	14 (5.0)	0.597	69 (25.1)	0.584	26 (7.1)	0.101
		Present	12 (6.0)		42 (20.9)		10 (4.7)
	Margins	Negative/close 25 (5.5)	0.313	108 (23.1)	0.286	35 (6.0)	0.793
		Positive	1 (25.0)		3 (50.0)		1 (12.5)
	Number of positive lymphnodes a 0	8 (3.7)	0.308 b	26 (12.5)	\0.001 b	9 (3.4)	\0.001 b
		1-3	12 (5.6)		41 (23.8)		13 (6.4)
		C4	5 (6.3)		43 (45.0)		13 (11.2)
	Histotype	Ductal	20 (6.1)	0.638	87 (26.9)	0.229	29 (8.0)	0.805
		Lobular	4 (

  Five-year cumulative incidences for local events were computed in a competing risk framework and compared by the Gray test. Five-year cumulative incidences for first events and deaths were compared by the log-rank test. Bolded entries indicate statistical significance (i.e.

	Breast Cancer Res Treat (2009) 113:577-583						581
	Estrogen receptors	Positive	18 (4.1)	0.003	84 (19.8)	\0.001	22 (3.6)	\0.001
		Negative	8 (16.1)		27 (48.5)		14 (24.2)
	Progesterone receptors	Positive	13 (2.8)	\0.001	71 (17.7)	\0.001	17 (3.1)	\0.001
		Negative	13 (15.6)		40 (43.6)		19 (16.9)
	Grading a	G1	0 (0.0)	0.046 c	6 (9.0)	\0.001 c	2 (3.1)	\0.001 c
		G2	13 (5.3)		42 (16.0)		11 (2.7)
		G3	10 (6.4)		59 (40.6)		22 (13.4)
	Her2/neu a	Overexpressed	7 (19.8)	\0.001	23 (56.9)	\0.001	10 (26.9)	\0.001
		Not overexpr.	9 (4.0)		49 (20.9)		16 (4.4)
	Ki-67 a	\20%	8 (3.5)	0.036	34 (14.4)	\0.001	11 (2.2)	0.013
		C20%	18 (7.9)		77 (32.4)		25 (10.2)
	Vascular invasion	Absent	14 (3.8)	0.135 d	57 (17.3)	\0.001 d 19 (4.1)	0.033 d
		Present	5 (8.1)		23 (32.5)		8 (9.5)
		Extensive	7 (8.3)		31 (40.3)		9 (12.1)
								P-value
	\ 0.05)
	a Information is not available for all patients
	b Positive vs. negative
	c G2-G3 vs. G1
	d Present/extensive vs. absent

Table 4

 4 Multivariate analysisHazard ratios and confidence intervals were calculated using the Cox Proportional Hazard model. For local events the Cox Proportional Hazard model was computed in a competing risk framework. The model for mortality was adjusted for age considered as a continuous variable. Bolded entries indicate statistical significance (i.e. P-value \ 0.05)

		Comparisons	Local events	All first events	Deaths
			HR (95% C.I.)	HR (95% C.I.)	HR (95% C.I.)
	pT	pT2/3 vs. pT1	-	1.17 (0.78; 1.75)	-
	Number of positive lymphnodes	1-3 vs. 0	-	1.86 (1.12; 3.08)	1.68 (0.70; 4.00)
		4+ vs. 0	-	2.73 (1.58; 4.75)	2.65 (1.06; 6.66)
	Er/Pgr receptors	Both absent vs. at least one positive	2.76 (1.06; 7.68)	2.40 (1.43; 4.05)	4.68 (2.10; 10.4)
	Her2/neu	Overexpressed vs. not overexpressed	3.18 (1.01; 10.0)	1.64 (0.92; 2.93)	1.82 (0.71; 4.67)
	Ki-67	C20 vs. \20	1.45 (0.57; 3.68)	1.45 (0.92; 2.27)	1.31 (0.59; 2.92)
	Vascular invasion	Present/extensive vs. absent	-	1.50 (0.98; 2.29)	1.86 (0.89; 3.88)

Breast Cancer Res Treat (2009) 113:577-583


M Botteri

C Rietjens

F Garusi

S Lorenzi

R Martella

Bosco

J Y Petit
email: jean.petit@ieo.it

Ae U Veronesi

Ae P Rey

Ae N Rotmensz

Ae E Botteri

Ae M Rietjens

Ae C Garusi

Ae F De Lorenzi

Ae S Martella

Ae R Bosco

Ae A Manconi

Ae A Luini

Ae V Galimberti

Ae P Veronesi

Ae G B Ivaldi

Ae R Orecchia

P Rey

Á M Rietjens

Á C Garusi

Á F De Lorenzi

Á S Martella

Á R Bosco

Á A Manconi

N Rotmensz

Á E Botteri

Á V Galimberti

Á P Veronesi

G B Ivaldi

Á R Orecchia

Nipple-sparing mastectomy: risk of nipple-areolar recurrences in a series of 579 cases

Keywords: Nipple-sparing, Mastectomy, Breast, Conservative treatment

Nipple-sparing mastectomy: risk of nipple-areolar

recurrences in a series of 579 cases

Introduction

Mastectomy is still required in around 20-25% of breast cancer patients, especially in case of multicentric, large tumours or recurrences after conservative treatment. The feeling of mutilation is increased because of the removal of the nipple areola complex (NAC) and remains despite the psychological improvement due to the NAC reconstruction [START_REF] Wellisch | The psychological contribution of nipple addition in breast reconstruction[END_REF]. The NAC seems to be the signature of the breast identity more than the volume or the shape which can be changed easily in esthetic surgery.

For these reasons we proposed in 2002 a new mastectomy technique preserving the Nipple Areolar Complex: the Nipple Sparing Mastectomy (NSM) [START_REF] Petit | The nipple sparing mastectomy: early results of a feasability study of a new application of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated[END_REF].

Since then we performed more than 700 NSM and we explore for the first time, in this paper, the oncological results of this type of intervention.

The most important risk of preserving NAC is retroareolar or areolar relapse. Number of articles studied the relation of nipple and areola involvement and the tumour site, the tumour size and the nodal status [START_REF] Cense | Nipple sparing mastectomy in breast cancer: a viable option?[END_REF][START_REF] Lambert | Parameters that predict nipple involvement in breast cancer[END_REF][START_REF] Gajdos | Subareolar breast cancers[END_REF][START_REF] Vyas | Prediction of nipple and areola involvement in breast cancer[END_REF][START_REF] Laronga | The incidence of occult nipple-areola complex involvement in breast cancer patients receiving a skin sparing mastectomy[END_REF][START_REF] Gerber | Skin-sparing mastectomy with conservation of the nipple-areola complex and autologous reconstruction is an oncologically safe procedure[END_REF] but we didn't find in the literature data about the risk of retroareolar recurrence in relation with the conservation of the NAC in case of breast cancer treatment. Most of these studies concluded that the NAC could be preserved only in the case of small tumours located at a distance from the areola, and with negative axillary nodes.

Based on the results of intra-operative radiotherapy (ELIOT) studies [START_REF] Petit | The nipple sparing mastectomy: early results of a feasability study of a new application of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated[END_REF][START_REF] Merrick | IORT for early breast cancer: a report on long-term results[END_REF][START_REF] Dubois | IORT in breast carcinomas[END_REF][START_REF] Battle | IORT for breast cancer[END_REF][START_REF] Veronesi | A preliminary report of intraoperative radiotherapy (IORT) in limited-stage breast cancers that are conservatively treated[END_REF][START_REF] Veronesi | Full-dose intraoperative radiotherapy with electrons durign breast conserving surgery. Experience with 590 cases[END_REF][START_REF] Veronesi | Focalised intraoperative irradiation after conservative surgery for early stage breast cancer[END_REF], we assumed that an intraoperative radiotherapy with electrons could avoid or decrease the risk of LR in selected patients with preserved NAC.

Technique

The surgical technique has been previously described in our pilot study [START_REF] Petit | The nipple sparing mastectomy: early results of a feasability study of a new application of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated[END_REF]. We consider very important the subcutaneous dissection under the areola that should remove the maximum of glandular and ductal tissue without compromising the NAC vascularization. The specimen should be carefully marked especially in the retroareolar area to allow a precise localization of the tumour by the pathologist. The ducts beneath the areola are taken separately to be analysed by frozen section: in case of positive histology the NAC should be removed. In case of poor blood supply and high risk of final necrosis, the plastic surgeon can also decide to remove the NAC before the delivery of radiotherapy.

ELIOT

The technique of ELIOT has been described in breast conserving surgery [START_REF] Dubois | IORT in breast carcinomas[END_REF][START_REF] Battle | IORT for breast cancer[END_REF][START_REF] Veronesi | Focalised intraoperative irradiation after conservative surgery for early stage breast cancer[END_REF]. A total dose of 16 Gy (at the point of maximum dose) is delivered in the region of the NAC. The biologic equivalent of a single intra-operative dose is felt to be 1.5-2.5 higher than the dose delivered with conventional fractionated irradiation. More precisely, equivalent doses can be estimated using radiobiological models to predict radiation effects. According to linearquadratic model and computing the surviving fraction of clonogenic units, a single dose of 16 Gy corresponds to a fractionated dose of about 45 Gy for early-responding tissue (tumour cells) and of 70-80 Gy for late-responding tissues (vessels, fat, nerves).

Breast reconstruction

The immediate breast reconstruction (IBR) was performed by a second team with different technique (expander, definitive prosthese or flap) depending on the size of the breast, the local situation, the possible contraindications and the wish of the patient.

Patients and methods

From March 2002 to January 2007, 810 patients were admitted at IEO with an indication of NSM.

Inclusion criteria were: multifocal and multicentric breast carcinoma, unicentric carcinoma not suitable to quadrantectomy for anatomic reasons, extensive intraductal neoplasia involving more than one quadrant.

Exclusion criteria were: tumours bigger than 3 cm, clinical invasion of the NAC, tumours closer than 1 cm from the areola, positive retroareolar frozen section.

Eighty-seven patients were excluded because of a positive retroareolar frozen section (83 cases) or NAC with insufficient vascularization (4 cases).

Seven hundred and three NSM were performed (579 for invasive cancer and 124 for in-situ disease), in 678 patients, 25 patients had bilateral NSM, 21 synchronous and 4 metachronous (Table 1).

In 169 patients ELIOT was not performed either for technical problems or for a poor vascularization of the nipple.

In 124 patients (127 NSM) the radiotherapy was delayed to the following days and performed by external beam and 45 patients never had radiotherapy because of the local conditions (36 cases) or because of a previous breast irradiation (9 cases).

Statistical methods

Disease-free survival was calculated from date of surgery to any local, regional or distant relapse, whichever occurred first, or to last visit date in case of no events. Other primary tumours were considered as competing events. Loco-regional disease-free survival was calculated from date of surgery to any local or regional relapse or to last visit date in case of no events. Other primary tumours and distant relapses were considered as competing events. Gray test was used to compare survival across different subgroups [START_REF] Gray | A class of k-sample tests for comparing the cumulative incidence of a competing risk[END_REF]. All analyses were performed with the SAS software (SAS Institute, Cary, NC). All tests were two sided.

Results

Patients characteristics and outcome are summarised in Table 2. The evaluation of the relapses was made considering only the carcinomas excluding all cases of pure Ductal Intraepithelial Neoplasia (DIN) and the four cases of metachronous tumour. The rate of local relapses is 0.9% per year.

Most local relapses (6/8 cases) were in the primary tumour bed, and no one occurred in the radiotherapy field. One local relapse was located in the opposite quadrant and another one was on the chest wall below the clavicle. All local regional events were treated by surgery and all patients are alive 7-34 months after the local relapse.

A full description of all events is given in Table 3: we observed 13 metastasis and 14 local or regional relapses. In addition we experienced three other primary tumours (one uterine cervix, one brain tumour and one renal cancer) and one breast cancer related death.

Cumulative incidence of any local, regional or distant relapse and overall survival curves are shown in Figs. 1 and2 respectively.

In our study the rate of positive retroareolar histology increased with the dimension of the tumour and was 12.6% for pT1, 23.3% for pT2 and 36.8% in case of pT3. These differences are significant (all P \ 0.0002). We didn't find any significant difference for the nodal status: 7% of positive retroareolar histology in case of pN0, 12.5% in case of pN1, 12.1% for the pN2 and 8.3% in case of pN3.

According with our internal guidelines all cases with positive retroareolar frozen section (83 cases) were considered not eligible and were excluded form the study. However, in 63 cases with negative frozen-section histology the definitive histology was positive for carcinoma. Out of these 63 patients, 7 underwent a secondary ablation of the NAC, while in 56 the NAC was saved. This decision was taken, after discussion with the patient, according to the hypothesis that radiotherapy would have destroyed any residual disease. In this series of 56 cases of conservation of the NAC (47 in situ lesions and 9 carcinomas) despite the final positive histology, we didn't experience any local relapse after 19 months of follow up.

Discussion

Our first concern, when we proposed the NSM [START_REF] Petit | The nipple sparing mastectomy: early results of a feasability study of a new application of perioperative radiotherapy (ELIOT) in the treatment of breast cancer when mastectomy is indicated[END_REF], was to avoid the retroareolar local relapses. For this reason the mammary tissue left in the retroareolar region was the minimal compatible with the survival of the nipple complex. Moreover a frozen section histology was always taken in the retroareolar region to be certain of its integrity. With these precautions and with the irradiation of the areola with high energy electron we did not observe a single recurrence in the retroareolar area. The number of local recurrences observed in other sites was low [START_REF] Gerber | Skin-sparing mastectomy with conservation of the nipple-areola complex and autologous reconstruction is an oncologically safe procedure[END_REF] and is consistent with the rate of LR after modified mastectomies (1% per year). Most of them were close to the primary tumour site despite the skin resection performed over the tumour bed. We have no comparative data to conclude that the absence of retroareolar recurrences is due to the ELIOT. However, we should stress that out of 579 patients with negative extemporaneous retroareolar examinations, 63 (10%) were positive at the final histology. The tumour was in situ in 51 cases (81%) and invasive in 12 cases (19%). Only 7 NAC were removed among the 63 positive, while 56 patients kept their NAC despite the positive histology. No recurrences were observed in the follow up of this group after 20 months. Gajdos et al. have already stressed the role of the radiotherapy to prevent the retroareolar recurrence [START_REF] Gajdos | Subareolar breast cancers[END_REF]. The risk of retroareolar recurrence appears very low even when no radiotherapy is delivered [START_REF] Cense | Nipple sparing mastectomy in breast cancer: a viable option?[END_REF][START_REF] Laronga | The incidence of occult nipple-areola complex involvement in breast cancer patients receiving a skin sparing mastectomy[END_REF][START_REF] Gerber | Skin-sparing mastectomy with conservation of the nipple-areola complex and autologous reconstruction is an oncologically safe procedure[END_REF], therefore a randomized trial would not reveal any difference without a huge accrual of patients. The nipple areolar invasion by the tumour cells is considered a major reason to avoid NAC conservation during mastectomy [START_REF] Smith | Involvement of the nipple and areola in carcinoma of the breast[END_REF][START_REF] Andersen | Spread to the nipple and areola in carcinoma of the breast[END_REF][START_REF] Cucin | Screening the nipple for involvement in breast cancer[END_REF][START_REF] Andersen | Involvement of the nipple and areola in breast cancer. Value of clinical findings[END_REF]. The relation between different preoperative parameters, and the presence of positive retroareolar histology is an important issue often underlined in the literature [START_REF] Schecter | Applicability of the nipple-areola complex-sparing mastectomy: a prediction model using mammography to estimate risk of nippleareola complex involvement in breast cancer patients[END_REF][START_REF] Vlajcic | Nipple-areola complex preservation: predictive factors of neoplastic nipple-areola complex invasion[END_REF][START_REF] Lambert | Parameters that predict nipple involvement in breast cancer[END_REF]. Most of these authors observe a relation between the tumour size, the tumour site, the lymph node metastases and the risk of retroareolar tumour involvement [START_REF] Cense | Nipple sparing mastectomy in breast cancer: a viable option?[END_REF][START_REF] Vyas | Prediction of nipple and areola involvement in breast cancer[END_REF][START_REF] Laronga | The incidence of occult nipple-areola complex involvement in breast cancer patients receiving a skin sparing mastectomy[END_REF]. Our study does not confirm the relation between the retroareolar tumour invasion and lymph node metastases. In our series, the risk of retroareolar involvement increases with the tumour size as like in the majority of the studies in the literature. We agree that the site of the tumour can influence the risk of NAC involvement, and the limits generally accepted vary from 1 to 4 cm from the areola margins. Gajdos et al. [START_REF] Gajdos | Subareolar breast cancers[END_REF] and Schecter include the mammogram to evaluate the distance between the tumour and the NAC. Schecter found an high rate (42%) of nipple involvement in patients who underwent a total mastectomy, which is very different with our rate of 10%. Friedmann et al. [START_REF] Friedman | Breast MR and the appearance of the normal and abnormal nipple[END_REF] made an interesting evaluation of the NAC invasion by MRI with an important diagnostic accuracy: In our series we didn't perform MRI as routine preoperative examination. Some authors proposed an histological Fig. 1 Cumulative incidence of any local, regional or distant relapse. The events occurred between 6 and 41 months after the first intervention [24] by a surgical biopsy and Govindarajulu et al. [START_REF] Govindarajulu | Preoperative mammotome biopsy of ducts beneath the nipple areola complex[END_REF] by a mammotome in place of the traditional frozen section. We didn't explore this possibility since we can have an histological response before the end of the mastectomy in order to decide the NAC conservation. Finally we note that most of the articles in the literature are focused on the possibility to predict a retroareolar invasion before surgery in order to select the best cases for a NSM. Our results demonstrate a very low rate of events even without strict criteria for inclusion in the study.

Conclusion

Our study shows the absence of local recurrences in the areolar or retroareolar region where a small proportion of glandular tissue has been purposely preserved. Moreover it confirms that the local recurrence rate in the NSM, completed with local radiotherapy on the NAC, is not higher than the usual rate observed in the modified mastectomy, as reported in the literature.

Fig. 2

 2 Fig. 2 Overall survival

Table 1

 1

	the study	Patients selection for	ELIOT of the nipple areola	External Radiotherapy	No Radiotherapy
		Nipple sparing mastectomy
		Patients	554	124		45
		Interventions	579	124		47
		RT dose	16 Gy	16 Gy
		Irradiated area: average	14.4 cm 2	18.6 cm 2
		Range	7.07-38.5	16-36
		Fractions	1	1-2
		Delay in days: average	0	2.5
				1-85
				94% B3
			Table 2 Patients characteristics and outcome
			Variable		Classification	No. (%)
			All patients			579
			Age		\35 years	78 (13.5)
					35-49 years	380 (65.6)
					C50 years	121 (20.9)
			Size of tumour		pT1	284 (49.8)
					pT2	240 (42.1)
					pT3	46 (8.1)
			Multifocality/multicentricity	Present	177 (31.0)
					Absent	394 (69.0)
			Number of positive lymphnodes	0	249 (43.0)
					1-3	227 (39.2)
					C4	103 (17.8)
			Histotype		Ductal	452 (78.1)
					Lobular	66 (11.4)
					Mixed	23 (4.0)
					Other	38 (6.6)
			Estrogen receptors		Positive	473 (82.0)
					Negative	104 (18.0)
			Progesteron receptors		Positive	402 (69.8)
					Negative	174 (30.2)
			Grading		G1	66 (12.7)
					G2	244 (47.1)
					G3	208 (40.2)
			Her2/neu		Overexpressed	139 (24.1)
					Not overexpressed	438 (75.9)
			Ki-67		\20%	282 (49.1)
					C20%	292 (50.9)
			Vascular invasion		Absent	377 (65.1)
					Present	202 (34.9)

Table 3

 3 Description of events

	Description of events		No.	Rate 9 100 patient years
	First event
	Local			8	0.9
	Regional			5	0.6
	Loco-regional		1	0.1
	Distant			13	1.5
	Other primary tumour		3	0.4
	Death			1	0.2
	40	5 yrs. Cumulative Incidence 15.5 (95% CI 9.2-23.4)
	30
	20
	10
	0
	Years:
	At risk:	579	356	175	61	18	0

Breast Cancer Res Treat (2009) 114:97-101


Ana Peixoto

Catarina Santos

Patrícia Rocha

Manuela Pinheiro

Sofia Príncipe

Deolinda Pereira

Helena Rodrigues

Fernando Castro

Joaquim Abreu

Leonor Gusmão

A E Patrı

´cia Rocha

A E Manuela

Pinheiro Ae

Sofia Prı

A E Deolinda

Pereira Ae

A E Fernando

Castro Ae

Ae Leonor Gusma

A E Anto

Amorim Ae

Manuel R Teixeira
email: mteixeir@ipoporto.min-saude.pt

Á C Santos

Á P Rocha

Á M Pinheiro

Á S Prı

Á M R Teixeira

Á H Rodrigues

Á J Abreu

L Gusma

Á A Amorim

A Amorim

The c.156_157insAlu rearrangement accounts for more than one-fourth of deleterious BRCA mutations in northern/central Portugal

Keywords: c.156_157insAlu BRCA2 mutation, Breast cancer, Hereditary predisposition

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

Introduction

Molecular analyses of the BRCA1 and BRCA2 genes in families suspected to have hereditary predisposition to breast/ovarian cancer have shown that most populations exhibit a wide spectrum of mutations throughout both genes. However, several founder mutations have been identified in individuals of different ancestries, including those of Ashkenazi Jewish (185delAG and 53832insC) [START_REF] Tonin | BRCA1 mutations in Ashkenazi Jewish women[END_REF], French Canadian (C4446T and 2953del3+C) [START_REF] Durocher | Mutation analysis of the BRCA1 gene in 23 families with cases of cancer of the breast, ovary, and multiple other sites[END_REF][START_REF] Tonin | Founder BRCA1 and BRCA2 mutations in French Canadian breast and ovarian cancer families[END_REF], Norwegian (1675delA and 1135insA) [START_REF] Dørum | Three per cent of Norwegian ovarian cancers are caused by BRCA1 1675delA or1135insA[END_REF], Dutch (2804delAA and Alu-mediated deletions encompassing exons 13 and 22) [START_REF] Peelen | The majority of 22 Dutch high-risk breast cancer families are due to either BRCA1or BRCA2[END_REF][START_REF] Petrij-Bosch | BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients[END_REF], Polish (5382insC, C61G, and 4153delA) [START_REF] Go ´rski | A high proportion of founder BRCA1 mutations in Polish breast cancer families[END_REF], Greek (5382insC and G1738R) [START_REF] Konstantopoulou | Greek BRCA1 and BRCA2 mutation spectrum: two BRCA1 mutations account for half the carriers found among high-risk breast/ovarian cancer patients[END_REF][START_REF] Anagnostopoulos | G1738R is a BRCA1 founder mutation in Greek breast/ovarian cancer patients: evaluation of its pathogenicity and inferences on its genealogical history[END_REF], Icelandic (999del5) [START_REF] Rafnar | BRCA2, but not BRCA1, mutations account for familial ovarian cancer in Iceland: a population-based study[END_REF], Spanish (330A[G, 6857_6858del, and 9254_9258del) [START_REF] Campos | Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain[END_REF][START_REF] Dı ´ez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF], and Swedish (3171ins5) [START_REF] Bergman | The western Swedish BRCA1 founder mutation 3171ins5; a 3.7 cM conserved haplotype of today is a reminiscence of a 1500-year-old mutation[END_REF] origins. We have previously reported several novel mutations in a first characterization of the mutational spectrum of the entire coding sequences and exon-intron boundaries of the BRCA1 and BRCA2 genes and large BRCA1 rearrangements in Portuguese breast/ovarian cancer families [START_REF] Peixoto | BRCA1 and BRCA2 germline mutational spectrum and evidence for genetic anticipation in Portuguese breast/ovarian cancer families[END_REF].

Recently, Teugels et al. [START_REF] Teugels | De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes[END_REF] identified an Alu insertion in BRCA2 exon 3 (c.156_157insAlu) in a Portuguese breast cancer patient living in Belgium. This mutation was not detectable using the conventional mutation screening techniques and originates skipping of BRCA2 exon 3 [START_REF] Teugels | De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes[END_REF]. A regional founder effect has recently been described for this rearrangement in hereditary breast/ovarian cancer families mostly originated from central/southern Portugal [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF]. In an attempt to gain insight into the ancestral origin and population spread of the c.156_157insAlu BRCA2 mutation, we estimated its frequency in breast/ovarian cancer families originated mostly from northern/central Portugal and analyzed the chromosomal background upon which this mutation occurs using 10 microsatellite markers surrounding the BRCA2 gene. Furthermore, because some doubts have been raised about the pathogenic effect of BRCA2 exon 3 skipping [START_REF] Dı ´ez | Caution should be used when interpreting alterations affecting the exon 3 of the BRCA2 gene in breast/ovarian cancer families[END_REF], we have performed segregation studies and mRNA expression analysis of c.156_157insAlu carriers and disease-free controls.

Materials and methods

Patients

This study comprised the 78 Portuguese breast/ovarian cancer families in whom no clearly deleterious BRCA1/ BRCA2 mutations were found in our previous study [START_REF] Peixoto | BRCA1 and BRCA2 germline mutational spectrum and evidence for genetic anticipation in Portuguese breast/ovarian cancer families[END_REF] and an additional 130 BRCA1/BRCA2 negative probands that had been selected using the following the criteria: (1) families with one or more breast cancers (one of them diagnosed before the age of 50 years) and one or more ovarian cancers (BC + OC); (2) families with two or more female breast cancers, one of them diagnosed before the age of 50 years (C2BC); (3) families with at least one case of breast cancer or ovarian cancer in association with at least one case of male breast cancer (MBC); (4) patients without family history of breast/ovarian cancer, but showing early-onset breast cancer (1BC, diagnosed before the age of 35 years), breast (diagnosed before the age of 60 years) and ovarian cancer (1BC + OC), bilateral breast carcinomas (1BlC, one of them diagnosed before the age of 50 years), or male breast cancer (1MBC, at any age). BRCAPRO mutation probabilities were estimated for all probands [START_REF] Parmigiani | Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2[END_REF].

DNA isolation and BRCA1/BRCA2 mutation screening

Genomic DNA was extracted from peripheral leucocytes according to standard procedures and subsequently amplified using primers described by van der Hout et al. [START_REF] Van Der Hout | A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting[END_REF]. Mutation screening of the entire coding regions of BRCA1 and BRCA2 was performed by Denaturing Gradient Gel Electrophoresis (DGGE), using the IngenyphorU system (Ingeny; Goes). Sequence analysis of genomic fragments with altered DGGE mobility pattern was carried out on an Applied Biosystems' 310 automated DNA sequencer (Foster City), using the dye terminator method. Multiplex Ligation-dependent Probe Amplification (MLPA) (MRC-Holland, Amsterdam) was used to detect large exonic BRCA1 and BRCA2 rearrangements (only 23 probands were analyzed for the latter gene) in cases that were negative for pathogenic BRCA1/BRCA2 point mutations.

Screening for the c.156_157insAlu BRCA2 mutation The c.156_157insAlu BRCA2 mutation was screened in 208 probands and in 262 control chromosomes (healthy blood donors). In the first PCR we used primers for BRCA2 exon 3 amplification (forward primer: GTCACTGGT TAAAACTAAGGTGGG and reverse primer: GAAGCCA GCTGATTATAAGATGGTT). The cycling conditions were 94°C for 1 min, 35 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 4 min, and a final extension of 72°C for 10 min. In the second PCR we used primers specific for the c.156_157insAlu BRCA2 mutation (forward primer: GACACCATCCCGGCTGAAA and reverse primer: CCC CAGTCTACCATATTGCAT). The cycling conditions were 97°C for 15 min, 6 cycles of 97°C for 1 min, 68°C for 1 min, and 72°C for 1 min, 6 cycles of 97°C for 1 min, 66°C for 30 s, and 72°C for 1 min, 6 cycles of 97°C for 1 min, 64°C for 30 s, and 72°C for 1 min, 22 cycles of 97°C for 1 min, 60°C for 30 s, and 72°C for 1 min, and a final extension of 72°C for 10 min. Sequence analysis of genomic fragments with the insertion was carried out on an Applied Biosystems' 310 automated DNA sequencer (FosterCity), using the dye terminator method.

RNA isolation and transcript analysis

Frozen cell pellets from nucleated cells of patients carrying the c.155_156insAlu BRCA2 variant and from control individuals (without known BRCA1 or BRCA2 mutations) were used to extract total RNA using 1 ml of Tripure isolation reagent (Roche Diagnostics, Indianapolis, USA), according to the manufacturer's instructions. After quantification using a NanoDrop ND-1000 spetrophotometer, One-Step RT-PCR was performed in all cases using the Superscript One-Step RT-PCR System as suggested by the manufacturer (Invitrogen, Carlsbad, USA). We used primers spanning from exon 1 to exon 6 of BRCA2 (forward primer B2Ex1F: GCTTACTCCGGCCAAAAAAGA and reverse primer B2Ex6R: GGTGTCTGACGACCCTTCACA). The cycling conditions were 50°C for 30 min, 95°C for 15 min, 40 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min, and a final extension of 72°C for 10 min. RT-PCR products were separated on a 2% agarose gel (SeaKem LE Agarose, Rockland, USA) and visualized with ethidium bromide in an image analyser ImageMaster VDS (Amersham Biosciences, Little Chalfont, UK). Individual bands were excised from the gel, purified using GFX TM PCR DNA and Gel Band Purification Kit (GE HealthCare) and sequenced as indicated above.

Microsatellite typing and haplotype analysis

A total of 14 probands and 14 family members were genotyped for polymorphic microsatellite markers flanking BRCA2 and for the c.-25G[A polymorphism. The order of the markers, the consensus repeat, and the distances relative to each other and BRCA2 are shown in Table 1. The physical distances of the genetic markers were derived from the Human Genome Browser assembly hg18 from the UCSC Bioinformatics (http://www.genome.ucsc.edu/). The consensus pattern was obtained with the software Tandem Repeats Finder (http://www.tandem.bu.edu/). The primer sequences for the amplification of the markers were derived from the Human Genome database (http:// www.gdb.org). All 10 markers were assayed by PCR using fluorescently end-labeled primers. PCR products were run on an ABI PRISM 310 Genetic Analyser (Applied Biosystems) together with a fluorescence labeled DNA fragment size standard. Genotyping of the c.-25G[A polymorphism was performed by sequencing. Haplotypes were reconstructed manually in the three informative families. The geographic origin of the c.156_157insAlu BRCA2 positive families was inferred from the birthplace of the oldest carrier or of the oldest affected family member most likely to be a carrier.

Results

Detection of the c.156_157insAlu BRCA2 mutation

We identified eight probands with the c.156_157insAlu mutation in our initial series of 78 patients in whom no deleterious BRCA1/BRCA2 mutation had been found by DGGE and MLPA. This finding, together with the 22 pathogenic mutations previously reported [START_REF] Peixoto | BRCA1 and BRCA2 germline mutational spectrum and evidence for genetic anticipation in Portuguese breast/ovarian cancer families[END_REF], raised the deleterious mutation detection rate to 30% (30/100) of all cases studied and to 37% (29/79) of those probands with a family history of breast/ovarian cancer. The c.156_157insAlu BRCA2 mutation accounts for 27% of the 30 probands with BRCA1/BRCA2 pathogenic mutations and Analysis of RNA transcripts RNA splicing was evaluated by amplifying the BRCA2 coding region from exon 1 through exon 6. When the products from these reactions were visualized on agarose gels, besides a normal transcript two shorter bands were common to both the patients and controls: a transcript representing an in frame skip of exon 3 (BRCA2-Dex3) and a transcript with deletion of exon 3 plus 109 bp of exon 4 (Fig. 1). However, the band representing the BRCA2-Dex3 transcript had much higher intensity in the patients with the c.156_157insAlu BRCA2 mutation. When these fragments were excised from the gel, amplified, and sequenced, and because the PCR were designed to generate fragments that contained the c.-25G[A polymorphism, we could demonstrate that the controls heterozygous for the c.-25G[A polymorphism showed heterozygosity in the full length and the BRCA2-Dex3 transcripts, whereas the patients heterozygous for that polymorphism presented mainly one allele in the full length transcript and the other allele in the BRCA2-Dex3 transcript (Fig. 1). These findings indicate that the normal mRNA for BRCA2 is produced only from one chromosome in the patient.

G/G G/G A/G A/G A/G G/G G/G G/G G/G A/G G/G G/G G/G G/G BRCA2 D13S1701

Haplotype analysis

Haplotypes were phased for most of the markers for only three out of the 14 families. The results of the haplotype analyses for the three informative families are shown in Fig. 2. In one informative family, the haplotype associated with the mutation could be inferred accurately as five carriers were genotyped. In the other two families the haplotype was inferred in most instances as two carriers per family were genotyped. One haplotype was found to segregate within two families (11 and 13). The shared haplotype covered eight markers, but varied for the D13S1701 and D13S1700 alleles.

In family 12 the disease associated alleles from the BRCA2 telomeric region were different from those associated with families 11 and 13. The genotypes found for the remaining probands are shown in Table 1 and the geographic origins of

Discussion

Our findings show that the specific detection of the c.156_157insAlu BRCA2 mutation in Portuguese breast/ ovarian cancer families allows a significant increase in the detection rate of BRCA1/BRCA2 mutations from 24.7% to 37% of those with a family history of cancer. This mutation rate is slightly above what has been reported in the majority of populations using similar criteria for referral to genetic counseling and testing [START_REF] Dı ´ez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF][START_REF] Claes | BRCA1 and BRCA2 germline mutation spectrum and frequencies in Belgian breast/ ovarian cancer families[END_REF][START_REF] Perkowska | BRCA1 and BRCA2 mutation analysis in breast-ovarian cancer families from northeastern Poland[END_REF][START_REF] Shih | BRCA1 and BRCA2 mutation frequency in women evaluated in a breast cancer risk evaluation clinic[END_REF], something that might reflect the fact that we are now able to detect a mutation that escaped conventional mutation screening techniques.

If we had used more strict selection criteria like those of the other major report on BRCA1/BRCA2 sixth being the c.156_157insAlu BRCA2 mutation [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF]), our mutation detection rate would have been 41%. Although this mutation rate can be seen as relatively high, it also indicates that other BRCA1/BRCA2 mutations undetectable by current-day screening methodologies may remain to be uncovered in high-risk breast/ovarian cancer families. On the other hand, five out of the 30 pathogenic mutations would have been missed had we used the generally recommended higher that 10% mutation probability criteria for genetic testing (8 of 30 if [25%). Careful selection of families after genetic counseling is therefore recommended to increase the chance of identifying all families that can benefit from genetic diagnosis of inherited predisposition to breast/ovarian cancer. Teugels et al. [START_REF] Teugels | De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes[END_REF] demonstrated that the c.156_157ins Alu mutation originates an in-frame deletion of exon 3 from the BRCA2 mRNA encoding a transcriptional activation domain. On the other hand, Dı ´ez et al. [START_REF] Dı ´ez | Caution should be used when interpreting alterations affecting the exon 3 of the BRCA2 gene in breast/ovarian cancer families[END_REF] issued a word of caution when interpreting as pathogenic alterations causing BRCA2 exon 3 skipping, as exon 3 splicing can also be detected in normal tissues. In order to better characterize the pathological and clinical significance of this mutation, we have looked for this mutation in disease-free controls, analyzed in more detail the mRNA in carriers and noncarriers, and reconstructed haplotypes in informative families. Three separate findings corroborate the conclusion that the c.156_157insAlu BRCA2 mutation is indeed deleterious. First, BRCA2 full length transcript is derived only from the wild type allele in patients carrying the c.156_157insAlu BRCA2 mutation, whereas the BRCA2-Dex3 transcript was transcribed mostly from the mutated allele. This was demonstrated using a heterozygous polymorphism to evaluate the relative contribution of each allele to normal and BRCA2-Dex3 transcripts in patients and controls [START_REF] Judkins | Single nucleotide polymorphisms in clinical genetic testing: the characterization of the clinical significance of genetic variants and their application in clinical research for BRCA1[END_REF], the latter showing both alleles giving rise to a small proportion of the BRCA2-Dex3 transcript. Second, if the abnormal transcript with the in-frame deletion of exon 3 is fully functional, as it was suggested by Dı ´ez et al. [START_REF] Dı ´ez | Caution should be used when interpreting alterations affecting the exon 3 of the BRCA2 gene in breast/ovarian cancer families[END_REF], then this variant would have to be considered a polymorphism given its high frequency (8%) in families suspected to have hereditary predisposition to breast/ovarian cancer. However, we did not find the c.156_157insAlu in 262 chromosomes from healthy blood donors, allowing us to dismiss the hypothesis that it is a neutral polymorphism. Third, we could show co-segregation of this alteration and disease in six affected (but not in healthy) family members belonging to the informative families. These pieces of information allow us to confidently conclude that the c.156_157insAlu BRCA2 mutation is associated with the hereditary predisposition to breast/ovarian cancer in these families.

Alu elements are believed to insert randomly in the host genome, so the fact that this particular Alu is inserted at exactly the same position in BRCA2 exon 3 in all 14 families is by itself sufficient evidence that this represents a founder effect and not a recurrent mutation [START_REF] Rowold | Alu elements and the human genome[END_REF]. Additionally, in all c.156_157insAlu mutation carriers heterozygous for the c.-25G[A polymorphism there was a physical association between the mutation and the allele that did not contribute to wild type transcript. Nevertheless, analysis of the chromosomal background upon which a mutation occurs can be used to reconstruct its origin and age. We have performed haplotype reconstruction with ten microsatellite markers in three informative families. In two of the three families one haplotype was shared for all but two markers, whereas in the third family all markers telomeric to BRCA2 differed from that observed in the other two. A recombination event around BRCA2 could account for the latter haplotype difference, whereas the variance in allele size of one tetranucleotide repeat unit within the D13S1701 allele in two families may have been The open circles indicate the origin of the 14 families here reported and the black circles the families previously described by Machado et al. [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF] caused by slippage during DNA replication of this repeat sequence. It is not possible to determine if this marker exhibits a higher mutation rate than other markers in the BRCA2 region, as it was suggested by other studies [START_REF] Oros | Haplotype analysis suggest common founders in carriers of the recurrent BRCA2 mutation, 3398delAAAAG, in French Canadian hereditary breast and/ovarian cancer families[END_REF], or if that mutation occurred early on during in the spreading of the population carrying the c.156_157insAlu BRCA2 mutation. If we compare the D13S1701 genotypes for all 14 c.156_157insAlu positive probands, it is tempting to say that all mutation carriers with unphased genotypes harbored the same 299 allele, but in two out of three informative families we observed that this allele did not segregate with the mutation. The possibility exists that this variation in D13S1701 is more frequent than that observed by Machado et al. [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF], so care should be taken when constructing haplotypes from independent families. If that is so, estimation based on the closest recombinant markers could greatly underestimate mutation age. Although Machado et al. [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF] claim that the c.156_157insAlu BRCA2 mutation has it origin 2,400-2,600 years ago in what is now central/southern Portugal, it is likely that it merely reflects their target population for genetic testing. In fact, we here show that the c.156_157insAlu BRCA2 mutation accounts for 27% of the BRCA1/BRCA2 deleterious mutations in northern/central Portugal (our main target population; Fig. 3), as compared to 16% in central/ southern Portugal [START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF]. Although all known c.156_157insAlu BRCA2 mutations have so far been identified in Portuguese breast/ovarian cancer families, we cannot rule out that this mutation may be present in other populations and so it might have been introduced in Iberian Peninsula from somewhere else.

In summary, our study provides significantly new evidence to substantiate the pathogenicity of the c.156_157insAlu BRCA2 rearrangement and shows that this mutation is responsible for more that one-fourth of deleterious BRCA1/BRCA2 mutations in northern/central Portugal. To further evaluate whether or not it constitutes a population-specific founder mutation, one needs to perform the screening of the c.156_157insAlu BRCA2 rearrangement in other populations, which will allow for a more accurate estimation of its distribution and age.

Fig. 1

 1 Fig. 1 RT-PCR analyses from exon 1 to exon 6 of the BRCA2 gene in c.156_157insAlu BRCA2 mutation carriers and controls. (a) Lane 1, 100 bp DNA standard. Lanes 2 and 3, patients with the c.156_157insAlu BRCA2 mutation. Lane 3, wild type control sample. A1 represents the full length transcript, A2 the BRCA2-Dex3 transcript and A3 the transcript with deletion of exon 3 plus 109 bp of exon 4 (data not shown). (b) Sequencing of the RT-PCR product (A1 band top and A2 band bottom) from a control individual with the c.-25G[A germline polymorphism (noted by an arrow), showing heterozygosity in the full length and the BRCA2-Dex3 transcripts. (c) Sequencing of the RT-PCR product (A1 band top and A2 band bottom) from a c.156_157insAlu BRCA2 mutation carrier with the c.-25G[A germline polymorphism (noted by an arrow), showing mainly one allele in the full length transcript and the other allele in the BRCA2-Dex3 transcript

Fig. 3

 3 Fig. 3 Map of Portugal showing the known geographical origin of the families with the c.156_157insAlu BRCA2 germline mutation.The open circles indicate the origin of the 14 families here reported and the black circles the families previously described by Machado et al.[START_REF] Machado | Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: evidence for a founder effect and analysis of the associated phenotypes[END_REF]

Table 1

 1 Microsatellite marker genotypes used to reconstruct the haplotypes in probands with the c.156_157insAlu BRCA2

	mutation

  53% of the 15 deleterious BRCA2 mutations identified in that series of 100 probands from breast/ovarian cancer families. Furthermore, screening of the c.156_157insAlu BRCA2 mutation in 130 additional probands in whom no germline BRCA1/BRCA2 mutations have been detected by DGGE and MLPA (data not shown) revealed six additional families with this mutation. Of the 14 probands with the c.156_157insAlu BRCA2 mutation, eight belonged to C2BC families, three to MBC families, two to BC + OC families, and one was a 1BC + OC patient. The mutation was also detected in six affected family members but neither in eight healthy relatives nor in the 262 chromosomes of the control population.

	299/299 295/299 299/299 291/299 295/299 291/299 295/299 295/299 291/299 299/299 295/299 299/307 299/307 291/299	230/230 230/240 230/240 230/230 226/230 226/230 230/230 230/230 230/240 230/230 230/230 226/226 230/240 230/230	242/250 242/250 242/250 242/246 240/242 242/250 236/246 242/250 242/250 242/258 242/250 250/252 242/250 242/254	228/234 228/234 228/228 234/236 228/232 228/234 234/234 228/232 234/234 228/234 228/234 234/234 228/234 234/238	144/144 144/144 144/144 148/148 144/144 144/144 148/166 144/148 144/150 144/148 144/144 148/150 144/150 144/148	156/158 144/156 156/158 144/150 156/158 156/158 144/150 144/150 144/144 146/158 150/158 144/158 146/158 144/156
	TTCC	TG	AC	TG	GT	TG
	0.17	D13S171 0.28	D13S1695 0.55	D13S1694 0.8	D13S310 0.81	D13S267 1.29	tel

Breast Cancer Res Treat (2009) 114:31-38