Isabel Chirivella

Begoña Bermejo

Amelia Insa

Alejandro Pérez-Fidalgo

Ana E Magro

Susana Rosello

Elisa García-Garre

Paloma Martín

Ana Bosch

Ana Lluch
email: ana.lluch@uv.es

A E Begon

Bermejo Ae

Amelia Insa Ae

Alejandro Pe ´rez-Fidalgo

Ana Ae

Rosello Susana

Elisa Ae

Garcı ´a-Garre

Paloma Ae

Martı

Ana Ae

Bosch

Á B Bermejo

Á A Insa

Á A Pe ´rez-Fidalgo

Á A Magro

Á S Rosello

Á E Garcı ´a-Garre

Á P Martı

Á A Bosch

Á A Lluch

Optimal delivery of anthracycline-based chemotherapy in the adjuvant setting improves outcome of breast cancer patients

Keywords: Dose-response effect, Anthracycline-based, Non-taxane, Adjuvant chemotherapy

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

Introduction

In the 1970s, it was first reported that patients with early breast cancer who received cyclophosphamide, methotrexate and fluorouracil (CMF) at full doses in the adjuvant setting had an improved disease-free survival (DFS) and overall survival (OS) compared with those patients who only underwent surgery [START_REF] Bonadonna | Combination chemotherapy as an adjuvant treatment in operable breast cancer[END_REF]. Three decades later, the benefits obtained by this patient group were still evident [START_REF] Bonadonna | 30 years' follow up of randomised studies of adjuvant CMF in operable breast cancer: cohort study[END_REF]. Later, the Spanish Breast Cancer Research Group (GEICAM) demonstrated that six cycles of fluorouracil, doxorubicin and cyclophosphamide (FAC) were superior to six cycles of CMF in the adjuvant treatment of operable breast cancer [START_REF] Martin | Doxorubicin in combination with fluorouracil and cyclophosphamide (i.v. FAC regimen, day 1, 21) versus methotrexate in combination with fluorouracil and cyclophosphamide (i.v. CMF regimen, day 1, 21) as adjuvant chemotherapy for operable breast cancer: a study by the GEICAM group[END_REF]. These findings were confirmed by the meta-analysis of the Early Breast Trialist's Collaborative Group (EBTCG), in which anthracycline-based combinations reduced the mortality of breast cancer patients, irrespective of the estrogen receptor and/or nodal status, in comparison with CMF schedules [START_REF]Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials[END_REF].

In spite of the strong evidence that supports the use of adjuvant chemotherapy, conflicting results have been reported about the relationship between chemotherapy dose and treatment response [START_REF] Henderson | Dose-response in the treatment of breast cancer: a critical review[END_REF]. Bonnadona et al. were the first to report a clear dose-response effect during the administration of CMF, particularly in the adjuvant setting [START_REF] Bonadonna | Dose-response effect of adjuvant chemotherapy in breast cancer[END_REF]. From that point, several retrospective analyses found similar improvements in outcomes [START_REF] Senn | Adjuvant chemoimmunotherapy with LMF + BCG in node-negative and node-positive breast cancer patients: 10 year results[END_REF][START_REF] Velez-Garcia | Postsurgical adjuvant chemotherapy with or without radiotherapy in women with breast cancer and positive axillary nodes: a South-Eastern Cancer Study Group (SEG) Trial[END_REF], whereas others reported no dose-response effect [START_REF] Howell | A controlled trial of adjuvant chemotherapy with melphalan versus cyclophosphamide, methotrexate, and fluorouracil for breast cancer[END_REF][START_REF] Ahmann | A preliminary assessment of factors associated with recurrent disease in a surgical adjuvant clinical trial for patients with breast cancer with special emphasis on the aggressiveness of therapy[END_REF]. However, the dose groups used in these analyses frequently varied from those used originally by Bonadonna. Using the same dose level groupings (C85%, 65-84%, and\65%) as the first study, a later study again showed that the dose level of CMF administered was an important prognosis factor in node-positive breast cancer patients. It was concluded that patients who received less than 65% of the prescribed CMF dose had a significantly worse outcome [START_REF] Colleoni | Doseresponse effect of adjuvant cyclophosphamide, methotrexate, 5-fluorouracil (CMF) in node-positive breast cancer. International Breast Cancer Study Group[END_REF].

Based on these findings, we considered it to be of interest to evaluate whether this dose-response effect reported for CMF administration was also true with anthracycline-based non-taxane schedules in the adjuvant treatment of early breast cancer patients. We retrospectively analysed the impact of chemotherapy delivery on DFS and OS at 10 years. The variables selected to assess chemotherapy delivery included not only the dose level administered, but also administration delays.

Methods

Study design

This was a retrospective database analysis performed in June 2007. Database was created in 1980. Since then, clinical data for all patients treated at the Hospital Clinico Universitario of Valencia (Spain) for breast cancer have been entered into this database.

Confidentiality of patients' data was maintained throughout the study. Data extraction was performed by two data managers. Four independent medical oncologists verified 15% of the extracted data against the original database to confirm the accuracy of the data extraction.

Study procedures

Inclusion criteria to be included in this retrospective analysis were to have a diagnosis of early breast cancer (stages I-IIIA) from January 1980 through December 2000, the primary treatment of the disease being a surgical procedure and an anthracycline-based non-taxane chemotherapy in the adjuvant setting. For each selected patient, the database included information about the schedule of chemotherapy given together with drug dosage and the quantitative and qualitative description of treatment delays and/or reductions performed. Patients who had to interrupt chemotherapy due to toxicity or any other reason were excluded from this analysis. To avoid poor quality data, patients not diagnosed in this hospital were also excluded.

In the adjuvant setting, patients could have received any of the following schedules: 3 cycles of doxorubicin (A, 30 mg/m 2 ) plus cyclophosphamide (C, 600 mg/m 2 ) every 21 days if they were staged with a T1N0M0 disease (39 AC); 8 cycles of the same schedule if they had a T2N0M0 disease (89 AC); or 3 cycles of A (50 mg/m 2 ), C (600 mg/m 2 ), 5-fluorouracil (F, 600 mg/m 2 ) and methotrexate (M, 30 mg/m 2 ) every 21 days followed by 5 cycles of FAC (600/40/600 mg/m 2 ) every 21 days (39 FAC-M ? 59 FAC) for those patients who had T3N0 and T1-1N1-2 disease. Tamoxifen was started after chemotherapy completion and continued for 5 years in all patients with hormone receptor-positive tumors. Radiotherapy was initiated within 4 weeks after the last cycle of chemotherapy in all patients who had undergone breastconserving surgery or had got a tumor size [5 cm or C4 lymph nodes affected Standard dose modification criteria were in place at the hospital. If the neutrophil count fell to \1.5 9 10 9 /l or platelets\100 9 10 9 /l, chemotherapy was delayed 5-7 days, but reintroduced upon recovery (neutrophil count [ 1.5 9 10 9 /l or platelets [ 100 9 10 9 /l) without any dosage modification. Granulocyte colony-stimulating factor (G-CSF) support was not administered to any of these patients. Chemotherapy was also delayed in the case of any grade 3/4 non-hematologic toxicity, and restarted when it improved to grade 1. If toxicity persisted, dosages were reduced to 75%. If toxicity still persisted after three weeks, chemotherapy was definitely interrupted.

Data extracted included patient age, year of diagnosis, tumor stage, histologic grade, as well as menopausal and hormonal receptor status. Other treatment-related data were extracted such as the mean percentage of administered dose throughout the cycles, the number of delayed chemotherapy cycles, the number of days of delay in chemotherapy administration and the last day of follow-up and any event (disease recurrence or death) that occurred to the patient during the follow-up period.

Statistical analysis

The primary objective of this analysis was to evaluate whether the optimal delivery of an anthracycline-based chemotherapy in the adjuvant setting could impact on DFS and OS at 10 years. OS at 10 years was defined as being alive 10 years after cancer diagnosis. Similarly, DFS at 10 years was defined as being alive, with no disease recurrence, 10 years after cancer diagnosis.

Three variables were chosen to assess the delivery of chemotherapy to the patient: the number of delayed cycles, the number of delayed days, and the RDI administered. The number of delayed cycles was based on whether the patient had more than 2 cycles with C3 days of delay with respect to the planned schedule (B2 delayed cycles, [2 delayed cycles). The number of delayed days during treatment administration was based on whether the patient's chemotherapy had to be delayed more than 14 days overall or not (\15 delayed days, C15 delayed days). Finally, the RDI was based on whether or not the patient's RDI was less than 85% (C85%, \85%). RDI was calculated as the mean percentage of administered dose throughout the entire treatment multiplied by the ratio of the number of treatment days as planned to the number of treatment days as planned plus the number of delayed days.

Descriptive statistical data evaluation was based on medians and standard deviations (SD) for continuous endpoints, and on absolute and relative frequencies with 95% confidence intervals (CI) for categorical variables. Dichotomous categorical variables were compared using a 2-sided log rank test. The survival analysis consisted of an overall analysis of DFS and OS and a Kaplan-Meier estimation for each outcome according to the three study variables. Stratification of the Kaplan-Meier estimation by previously defined clinically relevant covariables such as the number of affected lymph nodes (0, 1-3, 4-10, [START_REF] Ahmann | A preliminary assessment of factors associated with recurrent disease in a surgical adjuvant clinical trial for patients with breast cancer with special emphasis on the aggressiveness of therapy[END_REF] and the hormonal status (estrogen and progesterone receptors) was also performed. Finally, a Cox regression analysis to investigate the effect of study variables on DFS and OS while controlling previously defined covariables was performed. Statistical significance was set at P \ 0.05.

Results

Of the 1,790 breast cancer patients included in the initial database, 793 were included in this analysis. The reasons to exclude 997 patients were the following: 263 (26%) patients because of the lack of important clinical data, 232 (23%) were participating in different clinical trials with taxanes or high-dose chemotherapy, 229 (23%) were treated with hormonal therapy only, 178 (18%) received neoadjuvant chemotherapy and 95 (10%) were treated with CMF in the adjuvant setting. Patient characteristics at baseline are shown in Table 1. The majority of patients were diagnosed and treated from 1985 to 1989 (36%) and from 1990 to 1994 (31%), and had stage II disease (50%) with a histologic grade II (74%). Forty-seven percent of patients did not have lymph node involvement, whereas 32% had 1-3, 17% had 4-10, and 4% of patients had more than 10 nodes affected. An almost equal percentage of patients were pre-or perimenopausal (48%) and postmenopausal (52%).

Impact of suboptimal chemotherapy delivery on DFS and OS

With a median follow-up period of almost 10 years for the entire study population, the median DFS was 8.30 years (95% CI: 7.95-9.10) and the median OS was 9.42 years (95% CI: 8.96-10.11). At 10 years, the probability of surviving without recurrence of the disease was 66% (95% CI: 63-70) and the probability of being alive, with or without disease recurrence, was 77% (95% CI: 73-80).

As shown in Table 2, the Kaplan-Meier DFS was significantly affected by the number of delayed cycles (HR: 2.07, 95% CI: 1.61-2.67, P \ 0.0001), the number of delayed days (HR: 1.77, 95% CI: 1.38-2.28, P \ 0.0001) and the RDI (HR: 1.65, 95% CI: 1.18-2.30, P = 0.0029). Thus, patients with delays in scheduled chemotherapy (counted as C15 delayed days or as [2 delayed cycles) as well as patients who received a reduced RDI (\85%) had significantly lower probability of survival without recurrence of the disease at 10 years (Fig. 1). The Kaplan-Meier DFS analysis was statistically significant when data was stratified by the number of lymph nodes affected and the hormonal receptor status (estrogen or progesterone receptors), with P values in all cases \0.05.

The Kaplan-Meier OS was also significantly affected by the number of delayed cycles (HR: 1.70, 95% CI: 1.24-2.33, P = 0.0008), the number of delayed days (HR: 1.49, 95% CI: 1.09-2.04, P = 0.0115) and the RDI (HR: 1.73, 95% CI: 1.17-2.55, P = 0.0055). Patients who experienced treatment delays (counted as C15 delayed days or [2 delayed cycles) as well as patients who received a reduced RDI (\85%) had a significantly lower probability of being alive, with or without recurrence of the disease, at 10 years (Fig. 2). When the data obtained from the Kaplan-Meier OS analysis were stratified by the number of nodes affected and the hormonal receptor status, the only study variable that maintained its significance with any of the covariables evaluated was the number of delayed cycles (B2 cycles,[2 cycles). The number of delayed days (\15 days, C15 days) did not keep significance with any of the clinically relevant covariables. Lastly, RDI (C85%,\85%) had a statistically significant impact on OS when stratification was performed by the number of lymph nodes affected (HR: 1.61, 95% CI: 1.09-2.39, P = 0.0128), but not when stratification was performed by estrogen receptors (HR: 1.57, 95% CI: 0.98-2.51, P = 0.0559) or progesterone receptors (HR: 1.56, 95% CI: 0.98-2.49, P = 0.0592), although in both cases there was a clear trend.

Cox regression models for DFS and OS

As shown in Table 2, the Cox regression models showed that, when other clinically relevant disease characteristics such as number of nodes affected and hormonal receptor status were controlled, the three main study variables maintained significance on DFS (for [2 cycles, HR: 1.64, 95% CI: 1.21-2.22, P = 0.001), (for C15 days, HR: 1.41, 95% CI: 1.04-1.90, P = 0.027), (for RDI \ 85%, HR: 1.57, 95% CI: 1.06-2.31, P = 0.023). However, when modeling OS by Cox regression analysis controlling for the clinically relevant disease characteristics, the three study variables did not show statistical significance.

Discussion

We have retrospectively evaluated whether the optimal delivery of an anthracycline-based non-taxane chemotherapy could improve DFS and OS in the adjuvant setting of early breast cancer patients. Our results showed that those patients with delays in scheduled chemotherapy or who received a reduced RDI had significantly lower probability of being alive, with or without recurrence of the disease, at 10 years.

These results are similar to those observed by two previous retrospective analyses performed with CMF in the adjuvant setting of breast cancer [START_REF] Bonadonna | Dose-response effect of adjuvant chemotherapy in breast cancer[END_REF][START_REF] Colleoni | Doseresponse effect of adjuvant cyclophosphamide, methotrexate, 5-fluorouracil (CMF) in node-positive breast cancer. International Breast Cancer Study Group[END_REF]. In the first study [START_REF] Bonadonna | Dose-response effect of adjuvant chemotherapy in breast cancer[END_REF], CMF was found to be effective when given in a full, or nearly full, dose (C85% of the planned dose). Within each dose level, the results were influenced by the number of axillary nodes involved but not by the menopausal status. In another study [START_REF] Colleoni | Doseresponse effect of adjuvant cyclophosphamide, methotrexate, 5-fluorouracil (CMF) in node-positive breast cancer. International Breast Cancer Study Group[END_REF], a better OS and DFS were observed in the intermediate dose level group (65-84%) in comparison with the lower (\65%) dose level group. These results were consistent within different patient subgroups (defined by menopausal status and estrogen receptor status). In the case of nodal status, differences remained only in the case of patients with 1-3 positive nodes.

In the present analysis, DFS remained statistically different for the three study variables within all patient subgroups (defined by the number of nodes affected and the hormonal receptor status). However, only the number of delayed cycles had a significant impact on OS within all patients subgroups.

Our results also support the idea that the modest results obtained with many cytotoxic agents in the adjuvant setting may be attributed, in part, to the sub-optimal administration of chemotherapy in which thresholds of dose intensity are not reached [START_REF] Gurney | How to calculate the dose of chemotherapy[END_REF][START_REF] Lyman | Incidence and predictors of low dose-intensity in adjuvant breast cancer chemotherapy: a nationwide study of community practices[END_REF][START_REF] Budman | Dose and schedule as determinants of outcomes in chemotherapy for breast cancer[END_REF]. In the specific case of obese patients, this seems to be a common practice that indeed has a detrimental effect on their outcome [START_REF] Poikonen | Effect of obesity on the leukocyte nadir in women treated with adjuvant cyclophosphamide, methotrexate, and fluorouracil dosed according to body surface area[END_REF][START_REF] Rosner | Relationship between toxicity and obesity in women receiving adjuvant chemotherapy for breast cancer: results from cancer and leukemia group B study 8541[END_REF][START_REF] Colleoni | Relation between chemotherapy dose, oestrogen receptor expression, and bodymass index[END_REF][START_REF] Goodwin | Body size and breast cancer prognosis: a critical review of the evidence[END_REF]. The practice of prolonged dose reductions or delays to restrict toxicity should be avoided [START_REF] Wood | Dose and dose intensity of adjuvant chemotherapy for stage II, node-positive breast carcinoma[END_REF]. In spite of the important results reported from this retrospective analysis, it is possible that there may be some bias [START_REF] Redmond | The methodologic dilemma in retrospectively correlating the amount of chemotherapy received in adjuvant therapy protocols with disease-free survival[END_REF]. In particular, patients who did not have to delay chemotherapy or to reduce RDI may have different clinical characteristics than those patients who did not. However, the Cox regression models performed during this study showed that, when the number of lymph nodes affected and the hormonal receptor status were controlled, the three study variables maintained their significance on DFS, although not for OS. Indeed, within the clinical characteristics that may affect our results, the number of lymph nodes remains the strongest predictive factor of breast cancer recurrence and survival [START_REF] Velez-Garcia | Postsurgical adjuvant chemotherapy with or without radiotherapy in women with breast cancer and positive axillary nodes: a South-Eastern Cancer Study Group (SEG) Trial[END_REF][START_REF] Ahmann | A preliminary assessment of factors associated with recurrent disease in a surgical adjuvant clinical trial for patients with breast cancer with special emphasis on the aggressiveness of therapy[END_REF].

In conclusion, the results of this retrospective analysis indicate that the dose-response effect is a crucial factor in the administration of anthracycline-based non-taxane schedules for the adjuvant treatment of early breast cancer. Thus, delays and/or reductions of chemotherapy should be avoided whenever possible to achieve the maximal benefit.

Fig. 1

 1 Fig. 1 Impact of study variables on disease-free survival: (a) Number of delayed cycles (B2 cycles, [2 cycles); (b) Number of delayed days (\15 days, C15 days); (c) RDI (C85%, \85%)

Fig. 2

 2 Fig. 2 Impact of study variables on overall survival: (a) Number of delayed cycles (B2 cycles, [2 cycles); (b) Number of delayed days (\15 days, C15 days); (c) RDI (C85%, \85%)

Table 1

 1 Patient characteristics at diagnosis

	Characteristics	N	%
	Age, years (n = 793)
	Median (range)	51 (21-79)
	Treatment period (n = 789)
	1980-1984	86	11
	1985-1989	285	36
	1990-1994	246	31
	1995-2000	172	22
	Tumor stage (n = 782)
	I	182	23
	II	388	50
	IIIA	212	27
	Histological grade (n = 684)
	I	6 5	9
	II	506	74
	III	113	17
	Lymph nodes involvement (n = 785)
	0	368	47
	1-3	248	32
	4-10	135	17
	[10	34	4
	Hormonal receptor status
	ER+ (n = 594)	332	56
	PR+ (n = 593)	305	51
	Menopausal status (n = 754)
	Pre-/Perimenopausal	360	48
	Postmenopausal	394	52
	Adjuvant chemotherapy (n = 793)
	3 cycles of AC	153	19
	8 cycles of AC	202	26
	3 cycles of FAC-M [ 5 cycles of FAC	438	55
	Chemotherapy administration
	Delayed cycles (n = 793)
	B2 delayed cycles	581	73
	[2 delayed cycles	212	27
	Delayed days (n = 793)
	\15 days	560	71
	C15 days	233	29
	RDI (n = 791)
	C85%	698	88
	\85%	93	12
	ER: estrogen receptor; PR: progesterone receptor; A: doxorubicin; C:
	cyclophosphamide; F: 5-fluorouracil; M: methotrexate; RDI: relative
	dose intensity

Table 2

 2

	Impact of
	chemotherapy delivery on
	disease-free survival and
	overall survival

Acknowledgements The authors acknowledge the work of Salutis (Barcelona, Spain) in carrying out the statistical analysis of the study, Dr. Beatriz Gil-Alberdi from HealthCo (Madrid, Spain) for assistance in the preparation of the manuscript, and Amgen SA Spain for financial support.


Patani Neill
email: neillpatani@hotmail.com

Kefah Ae

Mokbel

N Patani

Á K Mokbel

K Mokbel
email: kefahmokbel@hotmail.com

The clinical significance of sentinel lymph node micrometastasis in breast cancer

Keywords: Breast cancer, Sentinel lymph node, Micrometastasis, Sentinel lymph node biopsy, Axillary lymph node dissection, Prognosis, Recurrence, Morbidity, Mortality, Evidence

Introduction The advent of sentinel lymph node biopsy (SLNB) and improvements in histopathological and molecular analysis have increased the rate at which micrometastases (MM) are identified. However, their significance has been the subject of much debate. In this article we review the literature concerning axillary lymph node (ALN) MM, with particular reference to SLNB. The controversies regarding histopathological assessment, clinical relevance and management implications are discussed. Methods Literature review facilitated by Medline and PubMed databases. Results Published studies have reported divergent results regarding the significance and implications of ALN MM in general and sentinel lymph node (SLN) MM in particular. Some studies demonstrate no associations, whilst others have found these to be indicators of poor prognosis, associated with non-SLN involvement, in addition to local and distant failure. Absolute consensus regarding the optimal analytical technique for SLNs has yet to be reached, particularly concerning immunohistochemical (IHC) techniques targeting cytokeratins and the utility of contemporary molecular analysis. Conclusion SLN MM are likely to represent an incremental detriment to prognosis and increased risk of non-SLN involvement, despite only modest up-staging within current classification systems. In the absence of level-1 guidance concerning the management of women with SLN MM, each case requires discussion with regard to other tumour and patient related factors in the context of the multidisciplinary team. Randomized studies are required to evaluate the prognostic significance and optimal management of each category of tumour burden within the SLN. The identification of MM remains highly dependent on the analytical technique employed and there exists potential for stage migration and impact on management decisions.

Introduction and background

ALN status at the time of diagnosis remains one of the most important prognostic indicators for women with breast cancer (BC) [START_REF] Carter | Relation of tumor size, lymph node status, and survival in 24, 740 breast cancer cases[END_REF]. In addition, lymph node status can influence management decisions regarding adjuvant local and/or systemic therapies. Axillary lymph node dissection (ALND) remains the gold standard for axillary staging, however the morbidity of this procedure can be high. Large randomized controlled trials have confirmed the efficacy and reduced morbidity associated with sentinel lymph node biopsy (SLNB) as an axillary staging procedure [START_REF] Veronesi | A randomized comparison of sentinel-node biopsy with routine axillary dissection in breast cancer[END_REF]. SLNB now represents the standard of care for women with early stage BC which is clinically node negative. SLNB is not routinely necessary for patients with ductal carcinoma in-situ (DCIS), although it has been offered to individuals undergoing mastectomy or those with large, palpable, highgrade or recurrent lesions, in view of the risk of accompanying invasive disease [START_REF] Patani | Current management of DCIS: a review[END_REF]. In patients where the sentinel lymph node (SLN) is free of tumour, ALND is considered unnecessary. In cases where there is evidence of SLN involvement with tumour, axillary treatment is indicated, either in the form of ALND or radiotherapy (RT). The aim of such treatment is to reduce the risk of local recurrence (LR) within the axilla.

In addition to the patient centered advantages of SLNB, the procedure has significantly reduced the number of lymph nodes sent to the pathologist for staging purposes, from an average of 15-20 from ALND to 1-3 nodes from SLNB. The analysis of this material has also been facilitated by accompanying advances in molecular biology. In combination, these facts have allowed the pathologist to examine each lymph node in greater detail using a variety of techniques. Despite incremental improvements in sensitivity and specificity, the outcome of histopathological analysis is not always binary. This can have potentially significant implications, both for the surgical and adjuvant treatment of patients. The majority of SLNs can be classified as positive or negative, based on the presence or absence of macrometastasis. The management of these patients is supported by a large evidence base. However, a significant minority of SLNs are found to contain a micrometastasis (MM) or isolated tumour cells (ITC) (Figs. 1 and2). These terms are defined by the American Joint Committee on Cancer (AJCC) as follows: macrometastasis 'greater than 2.0 mm', MM 'greater than 0.2 mm but not larger than 2.0 mm' and ITC 'no larger than 0.2 mm' [START_REF]WHO histological classification of tumours of the breast[END_REF][START_REF] Greene | AJCC Cancer Staging Manual[END_REF]. Small volume disease has also been incorporated into the TNM (UICC) classification and staging system, with pN1a or pNmi reflecting the presence of MM and pN0(i+) representing ITC [START_REF] Greene | AJCC Cancer Staging Manual[END_REF]. However, the significance of SLNs which contain MM and/or ITC in the absence of macrometastasis has been the subject of much debate. In this article we review the literature concerning ALN MM, with particular reference to SLNB. The controversies regarding histopathological assessment, clinical relevance and management implications are discussed.

Search strategy and selection criteria

Articles were identified by searches of MEDLINE and PubMed up to October 2007 using the terms: ''breast cancer'' or ''sentinel lymph node'' or ''axillary lymph node'' or ''lymph node'' and ''micrometastasis'' or ''micrometastases'' or ''micro metastasis'' or ''micro metastases'' or ''micro-metastasis'' or ''micro-metastases'' and ''evidence'' or ''prognosis'' or ''morbidity'' or ''mortality'' or ''recurrence''. Studies identified were screened for those that focused on SLN micrometastasis. All randomized controlled trials and large retrospective series were included. The reference articles in this review were selected to provide a balanced and representative overview of a complex subject with an extensive base of published work.

Analysis of the sentinel lymph node

Various methods of histopathological analysis have been described in the literature and technological advances continue to drive the evolution of this process. Analysis of the SLN is often completed in two stages, firstly, intraoperative assessment is performed in order to provide immediate guidance to the surgeon with regard to the need for ALND. This is followed by the delayed definitive examination of permanent sections.

Intra-operative analysis of the SLN Intra-operative assessment often involves frozen section analysis [START_REF] Khalifa | The accuracy of intraoperative frozen section analysis of the sentinel lymph nodes during breast cancer surgery[END_REF], based on haematoxylin and eosin staining (HES) or the cytological assessment of touch-imprints [START_REF] Tew | Metaanalysis of sentinel node imprint cytology in breast cancer[END_REF]. Intra-operative frozen section analysis is cost effective and affords many advantages to both the patient and the surgeon. However, the technique is more demanding, requiring a skilled pathologist, and less accurate than formal examination of fixed material. In view of the falsenegative rate associated with intra-operative techniques and subjective variations relating to their interpretation, attempts have been made to create more sensitive, accurate and objective methods of rapid detection. The intention is to avoid situations where the final histology differs from the intra-operative assessment, necessitating a further procedure for the patient. Recently, advances in molecular biology have enabled highly sensitive techniques to be applied to SLN analysis. Schoenfeld et al. [START_REF] Schoenfeld | Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction[END_REF] have used the reverse transcriptase polymerase chain reaction (RT-PCR) for cytokeratin 19 mRNA and found positive results in 15% of SLNs previously deemed to be negative. Kurosumi et al. [START_REF] Kurosumi | Significance and problems of histopathological examination and utility of real-time reverse transcriptase-polymerase chain reaction method for the detection of sentinel lymph node metastasis in breast cancer[END_REF] have recently reported the utility of a real-time RT-PCR method for the detection of SLN MM with a sensitivity and specificity of 89.5% and 96.7%, respectively, employing a frozen section based protocol. Using a one-step nucleic acid amplification (OSNA) based approach, Tsujimoto et al. [START_REF] Tsujimoto | One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients[END_REF] have established thresholds for cytokeratin 19 mRNA which can distinguish between macrometastasis and MM. Concordance with histopathological assessment was reported to be approximately 96% with no false positives. Ishikawa et al. [START_REF] Ishikawa | Transcription-reverse transcription concerted reaction and minimal residual disease in axillary sentinel nodes of breast cancer[END_REF] have recently employed a transcription-reverse transcription concerted reaction (TRC), targeted at carcinoembryonic antigen (CEA) mRNA, in order to reduce the false-negative rate associated with the routine analysis of SLNs. However, concerns exist regarding the utility of these approaches. Particular disadvantages include the fact that many of them do not permit morphologic correlation and often require destructive processing of the sample. False positive results can be caused by benign epithelial inclusions, benign mechanical transport of breast epithelium or extrinsic contamination and so there is potential for erroneous upstaging [START_REF] Treseler | Pathologic examination of the sentinel lymph node what is the best method?[END_REF][START_REF] Carter | Benign transport of breast epithelium into axillary lymph nodes after biopsy[END_REF]. On the other hand, these amplification based techniques offer the potential to detect metastatic disease which can not be identified by conventional staining or immunohistochemistry (IHC).

Definitive analysis of the SLN Advances have also been made in the definitive assessment of fixed SLNs. Efforts have been made to reduce significant differences in the pathological processing of lymph node blocks. Various methods of histopathological examination with HES have been described including single level analysis, step sectioning and serial sectioning [START_REF]European Working Group for Breast Screening Pathology (2003) Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines[END_REF]. More recently the development of IHC techniques targeting epithelial cytokeratins has revolutionized the definitive assessment of SLNs, particularly those which appear negative with conventional HES based analysis. These approaches have enabled the detection of otherwise occult metastases and additional extremely small lesions which would otherwise have been missed. The Philadelphia consensus meeting [START_REF] Schwartz | Proceedings of the consensus conference on the role of sentinel lymph node biopsy in carcinoma of the breast, April 19-22, 2001[END_REF] recommended that serial sections, less than 2 mm, were required for the reliable detection of macrometastasis. Significantly, the addition of IHC for cytokeratins (CK 19, CK AE1/3) was not routinely recommended. This is consistent with guidance from the American Society of Clinical Oncology [START_REF] Lyman | American society of clinical oncology guideline recommendations for sentinel lymph node biopsy in early breast cancer[END_REF]. The European recommendations [START_REF] Cserni | Pathologic work-up of sentinel lymph nodes in breast cancer: Review of current data to be considered for the formulation of guidelines[END_REF] suggest screening for MM ([0.2 mm) and advise against routine IHC analysis. Using a geometrical model of spherical metastases randomly distributed within a lymph node, Cserni [START_REF] Cserni | A model for determining the optimum histology of sentinel lymph nodes in breast cancer[END_REF] has suggested that sections taken 1 mm apart would provide a reasonable means of detecting almost all macrometastases. In addition, a step sectioning protocol with levels of 200 microns would be sufficient to screen for MM. Such narrow sections are technically more difficult and labour intensive to prepare. However, in support of this, serial sectioning of the entire SLN with cytokeratin staining at narrow intervals has been reported to identify occult metastases in an extra 25% of patents [START_REF] Dowlatshanhi | Occult metastases in sentinel nodes of 200 patients with operable breast cancer[END_REF]. This is consistent with other estimates in the literature which relate to the relative upstaging of previously negative lymph nodes [START_REF] Cote | Role of immunohistochemical detection of lymph-node metastases in management of breast cancer[END_REF][START_REF] Rivera | Minimal involvement of sentinel lymph node in breast carcinoma: prevailing concepts and challenging problems[END_REF]. The addition of the aforementioned PCR based methods can significantly increase the apparent 'false-negative' rate of conventional analysis [START_REF] Mori | Detection of cancer micrometastases in lymph nodes by reverse transcriptasepolymerase chain reaction[END_REF]. Multi-parameter flow cytometry, applied to permanently fixed sections, has also been shown to detect MM within SLNs [START_REF] Leers | Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer[END_REF]. Adjuncts to the conventional human assessment of specimens have been shown to be effective. Mesker et al. [START_REF] Mesker | Supervised automated microscopy increases sensitivity and efficiency of detection of sentinel node micrometastases in patients with breast cancer[END_REF] demonstrated that supervised automated microscopy can increase the sensitivity and efficiency of detection of SLN MM and Weaver et al. [START_REF] Weaver | Comparison of pathologist-detected and automated computer-assisted image analysis detected sentinel lymph node micrometastases in breast cancer[END_REF] have described the utility of automated computer-assisted image analysis.

The clinical significance of micrometastasis

Published studies have reported divergent results regarding the significance and implications of MM in BC. In the following, we consider the evidence for and against the clinical significance of ALN MM in general and SLN MM in particular.

Evidence in favour

Intuitively one expects that the presence of metastases, regardless of size, will have a detrimental effect on patient outcome. Furthermore, the indication for many adjuvant local and/or systemic therapies is now based on the estimated risk of local and/or distant failure. Proponents argue that minimal volume disease should be actively sought and factored into making management decisions. Some of the earliest studies comparing node negative women to those with ALN MM found associations with poorer prognosis [START_REF] Huvos | Significance of axillary macrometastases and micrometastases in mammary cancer[END_REF]. Fisher et al. [START_REF] Fisher | The evolution of breast cancer surgery: past, present, and future[END_REF][START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol No. 4). V. Significance of axillary nodal microand macrometastases[END_REF]] also reported higher rates of recurrence and death from BC in those with MM compared to those with negative nodes after an average follow-up of 4 years. More recently, Chen et al. [START_REF] Chen | The prognostic significance of micrometastases in breast cancer: a SEER population-based analysis[END_REF] have utilized the surveillance, epidemiology and end results (SEER) database, including over 200,000 patients, to demonstrate that nodal MM carries a prognosis intermediate to N0 and N1 disease, even after adjusting for patient and tumour related variables. The tumour burden of ALNs appears to be inversely correlated with prognosis, even when the tumour burden is very small [START_REF] Chen | The prognostic significance of micrometastases in breast cancer: a SEER population-based analysis[END_REF]. A large retrospective study involving over 10,000 patients with BC over 25 years found that patients with a solitary MM had a poorer prognosis than those without, although this only reached statistical significance in the group who did not have systemic adjuvant treatment. Furthermore, in patients who did not receive adjuvant systemic therapy, the prognosis of a single MM was comparable to a single macrometastasis [START_REF] Kuijt | The prognostic significance of axillary lymph-node micrometastases in breast cancer patients[END_REF]. Negative prognostic impact has also been reported by Reed et al. [START_REF] Reed | Occult metastases in axillary ymph nodes as a predictor of survival in node-negative breast carcinoma with long-term follow-up[END_REF]. In their study of 385 women with BC, poorer prognosis was found in those 45 cases with occult metastases compared to their truly node negative counterparts. Cote et al. [START_REF] Cote | Role of immunohistochemical detection of lymph-node metastases in management of breast cancer[END_REF] found that patients which were retrospectively found to have ALN MM, had a worse overall survival than others enrolled in the same adjuvant therapy trial who were node negative. A case controlled study of 96 patients with 15 year follow-up has found that occult MM in ALNs appears to predict subsequent distant metastases in patients with stage 1 BC [START_REF] Susnik | Occult micrometastases in axillary lymph nodes predict subsequent distant metastases in stage I breast cancer: a case-control study with 15-year followup[END_REF]. There are several other studies which have found a significantly poorer prognosis associated with MM [START_REF] Rosen | Axillary micro-and macrometastases in breast cancer: prognostic significance of tumor size[END_REF][START_REF] Clayton | Pathologic correlates of prognosis in lymph node-positive breast carcinomas[END_REF]. In a prospective study of women with stage 1 BC, the International Ludwig Breast Cancer Study Group reported a 5-year disease free survival of 58% in patients with MM compared with 74% in those without and overall survival rates of 79% and 88%, respectively [START_REF] International | Breast Cancer Study Group (1990) Prognostic importance of occult axillary lymph node micrometastases from breast cancers[END_REF]. In a study of 1,959 consecutive patients, Calleoni et al. [START_REF] Colleoni | Size of breast cancer metastases in axillary lymph nodes: clinical relevance of minimal lymph node involvement[END_REF] reported a significantly reduced disease-free survival and increased risk of distant metastases in patients with MM after a median follow-up of approximately 50 months. Umekita et al. [START_REF] Umekita | Clinical significance of occult micrometastases in axillary lymph nodes in ''node-negative'' breast cancer patients[END_REF] have demonstrated that disease free survival and overall survival, after a median follow-up of 98.5 months, were significantly better in patients without MM, 71% vs. 93% and 76% vs. 96%, respectively. Other reviews have also concluded that patients with MM detected by serial sections and IHC performed on HES negative nodes remained at a definite survival disadvantage [START_REF] Dowlatshahi | Lymph node micrometastases from breast carcinoma. Reviewing the dilemma[END_REF]. A metaanalysis of all studies from 1966 to 2003 has concluded that the majority of studies have found MM to be associated with a significantly poorer prognosis and in some cases this difference could only be identified after prolonged follow-up. The presence of MM should therefore be taken into consideration when making decisions about patient management [START_REF] Sakorafas | The clinical significance of axillary lymph node micrometastases in breast cancer[END_REF].

The advent of SLNB, alongside advances in analytical techniques, now enable the histopathologist to perform a more detailed evaluation of considerably fewer lymph nodes which are most likely to contain metastasis. Consequently, the diagnosis of SLN MM has significantly increased in frequency [START_REF] Chen | The prognostic significance of micrometastases in breast cancer: a SEER population-based analysis[END_REF]. However, the clinical significance and implication of such small volume disease has been the subject of much debate. Studies which have found SLN MM (as defined by the AJCC) to have particular prognostic utility are listed in Table 1. Mullenix et al. [START_REF] Mullenix | The association of cytokeratin-only-positive sentinel lymph nodes and subsequent metastases in breast cancer[END_REF] performed cytokeratin IHC on SLNs which appeared negative on HES staining and found that this small proportion of patients (4.4%) had a significantly increased risk of distant metastases. The risk of non-SLN involvement has been found to be significantly higher in women with SLN MM [START_REF] Van Rijk | Additional axillary metastases and stage migration in breast cancer patients with micrometastases or submicrometastases in sentinel lymph nodes[END_REF]. SLN MM and increasing MM size have been identified as significant predictors of non-SLN metastasis [START_REF] Viale | Predicting the risk for additional axillary metastases in patients with breast carcinoma and positive sentinel lymph node biopsy[END_REF]. This relationship can be considered as a continuous variable where SLN MM are significantly associated with non-SLN involvement [START_REF] Turner | Pathologic features associated with nonsentinel lymph node metastases in patients with metastatic breast carcinoma in a sentinel lymph node[END_REF]. In fact, Viale et al. [START_REF] Viale | Histologic detection and clinical implications of micrometastases in axillary sentinel lymph nodes for patients with breast carcinoma[END_REF] reported the overall rate of non-SLN positivity to be 16% in those patients with SLN MM less than 1 mm compared to 32% for those with SLN MM [ 1 mm. Dabbs et al. [START_REF] Dabbs | Sentinel lymph node micrometastasis as a predictor of axillary tumor burden[END_REF] found 10 of 112 patients (9%) with SLN MM to have non-SLN macrometastasis. In keeping with this, Leidenius et al. [START_REF] Leidenius | The prevalence of nonsentinel node metastases in breast cancer patients with sentinel node micrometastases[END_REF] found 25% of patients with SLN MM to have residual disease in the axilla. Schrenk et al. [START_REF] Schrenk | Prediction of non-sentinel lymph node status in breast cancer with a micrometastatic sentinel node[END_REF] have compared patients with SLN MM to those with SLN macrometastases undergoing ALND. Non-SLNs were found to be positive in 18% of those with SLN MM compared to 51.1% of those with macrometastasis. The size of MM and the presence of lymphovascular invasion were significantly related of the risk of finding non-SLN involvement in women with SLN MM. Even in patients with small MM (\0.5 mm) and no evidence of lymphovascular invasion, the risk of non-SLN involvement was 8.5%. In their study of 542 women with BC, Giard et al. [START_REF] Giard | Surgical implications of sentinel node with micrometastatic disease in invasive breast cancer[END_REF] identified 55 cases of SLN MM of which 40 underwent ALND and 6 were found to harbour additional metastatic foci. Similarly, den Bakker et al. [START_REF] Den Bakker | Non-sentinel lymph node involvement in patients with breast cancer and sentinel node micrometastasis; too early to abandon axillary clearance[END_REF] identified one or more involved non-SLN in approximately one-third of their 32 patients with SLN MM. Furthermore, within those with SLN MM no subgroup of tumour size or grade could be identified that did not have non-SLN involvement. Interestingly, Houvenaeghel et al. [START_REF] Houvenaeghel | Groupe des Chirurgiens de la Federation des Centres de Lutte Contre le Cancer. Micrometastases in sentinel lymph node in a multicentric study: predictive factors of nonsentinel lymph node involvement-Groupe des Chirurgiens de la Federation des Centres de Lutte Contre le Cancer[END_REF] found that the method employed for MM detection was a significant predictive factor for non-SLN involvement. Furthermore, Tommaso et al. [START_REF] Tommaso | Anatomic location of breast cancer micrometastasis in sentinel lymph node predicts axillary status[END_REF] demonstrated that the anatomical location of MM within the SLN was an independent predictor of non-SLN involvement, with intranodal lesions being more strongly associated with non-SLN metastasis than sinusal lesions.

Evidence against

Some authors have called into question the clinical relevance of such small metastatic foci [START_REF] Rutgers | Sentinel node micrometastasis in breast cancer[END_REF][START_REF] Broekhuizen | The incidence and significance of micrometastases in lymph nodes of patients with ductal carcinoma in situ and T1a carcinoma of the breast[END_REF], advising caution in their use when making decisions about offering or withholding established therapies. Many of these local and/or systemic therapies are based on a large body of level-1 evidence generated prior to the MM era. Considering the evidence against ALN MM in general, Tjan-Heijen et al. [START_REF] Tjan-Heijen | Micro-metastases in axillary lymph nodes: an increasing classification and treatment dilemma in breast cancer due to the introduction of the sentinel lymph node procedure[END_REF] in their review of eight studies, each with at least 100 patients and 5 years follow-up, concluded that there was insufficient evidence to confirm that MM were of prognostic utility. However, it is noteworthy that none of the studies reviewed employed the presently accepted definition of MM. Indeed, there are many examples in the literature where the definition of MM has differed; some authors have based the definition on the percentage of the cross-sectional area of the SLN involved, in addition to various references to occult metastasis [START_REF] Treseler | Pathologic examination of the sentinel lymph node what is the best method?[END_REF]. De Mascarel et al. [START_REF] De Mascarel | Prognostic significance of breast cancer axillary lymph node micrometastases assessed by two special techniques: reevaluation with longer follow-up[END_REF] compared 120 women with MM to those with N0 disease, median follow-up 7 years, and found that statistically significant associations with survival which were identified on univariate analysis did not remain after multivariate analysis. A lack of significant associations has also been reported by several other studies [START_REF] Colpaert | Early distant relapse in ''node-negative'' breast cancer patients is not predicted by occult axillary lymph node metastases, but by features of the primary tumour[END_REF][START_REF] Millis | Occult axillary lymph node metastases are of no prognostic significance in breast cancer[END_REF][START_REF] Kahn | Biological significance of occult micrometastases in histologically negative axillary lymph nodes in breast cancer patients using the recent American Joint Committee on Cancer breast cancer staging system[END_REF].

Several authors have also found MM to have limited utility with particular reference to SLNB. Studies which have not found SLN MM (as defined by the AJCC) to have clinical utility are listed in Table 2. A prospective study of 150 patients undergoing SLNB alone found no difference in the development of axillary recurrence between those with MM and those without after a median follow-up of 42 months [START_REF] Langer | Axillary recurrence rate in breast cancer patients with negative sentinel lymph node (SLN) or SLN micrometastases[END_REF]. In a study of 39 patients with SLN MM who did not undergo ALND, median follow-up of 32 months, Guenther et al. [START_REF] Guenther | Axillary dissection is not required for all patients with breast cancer and positive sentinel nodes[END_REF] have found no axillary recurrence and only a single case of distant failure. Similarly, in a series of 27 patients followed up for 30 months, Fant et al. [START_REF] Fant | Preliminary outcome analysis in patients with breast cancer and a positive sentinel lymph node who declined axillary dissection[END_REF] found no cases of axillary recurrence in those with SLN MM. In a smaller study including 15 patients with MM, 11 of which underwent ALND, no cases of local or systemic relapse were identified after a median follow-up of 13.5 months [START_REF] Liang | Is a completion axillary dissection indicated for micrometastases in the sentinel lymph node?[END_REF]. In a retrospective review, Rutledge et al. [START_REF] Rutledge | Sentinel node micrometastasis in breast carcinoma may not be an indication for complete axillary dissection[END_REF] found that the risk of finding non-SLN positivity was significantly lower in patients with SLN MM (3%) compared to SLN macrometastasis (63%). The authors concluded that careful assessment of risk versus benefit should be undertaken in view of the known morbidity of ALND. Hence in some studies the incidence of non-SLN metastasis in patients with SLN MM appears to be comparable to the false negative rate of SLNB itself. These findings are consistent with those of Fournier et al. [START_REF] Fournier | Micrometastasis in the sentinel lymph node of breast cancer does not mandate completion axillary dissection[END_REF] who reported only 1 case of non-SLN metastasis in 16 women undergoing ALND for SLN MM and no cases of local recurrence in any of the patients with SLN MM, including [START_REF] Wong | The use of cytokeratin staining in sentinel lymph node biopsy for breast cancer[END_REF] have found that SLNs positive only on IHC were not of prognostic significance. However, it is noteworthy that many of the studies which have failed to assign significance to SLN MM are comparatively small in patient number and hence may not have been adequately powered to identify any difference. In some cases the length of follow-up may also have been inadequate for the detection of local or distant recurrence.

In DCIS, IHC can identify MM in up to 9% of patients [START_REF] Klauber-Demore | Sentinel lymph node biopsy: Is it indicated in patients with high-risk ductal carcinoma-insitu and ductal carcinoma-in-situ with microinvasion?[END_REF] which is clearly in excess of the accepted rate of lymph node involvement. In another study of 102 patients with DCIS and negative axillary nodes, MM were identified in 13% on retrospective analysis. After 10-28 years of follow-up, the overall disease recurrence rate was 12% although none of these patients had been found to harbour MM and the recurrence site was not found to be the axilla in any case [START_REF] Lara | The relevance of occult axillary micrometastasis in ductal carcinoma in situ: a clinicopathologic study with long-term follow-up[END_REF]. The low rate of local recurrence and excellent overall survival in these patients has been suggested to provide further circumstantial evidence against the significance of MM. However, DCIS is a clinically heterogeneous lesion, the natural history of which remains elusive [START_REF] Patani | Current management of DCIS: a review[END_REF]. Hence, it may not be appropriate to make inferences about MM, given the extent to which the tumour biology of DCIS is likely to differ from BC.

Discussion

The last two decades have seen a paradigm shift in the management of women with breast cancer. Late presentation, delayed diagnosis and radical ablative surgery to the breast and axilla have been replaced by an effective national screening programme, prompt diagnosis, breast conservation and a more targeted approach to the axilla. One of the key drivers of this change has been an improved understanding of the natural history and tumour biology of BC. The 'mechanistic' model for progression hypothesized the centrifugal spread of metastases from station to station in a cumulative manner and this provided the justification for type of surgery advocated by Halsted [START_REF] Bland | The Halsted mastectomy: present illness and past history[END_REF]. More recently, we have come to accept a more 'biological' model which considers BC as a systemic disease with disseminated MM at the time of diagnosis [START_REF] Fisher | The evolution of breast cancer surgery: past, present, and future[END_REF][START_REF] Fisher | Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol No. 4). V. Significance of axillary nodal microand macrometastases[END_REF]. It has been suggested that the behavior of these MM is a key determinant of overall patient prognosis. Hence, modern surgical intervention aims to provide local control and no longer aspires to eradicate all disease. It is noteworthy that additional foci of carcinoma have been identified in over 60% of mastectomy specimens, approximately 80% of which lie outside the index quadrant [START_REF] Vaidya | Multicentricity of breast cancer: whole organ analysis and clinical implications[END_REF][START_REF] Baum | Multicentricity and recurrence of breast cancer[END_REF]. Landmark trials such as the NSABP-B06 suggest that following breast conserving surgery, such remote foci may be adequately treated by adjuvant radiotherapy [START_REF] Fisher | Twenty-year followup of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF]. The implication is that these incidental lesions may be of low malignant potential and uncertain clinical relevance. By providing local control, surgical intervention reduces patient morbidity in the long term and supplies material for staging purposes. The analysis of surgical specimens serves to categorize patients into those with 'good' or 'poor' prognosis and their risk of local or distant failure provides the justification for adjuvant local or systemic treatments. These therapies can target systemic micrometastatic disease and thereby afford improvements in survival. Although the concept of SNLB is largely a product of the 'mechanistic' model, in modern surgical practice it is employed purely as a staging procedure to guide ALND or axillary RT. Advances in molecular biology and IHC, combined with the practicality of performing a more detailed analysis on a single SLN, have increased the likelihood of finding MM. In fact the frequency of this diagnosis has increased substantially and is likely to continue doing so with each advance in analytical techniques. It seems somewhat paradoxical that women are now presenting with earlier stage primary tumours and yet there is a trend to increase the apparent stage at diagnosis by improvements in SLNB analysis. The impetus to determine the significance these lesions has also increased. MM found within the SLN could influence decisions regarding the further management of the axilla. However, in the era when ALND was the standard of care, MM found incidentally within the clearance specimen would be unlikely to have a significant impact on patient management. Controversy exists surrounding the best management of patients with SLN MM, in terms of further axillary surgery or radiotherapy and/or systemic adjuvant treatment. The fundamental question arises as to why we should consider treating micrometastatic disease within the SLN differently to that found elsewhere. It may be rational to rely on systemic adjuvant treatments to perform in the axilla as they do elsewhere and SLN MM could simply represent an indication for systemic adjuvant therapy. The counter argument is that ALND or axillary radiotherapy should reduce loco-regional recurrence in patients with SLN MM, given their association with non-SLN involvement. Locoregional recurrence can be particularly problematic to manage and is associated with poor prognosis. In fact, all reviewed studies including more than 70 patients with SLN MM support the argument that such small volume disease should be actively sought and incorporated into our axillary management strategy (Table 1 and2). The added value of further axillary surgery in the form of ALND in women with SLN MM, now needs to be formally quantified by appropriately powered studies. The relative reduction in the number of ALNDs performed over the last decade is supported by a large evidence base and improvements in the detection of SLN MM have the potential to drive management back towards an increased use of ALND. Since SLN MM are most commonly confirmed following complete pathological assessment, any further axillary surgery would need to take place on a second occasion.

An alternative could be to offer patients with SLN MM axillary radiotherapy. There is some evidence to support the management of women who are clinically node negative with axillary radiotherapy only. A study with 15 years of follow-up has found no difference in survival between clinically node negative women with early breast cancer managed with ALND or axillary radiotherapy, although a slightly higher rate of axillary recurrence was noted [START_REF] Louis-Sylvestre | Axillary treatment in conservative management of operable breast cancer: dissection or radiotherapy? Results of a randomized study with 15 years of follow-up[END_REF]. However this may be an equitable solution, in view of the fact that MM are likely to represent a lower risk of local and distant failure than macrometastasis and women could be spared the morbidity of ALND. However, at present, further intervention for women with SLN MM is primarily surgical in the form of ALND, rather than axillary radiotherapy. Randomised controlled trials are required to clarify the optimal management of patients with SLNs which contain MM in the absence of macrometastasis. In addition to their association with non-SLN involvement, SLN MM have been found to be indicators of poor prognosis, associated with local and distant failure. Tumour burden within the SLN is likely to represent a continuous variable extending from the presence of ITC to MM and macrometastasis. The relative importance of SLN tumour burden now needs to be determined in the context of other established patient and tumour related prognostic variables in order to provide an overall 'risk estimate'. This could be used to inform decisions regarding the risk/benefit of potential local and/or system interventions. Such information will need to be carefully considered in the context of the multidisciplinary team in order to make decisions regarding patient management.

In addition to the importance of SLN identification and retrieval during surgery, the reliable histological assessment and accurate determination of SLN involvement is obviously of critical significance. It is therefore somewhat surprising that whilst surgical technique has been relatively well standardized, absolute consensus regarding the optimal analytical technique has yet to be reached. The most effective technique for assessing the SLN will depend on the nature of the lesion that needs to be identified, hence decisions need to be made about the necessity of identifying MM and ITC [START_REF] Treseler | Pathologic examination of the sentinel lymph node what is the best method?[END_REF]. Cserni et al. [START_REF] Cserni | Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European Working Group for Breast Screening Pathology[END_REF] has demonstrated discrepancies in the current practice of SLN evaluation, in addition to inconsistency regarding the categorization of MM amongst pathologists presented with identical digital images. As HES is increasingly complemented by IHC and molecular amplification techniques, including PCR and RT-PCR, subgroups of patients will be identified in which MM are only detectable by one modality or the other. 'Positivity' can therefore be categorized according to the modality employed. Some authors have assigned greater significance to MM which are visible on HES rather than those which require IHC for detection [START_REF] Mascarel | Prognostic significance of immunohistochemically detected breast cancer node metastases in 218 patients[END_REF]. However the significance of MM detectable by particular modalities has not been determined and hence this information is not currently included in the TNM classification [START_REF] Singletary | Classification of isolated tumor cells: clarification of the 6th edition of the American Joint Committee on Cancer Staging Manual[END_REF]. Current prospective studies include ACOSOG Z0010, in which patients with MM found only by SLN IHC will not undergo further axillary surgery, but may have breast radiotherapy or systemic adjuvant treatment. Results from the NSABP B-32 trial will also be relevant as patients were randomized to SLNB (proceeding to ALND for cases which were positive on HES) or routine ALND. Postoperative IHC analysis will identify some additional patients with MM whose long term follow-up with regard to local and distant relapse will be of particular interest. For the time being the further management of the axilla is likely to remain dependent on the extent of SLN 'positivity' or tumour burden rather than the technique employed for detection.

It is also noteworthy that several reports have identified associations between percutaneous biopsy of the primary tumour and SLN involvement with tumour, including MM [START_REF] Hansen | Manipulation of the primary breast tumor and the incidence of sentinel node metastases from invasive breast cancer[END_REF][START_REF] Peters-Engl | The impact of preoperative breast biopsy on the risk of sentinel lymph node metastases: analysis of 2502 cases from the Austrian Sentinel Node Biopsy Group[END_REF]. Moore et al. [START_REF] Moore | Immunohistochemically detected tumor cells in the sentinel nodes of patients with breast carcinoma[END_REF] found that the presence of MM was significantly related to the method of breast biopsy, however this relationship was only observed for IHC detected MM rather than those identified by HE staining. However, in a study of 89 patients, Kinoshita et al. [START_REF] Kinoshita | Impact of fine-needle aspiration on long-term survival rate and axillary lymph node micrometastasis in patients with early breast cancer[END_REF] have recently reported no association between preoperative FNA and the frequency of MM or survival of patients with early breast cancer. The spread of epithelial cells to axillary lymph nodes may also be attributable to benign mechanical transport [START_REF] Diaz | Benign mechanical transport of breast epithelial cells to sentinel lymph nodes[END_REF].

Conclusions and recommendations for practice

The prognosis for women with MM within the SLN should not be considered to be the same as those without. SLN MM are likely to represent an incremental detriment to prognosis and increased risk of non-SLN involvement, despite only modest up-staging within current classification systems. Management need not be altered for women in whom further axillary treatment with ALND or radiotherapy and systemic adjuvant treatment are already indicated. However, in the absence of level-1 guidance concerning the management of women with SLN MM, each case requires discussion with regard to other tumour and patient related factors in the context of the multidisciplinary team. Whilst there may exist a minimum tumour burden within the SLN for which further treatment can be safely omitted, this critical amount has yet to be defined. If the outcomes for current trials, including the NSABP B-32 and ACO-SOG Z0010, do not provide authoritative guidance, they may at least provide ethical and clinical justification for further randomized studies to evaluate the prognostic significance and optimal management of each category of tumour burden within the SLN.

The identification of MM remains highly dependent on the analytical technique employed and there exists potential for stage migration and impact on management decisions. Guidance relating to the histopathological analysis of SLNs has not been consistent, particularly concerning IHC techniques targeting cytokeratin, despite the fact that minimal metastatic disease is unlikely to be detected unless they are employed. Molecular technologies for the detection of MM which are 'missed' by conventional methods and/or IHC should only be used for research purposes at present.

Fig. 1

 1 Fig. 1 Micrometastasis within a sentinel lymph node, haematoxylin and eosin stained: (a) low power and (b) high power

Fig. 2

 2 Fig. 2 Isolated tumour cell within a sentinel lymph node, haematoxylin and eosin stained

Table 1

 1

	Studies providing evidence supporting the	Author [Ref.]	Year Total patient no. Cases with SLN MM Outcome measure
	significance of SLN MM	Mullenix [40]	2005	334	4	Local recurrance/distant failure
		van Rijk [41]	2006 2,150	148	Non-SLN involvement
		Houvenaeghel [50] 2006	700	700	Non-SLN involvement
		Di Tommaso	2006	540	62	Non-SLN involvement
		Schrenk [51]	2005	379	138	Non-SLN involvement
		Leidenius [46]	2005	84	84	Non-SLN involvement
		Viale [42]	2005 1,228	318	Non-SLN involvement
		Giard [48]	2004	542	55	Non-SLN involvement
		Dabbs [45]	2004	445	49	Non-SLN involvement
		den Bakker [49]	2002	32	32	Non-SLN involvement
		Viale [44]	2001	634	109	Non-SLN involvement
		Turner [43]	2000	194	93	Non-SLN involvement

Table 2

 2

	Studies providing significance of SLN MM evidence against the	Author [Ref.]	Year	Total patient no.	SLN MM Cases with	Outcome measure
		Nagashima [82]	2006	375	19	Local recurrance/distant failure
		Langer [59]	2005	234	27	Local recurrance/distant failure
		Guenther [60]	2003	46	16	Local recurrance
		Fant [61]	2003	31	27	Local recurrance
		Rutledge [63]	2005	358	29	Non-SLN involvement
		Fournier [64]	2004	194	21	Non-SLN involvement
		Ganaraj [83]	2003	305	41	Non-SLN involvement
		Liang [62]	2001	227	15	Non-SLN involvement
		Chu [81]	1999	157	69	Non-SLN involvement

Breast Cancer Res Treat (2009) 114:393-402

Breast Cancer Res Treat (2009) 114:393-402


Alessandra Gennari
email: alessandra.gennari@istge.it

Michele Tursi

Consiglia Carella

Enrico Ricevuto

Cinzia Orlandini

Antonio Frassoldati

Pierfranco Conte

Paolo Bruzzi

Stefano Iacobelli

Alessandra Gennari Ae

Michele De Tursi

A E Consiglia

Carella Ae

A E Cinzia

Orlandini Ae

Antonio Frassoldati Ae

A E Paolo

Bruzzi Ae

Á C Carella

Á S Iacobelli

Á P Conte

Epirubicin plus low-dose trastuzumab in HER2 positive metastatic breast cancer

Keywords: Low-dose trastuzumab, Epirubicin, Metastatic breast cancer

Introduction

Although metastatic breast cancer (MBC) remains an incurable disease, a wide range of treatment options is available, and many further substances are currently evaluated in the clinical and preclinical setting. The decision to choose a certain therapy is usually based on tumor biology, symptoms, metastatic pattern, time to disease recurrence, and patient desire.

In human epidermal growth factor 2 (HER-2)-positive MBC, the use of trastuzumab (T) is firmly established. Trastuzumab is a recombinant monoclonal antibody that targets the extracellular domain of HER-2 protein. Preclinical models support several potential mechanisms of action of T, including inhibition of abnormal signaling by HER-2 overexpressing cells, activation of antibody-dependent cellular cytotoxicity, and inhibition of angiogenesis [START_REF] Vogel | Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer[END_REF][START_REF] Hudis | Trastuzumab-mechanism of action and use in clinical practice[END_REF].

The addition of trastuzumab to first line chemotherapy with either paclitaxel single agent or doxorubicin plus cyclophosphamide (AC regimen) resulted into an improved activity and efficacy as compared with chemotherapy alone. Unfortunately, despite its clinical benefit, the combination of AC plus trastuzumab, was associated with an overall rate of cardiotoxicity of 27%, with approximately two-third of cases belonging to New York Heart Association class III or IV [START_REF] Slamon | Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[END_REF].

Early clinical trials which evaluated the safety, pharmacokinetics and clinical activity of trastuzumab, used a loading dose of 4 mg/kg followed by a weekly maintenance dose of 2 mg/kg: this dose was required to obtain the minimum serum trough concentration (C min ) of 10-20 lg/ml which was shown to be associated with preclinical activity [START_REF] Vogel | Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer[END_REF][START_REF] Cobleigh | Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease[END_REF]. The three-weekly schedule, based on higher doses and longer intervals, consisting in a loading dose of 8 mg/kg, followed by an every-3-week maintenance dose of 6 mg/kg, yielded steady state serum trough concentration roughly similar to those obtained with weekly dosing [START_REF] Leyland-Jones | Pharmacokinetics, safety, and efficacy of trastuzumab administered every three weeks in combination with paclitaxel[END_REF][START_REF] Carter | Humanization of an anti-p185HER2 antibody for human cancer therapy[END_REF]. However, with both schedules, trastuzumab achieves steady state concentrations in the range of 60 lg/ml and a half life in excess of 3 weeks, thus exceeding the target concentration associated with clinical activity. Therefore, it can be hypothesised that the target concentration of 10-20 lg/ml might be achieved with lower trastuzumab doses, i.e. half of those conventionally administered. With these premises, in the attempt to improve the therapeutic index of the anthracycline/trastuzumab combination in MBC, we performed an exploratory study to evaluate the activity and cardiac safety of epirubicin combined with low-dose trastuzumab in patients with HER-2-positive disease.

Patients and methods

Study population

Patients with histologically proven HER-2-positive MBC were enrolled into the study. HER-2 positivity was defined as either an immunohistochemical staining of 3? by Herceptest (Dako) or gene amplification by fluorescence in situ hybridization (FISH). In patients with tumors scored as 2? by Herceptest, confirmatory FISH was performed. The patients were required to have measurable disease according to WHO criteria, and an Eastern Cooperative Oncology Group (ECOG) performance status B2. Lactating or pregnant women were excluded from the study. Patients of child-bearing potential were required to have a negative pregnancy test and to make use of effective contraception. Patients were excluded if they had received prior therapy with trastuzumab, prior chemotherapy for MBC or any investigational agent within 30 days before the study began. Prior adjuvant/neoadjuvant chemotherapy was allowed if terminated at least 1 year before study entry. Patients could have been treated up to a maximum of 480 mg/sqm of epirubicin and 240 mg/sqm of adriamycin. Prior endocrine therapy for adjuvant and/or for metastatic disease and prior radiation therapy were allowed. Patients had to be 18 years old, have a life expectancy longer than 6 months, and a left ventricular ejection fraction (LVEF) [50% by echocardiography or multigated radionuclide angiography (MUGA) scan. Baseline normal haematological, renal, liver enzymes were also required.

All patients provided signed informed consent. The study was conducted in accordance with the ethical principles that originated in the Declaration of Helsinki and with local research ethics board approval at each participating centre.

Treatment

All patients received epirubicin at 90 mg/sqm as a short i.v. bolus on day 1 every 3 weeks and trastuzumab at 2 mg/kg i.v. on day 1 of week 1 and thereafter at 1 mg/kg once weekly (LD-T). After a maximum of eight cycles of epirubicin, patients received trastuzumab LD-T as a single agent for up to 52 weeks.

Treatment was discontinued for disease progression, excessive toxicity or in patients developing protocoldefined cardiotoxicity. Chemotherapy was delayed for a maximum of 2 weeks if the absolute neutrophil count was \1.5 9 10 9 /l, if the platelet count was \100 9 10 9 /l or the haemoglobin was \10 g/dl on the day of the planned chemotherapy administration. In case of anemia, thrombocytopenia or non-hematologic grade 3 toxicity, subsequent chemotherapy was administered with a 25% dose reduction of epirubicin, within a maximum of 2 weeks. If grade 3 toxicity did not resolve within this period, patients were withdrawn from the study. In case of any non-hematological toxicity of grade 4, epirubicin was reduced by 50% or hold at physician's judgment. Cardiotoxicity was defined as signs or symptoms of congestive heart failure (CHF), a decline of LVEF C15% without symptomatic CHF, or a decline of LVEF less than 15% to below lower limits of normal (50%), without symptoms. Physical examination, routine blood chemistry, chest radiography, bone scan, liver ultrasound, electrocardiogram (ECG) and echocardiogram or MUGA scan were conducted at baseline and at regular intervals throughout the study. In particular, ECG was repeated before each treatment cycle, and echocardiogram or MUGA scan was repeated every other cycle during chemotherapy and every 2 months during trastuzumab alone, or as clinically indicated. Toxicity of treatment was categorized according to NCI-CTC version 2 grading system.

Study outcome variables

The primary study objective was to evaluate overall response rate (RR) in patients treated with epirubicin plus LD-T. Imaging procedures to assess tumor response were repeated every 6 weeks. Response was evaluated via bi-dimensional measurement changes using RECIST criteria [START_REF] Therasse | New guidelines to evaluate the response to treatment in solid tumors[END_REF]. Patients who achieved a complete or partial response were re-imaged 4 weeks later to confirm the initial observation. Stable disease was defined as failure to achieve a response or failure to demonstrate progressive disease following two cycles of treatment. Patients were evaluable for tumor response if they received at least 2 cycles of chemotherapy. Duration of response was defined as the length of time from date of objective response to disease progression or death due to any cause. Progression free survival (PFS) was calculated from the time of initial treatment to first evidence of progression or death due to any cause. Overall survival (OS) was determined from the time of initial treatment with study drugs to death due to any cause.

Statistical analysis

The sample size was calculated according to the Simon's two-step optimal design [START_REF] Simon | Optimal two-stage designs for phase II clinical trials[END_REF], to obtain a 90% power in order to test the null hypothesis of an ORR of 50% versus the alternative hypothesis of ORR of C70% (one-sided test, a = 0.05). This required a total two-stage sample of 45 patients with rejection of the null hypothesis if 26 or more patients achieved an objective response.

Under this hypothesis, 21 patients were enrolled on a first step. If \12 responses were observed, this step had to be performed increasing the dose of trastuzumab to 2 mg/kg. Moreover, this regimen was considered feasible if the cumulative probability of developing symptomatic cardiotoxicity (CHF) was B10% at a cumulative epirubicin dose of 720 mg/sqm and B20% at a cumulative epirubicin dose of [720 B 1,000 mg/sqm.

The cumulative probability of developing cardiac toxicity, while taking into account the different number of patients at the various cumulative dose levels of epirubicin, was computed as follows. According to the life-table method, for each cumulative dose level, the number of patients who subsequently experienced cardiac events and had not been treated with higher cumulative doses was divided by the total number of patients treated at that level (including patients who had been treated with higher doses). Patients who received anthracyclines in the adjuvant setting were entered at the corresponding cumulative dose. Analyses were performed using SPSS/PC ? 11.5 statistical software (LEAD Technologies, USA).

Results

Forty-five consecutive patients (median age, 55 years; range, 34-71 years) with HER2-positive MBC were enrolled in four centres in Italy. Twenty-three (51.1%) patients had ER and/or PR positive tumors. Twenty-four (53.3%) patients received prior radiotherapy, and 21 (46.7%) prior endocrine therapy. Twenty-three (51.1%) patients received prior anthracyclines. The majority of patients (82.2%) had visceral involvement. No difference in patients characteristics were observed between patients enrolled in the different centres. Table 1 lists the characteristics of the included patients.

Activity

Overall, 44 out of 45 patients were evaluable for response. One patient had HER2-negative tumor and was not eligible for the study. In the first step of the study, 12 responses among 21 patients were recorded. These results met the requirements for study continuation with trastuzumab at the dose of 1 mg/kg once weekly. Overall objective responses were recorded in 27 out of 44 patients (61.4%); one patient (2.3%) achieved a complete response, and 26 (59.1%) achieved a partial response. Eleven patients (25.0%) had stable disease following at least two cycles of epirubicin plus LD-T. Details of treatment outcome are reported in Table 2.

At a median follow-up of 11.8 months (range 1-47.4 months), 41 patients (93.2%) have progressed, and 1).

Adverse events

Forty-four out of 45 patients were evaluable for toxicity.

One patient never started the planned treatment and was not considered for toxicity. Most adverse events were mild to moderate. Grade 3/4 neutropenia, thrombocytopenia and anemia occurred in 25%, 7% and 2% of the patients, respectively. One case of febrile neutropenia was registered. Non-hematologic toxicity was generally mild; grade 3 alopecia occurred in 46% of the patients (Table 3). Patients received a median of 6 (range 1-8) threeweekly administrations of epirubicin and 32 weekly administrations of LD-T. Thirty-three (75.0%) patients received at least six courses of epirubicin. Of the 326 courses of epirubicin administered, dose reductions or delays were necessary in 4 and 39 cycles, respectively.

Cardiotoxicity

The mean LVEF at baseline was 67.3% (SD = 8.0%; median 68.0%). Mean LVEF was 53.5% (SD = 12.1%; median 60%) after 6-12 months, for an absolute decline in LVEF of 20.5% from baseline (P \ 0.002 by Mann-Whitney) (Fig. 2).

Overall, eight episodes of protocol-defined cardiotoxicity were observed: six patients (13.6%) showed a decline in LVEF C 15% as compared to baseline, while maintaining an absolute LVEF greater than 50%. Two patients (4.5%) developed symptomatic CHF after a median cumulative epirubicin dose of 720 mg/sqm: both patients had not received anthracyclines in the adjuvant setting. The first patient was a 43-year-old woman with family history for cardiac disease and a LVEF of 60% at baseline; she developed symptomatic CHF (class III NHYA) with an LVEF of 30% after eight cycles of epirubicin plus LD-H Treatment was stopped and she was treated with diuretics plus an ACE-inhibitor. Two months later LVEF was 50% and symptoms improved. The cumulative risk of developing CHF was estimated as 8.7% at the cumulative epirubicin dose of 720 mg/m2, and the overall cumulative risk of any protocol-defined cardiotoxic event was 34% (Fig. 3).

Discussion

For more than 30 years, regimens containing anthracyclines have been considered among the mainstays of treatment for advanced breast cancer, with a clear therapeutic advantage over those not including anthracyclines [START_REF] Fossati | Cytotoxic and hormonal treatment for metastatic breast cancer: a systematic review of published randomized trials involving 31,510 women[END_REF]. However, the high cumulative risk of cardiotoxicity remains the major limitation to the use of these drugs [START_REF] Jones | Anthracycline cardiotoxicity[END_REF].

The addition of trastuzumab to anthracyclines in patients with HER-2 positive breast cancer, although associated with a significantly higher clinical benefit, further increased the incidence of cardiotoxicity [START_REF] Ewer | The anthracycline-trastuzumab interaction: upregulated binding may provide vital mechanistic insight[END_REF][START_REF] Gianni | Anthracycline cardiotoxicity in breast cancer patients: synergism with trastuzumab and taxanes[END_REF].

In the present study, in the attempt to minimize cardiotoxicity, while maintaining antitumor activity, trastuzumab was administered at a dose (1 mg/kg) that is half of the recommended one in combination with epirubicin 90 mg/sqm. The observed response rate (61.4%) is in the range of the activity reported with the combination of anthracyclines and trastuzumab [START_REF] Slamon | Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[END_REF]. However, two out of 44 patients (4.5%) developed CHF, after a cumulative epirubicin dose of 720 mg/sqm, for a cumulative risk of developing CHF at this dose level of 8.7%. This rate is lower than the protocoldefined threshold of 10%, which was chosen as maximal acceptable toxicity. Moreover, it is lower than that reported in the trial by Slamon, where the combination of anthracyclines and trastuzumab was associated with a 16% incidence of CHF. Preclinical models have shown that HER2 plays a pivotal role in limiting the left ventricular dysfunction promoted by anthracyclines. In particular, HER-2 signalling is required for the growth, repair and survival of cardiomyocytes, thus helping in maintaining cardiac contractility and function, and preventing dilated cardiomiopathy [START_REF] Chien | Herceptin and the heart-a molecular modifier[END_REF]. Under this hypothesis, one could speculate that the apparently decreased incidence of CHF episodes observed in the present study, can be related to an incomplete bock of the cardiac HER2 rescue pathway, occurring with reduced trastuzumab doses. Moreover, although the relationship between dose of trastuzumab and cardiac toxicity is not supported by available clinical data, in vitro data from chick embryos indicate that the drug consistently produced arrhythmia in a dose-dependent fashion [START_REF] Yoshiyama | Cardiotoxicity of trastuzumab (herceptin) in chick embryos[END_REF]. Recently, updated results on the cardiac profile of the combination of epirubicin and full-dose trastuzumab as first line therapy in MBC reported a 5% incidence of CHF, i.e. similar to our study, with an activity in the range of 60% [START_REF] Untch | Cardiac safety of trastuzumab in combination with epirubicin cyclophosphamide as first-line therapy in patients with HER2 positive metastatic breast cancer[END_REF]. Yet, a cardiotoxicity risk of this order of magnitude, cannot longer be considered acceptable at the light of the availability of new anthracycline formulations (e.g. liposomal doxorubicin) [START_REF] Chia | Pegylated liposomal doxorubicin and trastuzumab in HER-2 overexpressing metastatic breast cancer: a multicenter phase II study[END_REF], new anti-HER-2 drugs (lapatinib) [START_REF] Burris | Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas[END_REF][START_REF] Moy | Lapatinib-associated toxicity and practical management recommendations[END_REF] and non anthracycline based regimens (e.g. vinorelbine plus trastuzumab or taxane plus trastuzumab) [START_REF] Burstein | Trastuzumab plus vinorelbine or taxane chemotherapy for HER2-overexpressing metastatic breast cancer: the trastuzumab and vinorelbine or taxane study[END_REF][START_REF] Esteva | Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer[END_REF][START_REF] Marty | Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as firstline treatment: the M77001 study group[END_REF], all endowed with a comparable activity, but with lower or no cardiac toxicity. In conclusion, the combination of epirubicin with LD-T has an activity comparable to that of anthracyclines with full-dose trastuzumab. However, this regimen is associated with a relatively high rate of cardiotoxicity, thus precluding further evaluation of this combination. Future trials in this setting should not include a control arm using conventional anthracyclines.

Fig. 1

 1 Fig. 1 (a) Progression free survival; (b) overall survival

Fig. 2

 2 Fig. 2 Left ventricular ejection fraction modifications over time

Fig. 3

 3 Fig.3Cumulative probability of CHF or ; L-VEF C15%

Table 1

 1 Baseline patients characteristics (N = 45)

	Characteristic	Patients
		No	%
	Median age, years (range) 55 (34-71)
	Performance status
	0	26	57.8
	1	17	37.8
	2	2	4.4
	Hormonal receptor status
	Positive (ER and/or PR)	23	51.1
	Negative	21	46.7
	Unknown	1	2.2
	Prior radiotherapy	24	53.3
	Prior endocrine therapy	21	46.7
	Prior adjuvant chemotherapy
	CMF	6	13.3
	Anthracyclines	23	51.1
	Metastatic sites
	Visceral	37	82.2
	Bone ± soft tissues	4	8.9
	Both	4	8.9
	Number of involved sites
	1	36	80.0
	2	7	15.6
	C3	2	4.4

Table 2

 2 Activity and efficacy (N = 44)

	Parameter	No.	%	95% CI
	Activity
	Overall response rate	27	61.4
	Complete response	1	2.3
	Partial response	26	59.1
	Stable disease	11	25.0
	Disease progression	5	11.4
	Not available a	1	2.3
	Progression free survival
	Median PFS (months)	7.3		5.8-9.3
	1-year PFS		32.8
	Overall survival
	Median OS (months)	32.8		17.1-48.5
	1-year OS		90.6
	2-year OS		86.8

a One patient never started the planned treatment

Table 3

 3 Incidence of hematological and not-hematological toxicities by grade (N = 44)

	Toxicity (% of patients) Grade 1 Grade 2 Grade 3 Grade 4
	Leucopenia	18.2	54.5	6.8	4.5
	Neutropenia	25.0	25.0	11.4	13.6
	Anemia	27.3	20.5	2.3	-
	Thrombocytopenia	4.5	-	6.8	-
	Nausea, vomiting	11.4	13.6	-	-
	Mucositis	6.8	-	4.5	-
	Cutaneous	-	2.3	2.3	-
	Diarrhoea	4.5	4.5	-	-
	Alopecia	6.8	25.0	45.5	-

Breast Cancer Res Treat (2009) 115:131-136