Liliana Santos

Sonia Marín

Vicent Sanchis

Antonio Ramos Girona

Av Antonio J Ramos
email: ajramos@tecal.udl.es

In vitro effect of some fungicides on growth and aflatoxins production by Aspergillus flavus isolated from Capsicum powder

The aim of this study was to determine the effect of some pre-harvest fungicides on growth 14 and aflatoxin (AF) production of three A. flavus strains found in Capsicum powder. Each 15 isolate, previously isolated from paprika, chilli and smoked paprika, was inoculated on Yeast 16 Extract Sucrose agar and on a 3% paprika extract agar medium supplemented with different 17 fungicides and incubated at 20 and 30 ºC during 7 days. Growth measurements were obtained 18 on days 3, 5 and 7, and the AF production was determined on day 7. The significance of the 19 effects of the factors (strain, medium, temperature, time and fungicides) and their interaction 20 over colony diameter and AF production was determined. Temperature constrained the 21 effectiveness of fungicides in reducing growth, the fungicides being most effective at 20 ºC.

22

The efficacy of the fungicides over AF production depended on the medium used and 23 temperature. The most effective fungicides in inhibiting growth and AF production, 24 regardless of the strain tested or applied conditions, were tebuconazole 25% and mancozeb 25

The possible presence in food and feed of aflatoxins (AF), fungal toxic compounds to animals 33 and man, has led to increased research aiming to prevent and detect such compounds, mainly 34 due to their carcinogenic, mutagenic and teratogenic properties. The Aspergillus section Flavi 35 is well known for its AF production. The more common AF-producers species are Aspergillus 36 flavus and A. parasiticus, though A. arachidicola, A. bombycis, A. minisclerotium, A. nomius, 37 A. parvisclerotigenus, A. pseudotamarii and A. toxicarius also have the ability to produce AF 38 [START_REF] Samson | Old and new concepts of species differentiation in 399 Aspergillus[END_REF]). The AF production may happen either in the field, during harvest and/or 39 transportation or storage. The AFB1, AFB2, AFG1 and AFG2 are the most studied AF, being 40 the AFB1 the most toxic.

41 Some species of the genus Capsicum are grown for their fruits, which can be consumed as 42 dried powder, usually named paprika and chilli. A. flavus, which have the ability to produce 43 AF, can be found in this kind of commodities [START_REF] Christensen | Microflora of 303 black and red pepper[END_REF]Mandeel, 2005; 44 [START_REF] Hashem | Contamination of common spice in Saudi Arabia markets with 339 potential mycotoxin-producing fungi[END_REF]. A review about mycotoxin contamination of Capsicum powder 45 indicated that out of 1027 samples, 61.4% were positive for AF [START_REF] Santos | Capsicum and Mycotoxin 401 Contamination: State of the Art in a Global Context[END_REF], the 46 contamination being in the range from limit of detection to 969 µg kg -1 [START_REF] Santos | Capsicum and Mycotoxin 401 Contamination: State of the Art in a Global Context[END_REF] 47 [START_REF] Bircan | Aflatoxin levels in dried figs, nuts and 297 paprika for export from Turkey[END_REF][START_REF] Hierro | Aflatoxins and ochratoxin 343 A in red paprika for retail sale in Spain: Occurrence and evaluation of a simultaneous 344 analytical method[END_REF][START_REF] O'riordan | A survey of the incidence and level of aflatoxin 384 contamination in a range of imported spice preparations on the Irish retail market[END_REF]Santos et al. 2010b).

48

The mycotoxin contamination of spices should not be underestimated despite the small of morphological identification, the 3 isolates were subjected to molecular analysis with 73 specific primers for this species. All genomic DNAs were tested for suitability for PCR 74 amplification using universal primers ITS1 and ITS4. Specific PCR assays were carried out 75 using primers FLA1 and FLA2 based on ITS1-5.8SITS2 region and the protocol described 76 elsewhere (González-Salgado et al., 2008). (3% paprika) were used for mould cultures. The 3% paprika medium was prepared by boiling 84 30 g of chilli powder in 1 L distilled water for 30 min. The resulting mixture was filtered 85 through a double layer of muslin and the volume was made up to 1 L [START_REF] Marín | Modelling of growth of aflatoxigenic A. 359 flavus isolates from red chilli powder as a function of water availability[END_REF] For inocula preparation, one plate of Malt Extract Agar (MEA) (Malt Extract 15 g; peptone 90 0.75 g; glucose 15 g; agar 9 g; H 2 O 750 mL to 1 L) per strain was inoculated and incubated 91 for 7 days at 25 ºC. On the seventh day, suspensions of 1-5•10 6 spores mL -1 (in sterile water 92 plus Tween 80) for each strain were prepared and adjusted using a Thoma counting chamber.

93

YES and 3% paprika plates were inoculated once in the centre, with an inoculation needle. respectively. An isocratic mobile phase of water-acetonitrile-methanol (60:25:15, v/v/v) was 114 used at a flow rate of 1.0 mL min -1 . Post-column derivatization was achieved by using a 115 photochemical reactor for enhanced detection (PHRED) (LCTech UVE, Dorfen, Germany).

116

For confirmation of the presence of AF, the PHRED was switched off and the decrease of the 117 AFB1 and AFG1 peaks was registered. For total AF (AFt), limit of detection (LD) was 0.6 ng parameters have been investigated separately but it must be noted that they all interact under 143 natural conditions [START_REF] Martins | Interaction of strains of non-toxigenic 362 Aspergillus flavus with Aspergillus parasiticus on aflatoxin production[END_REF]. Some strains of A. flavus are known to be non-144 toxigenic or to produce small quantities of AF, as the isolate UdLTA 3.215. The ability to 145 produce AF relies on the metabolic system of each individual [START_REF] Martins | Interaction of strains of non-toxigenic 362 Aspergillus flavus with Aspergillus parasiticus on aflatoxin production[END_REF]. Those A.

146 flavus that produce AF, usually produce only B-type AF, however there are some strains also 147 capable to produce G-type AF. All the isolates used in this study produced B-type and G-type 148 AF, depending on the conditions. Like us there are other authors who also found A. flavus G-149 type producers (Gabal et al., 1994;Ehrlich et al., 2007). The toxigenicity of A. flavus also 150 varies with strain, substrate and geographic origin [START_REF] Klich | Environmental and development factors influencing aflatoxins production 349 by Aspergillus flavus and Aspergillus parasiticus[END_REF]. As mentioned before, each 151 strain derived from different a substrate.

153

Usually, A. flavus growth is possible in the range of 10 ºC or a little higher to about 43º, being 154 the optimal growth at temperatures slightly exceeding 30 ºC (ICMSF 1996). The usual 155 optimal temperature for AF production by A. flavus is 20-35ºC and AF production decreases 156 as temperature goes down or above these levels [START_REF] Schroeder | Aflatoxins: production of the toxin in vitro in relation to 409 temperature[END_REF], Northolt et al. 157 1977[START_REF] Gqaleni | Effects of temperature, water activity, and 333 incubation time on production of aflatoxins and cyclopiazonic acid by an isolate of 334 Aspergillus flavus in surface agar culture[END_REF][START_REF] Ogundero | Temperature and aflatoxins production by Aspergillus flavus and A. 387 parasiticus strains from Nigeria groundnuts[END_REF]. There are some authors who have observed that 158 A. flavus can also have an optimal temperature for AF production at 13-16 ºC or 16-24 ºC, 159 depending on the strain [START_REF] Northolt | Differences between Aspergillus flavus strains 381 in growth and aflatoxins B1 production in relation to water activity and temperature[END_REF]. In our study, although only two temperature 160 levels were assayed, it was observed that A. flavus grew better and produced more AF at 30 161 ºC.

163

In the controls where no fungicide was added, growth and AFt, AFB1 and AFB2 production 164 were significantly higher in YES than in 3% paprika medium. In general, the three strains 165 grew significantly faster and produced significantly more AF at 30ºC than at 20ºC in both 166 media. Regarding the AFB2 production, the temperature only influenced the strain UdLTA In the controls when comparing strains and regardless of the medium used, it was possible to 172 observe that the UdLTA 3.217 strain grew significantly faster (P<0.05). The UdLTA 3.193 173 produced significantly more AFt, AFB1 and AFB2 than the other strains, and no significant 174 differences were found among the strains for the production of AFG1 and AFG2 (P<0.05).

175

The ability to produce AF relies on the metabolic system of each individual strain (Martins et 176 al. 2000). Thus, some strains of A. flavus are non-toxigenic or produce less quantity of AF, as For statistical purposes, an AF production of 0.0 ng AF/g of agar was assigned to no-growth 206 observations. Moreover, aflatoxin results for <5 mm growth observations were omitted. Thus The efficacy of the fungicides over AF production depended on the medium used and 213 temperature (P<0.05). Regarding AFt production in 3% paprika the only effect observed was 214 a significant increase of the AFt production for the strain UdLTA 3.217 at 30 ºC and for the 215 strain UdLTA 3.215 at 20 ºC when using triadimenol 31.2% (F4) (P<0.05). The opposite was 216 only observed in YES. The fungicides had a significantly inhibitory effect over the AFt In the present study, the most effective fungicides in inhibiting growth and AF production, 232 regardless of the strain, mycotoxin or applied conditions, were tebuconazole 25% (F6) and 233 mancozeb 80% (F7). Tebuconazole and mancozeb fungicides belong to the triazole and 234 dithiocarbamates groups, respectively. The dithiocarbamates action has direct effect on 235 moulds by interfering with sporulation, inhibiting some enzymatic process and disrupting the 236 lipids metabolism [START_REF] Yoneyama | Studies on the fungicidal action of dithiocarbamates 420 3. Effect of sodium dimethyldithiocarbamate on the fatty acid synthesis of Xanthomonas 421 oryzae[END_REF], whereas tebuconazole inhibits the biosynthesis of ergosterol which had an effect on mycelia growth (Godwin et al. 1992). The same active 238 ingredients of these fungicides (mancozeb and tebuconazole) proved to be effective in 239 reducing growth and sometime in inhibiting mycotoxin production: the use of mancozeb was 240 shown to be effective in controlling the Alternaria, Fusarium and Botrytis growth 241 [START_REF] Boughalleb | Effect of fungicides on in vitro infestation level 299 of radish, carrot and pepper seeds[END_REF][START_REF] Nallathambi | Post-harvest management 378 of ber (Ziziphus mauritiana Lamk) fruit rot (Alternaria alternata Fr. Keissler) using 379 Trichoderma species, fungicides and their combinations[END_REF]. Mancozeb was also capable to inhibit 242 effectively the growth of A. niger, A. carbonarius, A. flavus, A. parasiticus and A. ochraceus, 243 but also AFB1 and ochratoxin A production [START_REF] Bellí | Impact of fungicides on Aspergillus 292 carbonarius growth and ochratoxin A production synthetic grape-like medium and on grapes. 293[END_REF][START_REF] Reddy | Efficacy of certain agrochemicals on 396 Aspergillus spp. and subsequent aflatoxins production in rice[END_REF]). The use of 244 tebuconazole was also effective in reducing the growth of some Fusarium species 245 [START_REF] Müllenborn | Effect of fungicides on the complex 376 Fusarium species and saprophytic colonizing wheat kernels[END_REF]. There are also other fungicides which are capable of inhibiting 246 growth of A. flavus, as well as the AFB1 production, such as bitertanol 25%, carbendazim 247 50%, carpropamid, hexaconazole 5%, propiconazole, propineb 70%, tricyclazole 75% and 248 carbendazin + mancozeb [START_REF] Reddy | Efficacy of certain agrochemicals on 396 Aspergillus spp. and subsequent aflatoxins production in rice[END_REF]. Diodine is also effective in blocking the growth 249 of A. flavus [START_REF] Luz | In vitro susceptibility to fungicides by invertebrate-353 pathogenic and saprobic fungi[END_REF]. 250 251 Some authors suggested that production of some mycotoxins is not associated with rapid 252 growth of the mould; rather, higher growth rates seem to restrict mycotoxin production 253 [START_REF] Häggblom | Production of ochratoxin A in barley by Aspergillus ochraceus and 336 Penicillium viridicatum: effect of fungal growth, time, temperature, and inoculums size[END_REF]. Although this was observed in paprika medium, in YES the strain UdLTA 254 3.193 grew well and also produced high levels of AF. Moreover, in presence of sulphur 80% 255 (F5) it grew well and AF production was further promoted.

257

The use of fungicides can be very attractive in reducing mycotoxigenic fungal load, but we 258 must be aware that in some cases it may reduce fungal growth and also stimulate the 259 mycotoxin production [START_REF] Medina | Effect of carbendazim 370 and physicochemical factors on the growth and ochratoxin A production of Aspergillus 371 carbonarius isolated from grapes[END_REF]). In our study this fact occurred in the presence of Finally, care should be taken in the choice of a suitable fungicide, because its effectiveness 264 depends on the strain, temperature and substrate used. It is also necessary to note that results

265

obtained in vitro are unlikely to be similar to those obtained in the field trials due to the 266 influence of several factors. When choosing a fungicide it is necessary to take into account 267 that not always the most efficient fungicides in reducing fungal growth is the best choice 268 because it can also promote the production of AF. Also it should be remembered that even if 269 fungal growth is not reduced, the AF production could still be promoted. Thus, the best 270 fungicide is the one which can prevent fungal growth and AF production at the same time.

5

 5 Three replicate plates per fungicide and strain plus controls (media without fungicides) were 96 incubated in the dark at 20 and 30 ºC. examined on days 3, 5 and 7 and the diameters of the growing colonies were 99 measured in two directions at right angles to each other. 100 Determination of AF production 101 On day 7 of incubation, a high performance liquid chromatography (HPLC) method was used 102 to determine the AF production. The extraction method was a modification of the Bragulat et 103 al. (2001) protocol. Three plugs (diameter 5 mm) were aseptically removed from the inner, 104 middle, and outer area of each colony. The plugs were placed into 4 mL glass vials for 105 extraction. Extraction was done adding 1 mL of acetronitrile and vortex-mixing for 5 seconds. 106 After 1 hour, the vials were shaken again and the solvent was filtered (Millex HV filter 0.45 107 mm, Millipore, Bedford, MA, USA) on a HPLC glass vial. After filtration, 300 µL of 108 acetonitrile extract and 700 µL water were mixed and 100 µL of this solution was injected in 109 the HPLC system (Waters, Milford, MA, USA) with a reverse-phase C18 silica gel column 110 (Waters Spherisorb® 3 µm ODS2 4.6 × 150 mm, Milford, MA, USA) kept in a column oven 111 at 40 ºC. Detection was achieved by fluorescence (Waters 2475 fluorescence detector, Waters, 112 Milford, MA, USA); excitation and emission wavelengths were set at 362 and 440 nm,

  113

  167

  , on YES. Moreover, AFG1 and AFG2 production was not influenced by either medium 168 or temperature. As expected, increasing colony diameters were observed with time, so all the 169 results described a posteriori were based on the results of the day 7.

  177the strains UdLTA 3.215 and UdLTA 3.217.178Growth response to fungicides 179 Regarding fungicide effects on growth of all strains, the fungicides were more efficient at 20 180 ºC and on 3% paprika (P<0.05). The data from the effects of fungicides treatment over growth 181 are showed in the Table2. Almost all fungicides reduced significantly the growth of all 182 strains, except F3 (copper oxychloride 50%) which never showed good results on YES, F4 183 (triadimenol 31.2%) and F5 (sulphur 80%) which showed a light effect, in particular for 184 isolates UdLTA 3.215 and UdLTA 3.217, and F9 (trifloxystrobin 50%), which was not 185 effective at 30 ºC. The fungicides F6 (tebuconazole 25%) and F7 (mancozeb 80%) totally 186 prevented growth of all isolates, regardless of medium or temperature. The fungicide F1 187 (cypronidil 37.5% + fludioxonil 25%) reduced growth of all strains in 95 to 100%, regardless 188 of the temperature and medium used. The fungicide F2 (dodine 40%) reduced growth in YES 189 over 99%, but in 3% paprika it had lower efficiency (over 20%), being lower at 30 ºC (20-The fungicide F8 (cyproconazole 10%) inhibited growth in rates of 31 to 83%, 191 depending on medium and temperature.192Copper oxychloride 50% (F3) and triadimenol 31.2% (F4) were the worst fungicides for 193 growth controlling of A. flavus and the best ones were tebuconazole 25% (F6) and mancozeb 194 80% (F7), regardless of isolate, medium and temperature.195It is important to note that temperature constrained the effectiveness of fungicides in reducing 196 growth, being the fungicides more effective at 20 ºC. For a proper election of the fungicides it 197 is also necessary to discern their effect on the AF production.

  198 AF production response to fungicides 199 Since fungicides are widely used to control crop diseases, it is pertinent to consider the effects 200 of these agents on mycotoxin production. In both laboratory studies with pure cultures of 201 moulds and field trials with crop plants the resulting evidence concerning the effectiveness of 202 fungicides is contradictory and in certain cases unexpected (D'Mello et al. 1998), as it is 203 possible to observe through the results of this study.204 205

207

  it was not possible to analyse the effect of the fungicides F1 (cypronidil 37.5% + fludioxonil 208 25%) over the AF production capacity of all strains at 30 ºC, and also the effect of the 209 fungicide F2 (dodine 40%) over the strain UdLTA 3.215 at 20 ºC in 3% paprika and over the 210 strain UdLTA 3.217 at 30 º C in YES. The data from the effects of fungicides treatment over 211 AF production are shown in the Tables 3, 4, 5 and 6.

  212

217

  production of 51 to near 100%, regardless of the strains and temperature (P<0.05), except for 218 strain UdLTA 3.193 at 20 ºC where fungicides had no significant effect. The fungicides effect 219 over AFB1 production was similar to the effect over AFt production. There was an absence of 220 any significant effect in 3% paprika and a significant inhibitory effect between 51 and near 221 100%, on YES, regardless of strain and temperature, except at 20 ºC for the UdLTA 3.193 222 strain in which no effect was observed (P<0.05). Regarding fungicide effect in AFB2 223 production, there was no significant effect in 3% paprika, while in YES the fungicides 224 reduced significantly the AFB2 production in the range 39-100% for the strain UdLTA 3.217, 225 regardless of the temperature level and for the strain UdLTA 3.193 at 30 ºC. For AFG1 and 226 AFG2 production, there was no significant effect, except in YES at 30 ºC for the strains 227 UdLTA 3.193 and UdLTA 3.215. However, there were some cases the presence of fungicides 228 F1 (cypronidil 37.5% + fludioxonil 25%), F2 (dodine 40%) and F9 (trifloxystrobin 50%) 229 promoted AF production.230 231

  260

  F2) in 3% paprika at 20 ºC for the isolate UdLTA 3.193, which reduced 79% of 261 growth but promoted a high level of AF production. 262 263

  (AFB1, AFB2, AFG1 and AFG2) = 0.2 ng g -1 ; Absence of values indicates analysis not done (growth < 5mm).

  The fungicides used in this work are those which are commonly used for the treatment of 79 peppers and are listed in Table1.

	77
	78	Fungicides
	80
	81	Medium
	82	Yeast Extract Sucrose agar (YES) (yeast extract 20 g; Czapek concentrate 7.5 mL; sucrose
	83	150 g; MgSO 4 •7 H 2 O 0.5 g; agar 20 g; H 2 O 885 mL to 1 L) and a 3% paprika extract agar

Table 1 -

 1 List of fungicides used and their concentrations.

	Fungicide code	Commercial name	Active substance	Recommended/Used dose
	F1	Switch	Cypronidil 37.5% + Fludioxonil 25%	1 g L -1
	F2	Syllit®flow	Dodine 40%	1.6 g L -1
	F3	Copper fungicide Copper oxychloride 50%	3 g L -1
	F4	Bayfidan SC312	Triadimenol 31.2%	0.4 mL L -1
	F5	Sufrevit	Sulfur 80%	5 mL L -1
	F6	Folicur 25WG	Tebuconazole 25%	0.75 g L -1
	F7	Micene ® WP	Mancozeb 80%	3.5 g L -1
	F8	Caddy 10	Cyproconazole 10%	0.20 g L -1
	F9	Flint	Trifloxystrobin 50%	0.25 g L -1

193 UdLTA 3.215 UdLTA 3.217 UdLTA 3.193 UdLTA 3.215 UdLTA 3.217

	Control	30.8 A	30.5 A	32.0 AB	74.7 A	71.2 A	76.2 A
	F1	0.0 E	0.0 E	0.0 E	2.8 F	2.2 DE	3.2 D
	F2	6.3 D	2.7 DE	0.0 E	58.0 C	56.8 AB	54.0 B
	F3	13.3 C	9.3 CDE	13.8 D	19.0 E	15.2 CDE	26.3 C
	F4	22.0 B	20.2 AB	30.0 B	49.5 D	31.8 BCD	50.2 B
	F5	23.8 B	11.2 BCD	34.7 A	66.3 B	38.7 BC	70.8 A
	F6	0.0 E	0.0 E	0.0 E	0.0 F	0.0 E	0.0 D
	F7	0.0 E	0.0 E	0.0 E	0.0 F	0.0 E	0.0 D
	F8	15.3 C	13.7 BC	19.2 C	47.0 D	37.3 BC	52.5 B
	F9	3.0 DE	6.3 CDE	11.7 D	47.3 D	39.3 BC	49.8 B
	Fungicides		20 ºC	YES		30 ºC

UdLTA 3.193 UdLTA 3.215 UdLTA 3.217 UdLTA 3.193 UdLTA 3.215 UdLTA 3.217

	Control F1	57.0 A 0.0 C	52.3 A 0.0 D F o r P e e r R e v i e w 53.3 A 84.0 A 85.0 A 85.0 A 0.0 E 2.7 E 4.0 E 3.0 C O n l y
	F2	00.0 C	0.0 D	0.0 E	0.0 E	0.0 E	0.7 C
	F3	49.3 A	46.7 B	50.0 A	85.0 A	85.0 A	85.0 A
	F4	28.5 B	11.5 C	21.8 C	50.7 C	30.7 D	48.5 B
	F5	32.5 B	0.0 D	41.3 B	69.5 B	0.0 E	83.7 A
	F6	0.0 C	0.0 D	0.0 E	0.0 E	0.0 E	0.0 C
	F7	0.0 C	0.0 D	0.0 E	0.0 F	0.0 E	0.0 C
	F8	9.7 C	11.2 C	14.2 D	43.2 D	37.2 C	48.0 B
	F9	5.2 C	1.3 D	13.2 D	81.2 A	70.8 B	85.0 A

Table 3 -

 3 Effects of fungicide treatments (Cypronidil 37.5% + Fludioxonil 25% (F1), Dodine 40% (F2), Copper oxychloride 50% (F3), Triadimenol 31.2% (F4), Sulfur 80% (F5), Tebuconazole 25% (F6), Mancozeb 80% (F7), Cyproconazole 10% (F8), and Trifloxystrobin 50% (F9)) over AF production (ng AF g of agar -1 ) of A. flavus (UdLTA 3.193) (n=3, means followed by different letters are significantly different, P<0.05, according toTukey HSD test).

	3 % paprika

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgments

The authors are grateful to the Spanish (Project AGL2007-66416-C05-03) and Catalonian

275

(XaRTA-Reference Network on Food Technology) Government for their financial support.

276

The authors would like to thank X. Ochoa de Eribe (R&D technician of SIPCAM INAGRA)

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

F o r P e e r R e v i e w O n l y

Table 4 -Effects of fungicide treatments (Cypronidil 37.5% + Fludioxonil 25% (F1), Dodine 40% (F2), Copper oxychloride 50% (F3), Triadimenol 31.2% (F4), Sulfur 80% (F5), Tebuconazole 25% (F6), Mancozeb 80% (F7), Cyproconazole 10% (F8), and Trifloxystrobin 50% (F9)) over AF production (ng AF g of agar -1 ) of A. flavus (UdLTA 3.215) (n=3, means followed by different letters are significantly different, P<0.05, according toTukey HSD test).

% paprika 20 ºC 30 ºC Fungicides


M.D.;Hemming Johansson Asgerdur Sverrisdottir

Ulla Johansson

M.D. PhD Jonas Bergh

M.D. PhD Samuel Rotstein

M.D. PhD Larserik Rutqvist

M.D. PhD Tommy Fornander

Interaction between goserelin and tamoxifen in a prospective, randomised clinical trial of adjuvant endocrine therapy in premenopausal breast cancer

Keywords: breast cancer, premenopausal, goserelin, tamoxifen, interaction, randomised

Ovarian ablation improves survival in premenopausal early breast cancer, but the potential added value by luteinizing hormone-releasing hormone (LHRH) agonists to tamoxifen, is still not clear. The purpose of our study is to examine the efficacy of the LHRH agonist goserelin for adjuvant therapy of premenopausal breast cancer, the role of interaction between goserelin and tamoxifen, and the impact of estrogen receptor (ER) content. A total of 927 patients were included in the Stockholm part of the Zoladex in Premenopausal Patients (ZIPP) trial. They were randomly allocated in a 2 x 2 factorial study design to goserelin, tamoxifen, the combination of goserelin and tamoxifen or no endocrine therapy for 2 years, with or without chemotherapy. This is formally not a preplanned subset analysis presenting the endpoint first event. In this Stockholm sub-study, at a median follow up of 12.3 years, goserelin reduced the risk of first event by 32 % (P = 0.005) in the absence of tamoxifen, and tamoxifen reduced the risk by 27 % (P = 0.018) in the absence of goserelin. The combined goserelin and tamoxifen treatment reduced the risk by 24 % (P = 0.021) compared to no endocrine treatment. In highly ER positive tumours, there were 29% fewer events among goserelin treated (P = 0.044) and a trend towards greater risk reduction depending on the level of ER content. The greatest risk reduction from goserelin treatment was observed among those not receiving tamoxifen (HR: 0.52, P = 0.007). In conclusion, goserelin as well as tamoxifen, reduces the risk of recurrence in endocrine responsive premenopausal breast cancer. Women with strongly ER positive tumours may benefit more from goserelin treatment. The combination of goserelin and tamoxifen is not superior to either modality alone. With the limitations of a subset trial, these data have to be interpreted cautiously.

Introduction

The potential added value by luteinizing hormone-releasing hormone (LHRH) agonists to tamoxifen in the adjuvant setting for premenopausal breast cancer, remains unclear. In addition, the optimal timing and duration of LHRH agonist treatment is yet to be defined [START_REF] Davidson | Chemoendocrine therapy for premenopausal women with axillary lymph node-positive, steroid hormone receptor-positive breast cancer: Results from int 0101 (e5188)[END_REF][START_REF] Gnant | Endocrine therapy plus zoledronic acid in premenopausal breast cancer[END_REF][START_REF] Cuzick | Use of luteinising-hormone-releasing hormone agonists as adjuvant treatment in premenopausal patients with hormone-receptor-positive breast cancer: A metaanalysis of individual patient data from randomised adjuvant trials[END_REF][4]. Nevertheless, it is clear that ovarian ablation improves survival in premenopausal women with early breast cancer [START_REF] Cuzick | Use of luteinising-hormone-releasing hormone agonists as adjuvant treatment in premenopausal patients with hormone-receptor-positive breast cancer: A metaanalysis of individual patient data from randomised adjuvant trials[END_REF]. Results from trials using LHRH agonists in the adjuvant setting have indicated significant benefit in terms of prolonged disease-free survival and improved survival with 2 years of treatment, regardless of other systemic treatment. Treatment induced ovarian ablation, whether by endocrine-or chemotherapy, radiation or surgery, results in increased disease-free survival and overall survival in premenopausal endocrine responsive breast cancer [4]. Remaining research questions include the benefit of LHRH agonists, in addition to tamoxifen, and if LHRH agonists have a role among those not achieving amenorrhea during cytotoxic chemotherapy. Moreover, efficacy of endocrine treament in relation to estrogen receptor (ER) status is mainly established among postmenopausal women, and the different endocrine milieu in premenopausal women may affect the predictive ability of ER status [START_REF] Fisher | Influence of tumor estrogen and progesterone receptor levels on the response to tamoxifen and chemotherapy in primary breast cancer[END_REF][START_REF] Rutqvist | The stockholm trial on adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor level and treatment effect[END_REF][START_REF] Viale | Prognostic and predictive value of centrally reviewed expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and tamoxifen adjuvant therapy for postmenopausal early breast cancer: Big 1-98[END_REF][START_REF] Khoshnoud | Immunohistochemistry compared to cytosol assays for determination of estrogen receptor and prediction of the long-term effect of adjuvant tamoxifen[END_REF].

Overview data on the use of LHRH agonists have not shown therapeutic benefit with combination endocrine therapy versus tamoxifen or goserelin alone in women treated with chemotherapy [START_REF] Cuzick | Use of luteinising-hormone-releasing hormone agonists as adjuvant treatment in premenopausal patients with hormone-receptor-positive breast cancer: A metaanalysis of individual patient data from randomised adjuvant trials[END_REF]. The ZIPP trial was included in the overview and it is the largest one assessing the integration of LHRH agonists, into hormonal therapy.

Results from the ZIPP trial have been reported and are recently updated [START_REF] Baum | Adjuvant goserelin in pre-menopausal patients with early breast cancer: Results from the zipp study[END_REF][START_REF] Hackshaw | Long-term effectiveness of adjuvant goserelin in premenopausal women with early breast cancer[END_REF]. At a median follow-up of 12 years, two years treatment with goserelin was as effective as two years of tamoxifen. In those not treated with tamoxifen, there was a large survival and recurrence benefit with goserelin (13.9% fewer events, 95% confidence interval (CI), -19.4 to -7.5), and the addition of tamoxifen to goserelin showed marginal but not statistically significant improvement (2.8% fewer events, CI -7.7 to 2.0) [START_REF] Hackshaw | Long-term effectiveness of adjuvant goserelin in premenopausal women with early breast cancer[END_REF]. One of the limitations of the ZIPP trial is that randomisation was not strict factorial at all participating study sites. Some centres gave tamoxifen electively and only randomised to goserelin or no goserelin. A recent Cochrane review pointed out that results from the trial have not been reported separately for patients who underwent randomisation to both an LHRH agonist and tamoxifen [START_REF] Goel | Lhrh agonists for adjuvant therapy of early breast cancer in premenopausal women[END_REF].

As the research question of the benefit of tamoxifen in addition to an LHRH agonist remains unsettled, it was considered reasonable to analyse the Stockholm part of the ZIPP trial separately, and examine if the indirect comparisons of the overall trial can be reproduced in the Stockholm cohort. The Stockholm study was initially planned as a separate trial, but as it was found to be underpowered as a stand-alone trial for survival analysis, collaboration with centres in Sweden, UK and Italy (ZIPP trial) was established. This late and formally not planned separate analysis of the Stockholm cohort, presents therefore only the endpoint event free survival (EFS). The Stockholm part of the trial is a strict randomised 2 x 2 factorial subset of the ZIPP trial. In other centres, the analysis made in comparison to the control group, is indirect concerning the tamoxifen treatment, since the drug was prescribed electively. In addition, the Stockholm cohort is the only one with complete information on nodal status and whether chemotherapy was given or not. Furthermore, ER status of the primary tumour was available in a high proportion of all randomised women and all assays performed at a single laboratory.

Patients and methods

The ZIPP trial was a collaboration between four research groups (Stockholm, South East Sweden, Cancer Research UK and UCL Cancer Trials Centre (CRC), United Kingdom and Gruppo Interdisciplinare Valutazione Interventi in Oncologia (GIVIO), Italy). The study was designed to determine whether the addition of goserelin and/or tamoxifen to adjuvant therapy provided benefit for premenopausal women with early breast cancer as has been described elsewhere [START_REF] Baum | Adjuvant goserelin in pre-menopausal patients with early breast cancer: Results from the zipp study[END_REF].

In Stockholm all patients were included in a 2 x 2 factorial randomisation to goserelin (3.6 mg sucutaneously every 28 days), tamoxifen (40 mg daily), combination of goserelin and tamoxifen or no endocrine therapy for 2 years. Node-positive women were allocated to adjuvant CMF chemotherapy (six cycles of cyclofosfamide 600 mg/m 2 , methotrexate 40 mg/m 2 and fluorouracil 600 mg/m 2 intravenously days 1 and 8, every 28 days), in addition to endocrine therapy. Patients with four or more positive lymph nodes received additional locoregional radiotherapy including the chest wall, axillary-and supraclavicular lymph nodes, 46 Gy through 4.5 weeks. Endocrine treatment was given concurrently with chemotherapy.

The randomisation was stratified in three groups based on nodal status and use of other adjuvant therapies: node negative patients who received no adjuvant chemotherapy, patients with 1-3 positive lymph nodes who received chemotherapy, and patients with four or more positive lymph nodes who received both chemotherapy and loco-regional radiotherapy (Figure 1). Randomisation was carried out by telephone to a central office where the patient identifiers were recorded before the allocated treatment was revealed to the responsible physician. Treatment allocation was based on balanced lists using the permuted block technique.

The inclusion criteria were: invasive breast cancer ≥ 10 mm, premenopausal menstrual status (defined as last menstruation < 6 months earlier), primary surgery consisting of a mastectomy or a wedge resection plus axillary node dissection, and no clinical evidence of distant metastases. The exclusion criteria were: inoperable breast cancer, previous radiotherapy or neo-adjuvant chemotherapy, and previous or concurrent endocrine therapy.

All hormone receptor analyses on tumour samples were performed at a single laboratory, which participated in repeated control studies for receptor determination, using the same technique. The ER content was determined by isoelectric focusing on polyacrylamide gel which has been described elsewhere [START_REF] Wrange | Cytosol estradiol receptor in human mammary carcinoma: An assay based on isoelectric focusing in polyacrylamide gel[END_REF]. The receptor values were normalised to DNA content as measured by Burton and expressed as the binding capacity of estradiol in femtomoles (fmol) per microgram (μg) of DNA [START_REF] Burton K | A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid[END_REF]. Tumours with a receptor content equal or more than 0.05 fmol/μg DNA, were classified as ER positive, whereas tumours with estrogen content less than 0.05 fmol/μg DNA, were classified as ER negative [START_REF] Rutqvist | The stockholm trial on adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor level and treatment effect[END_REF]. The ER positive tumors were further sub divided into intermediate ER: 0.05 -0.59 fmol/μg DNA and high ER: ≥ 0.60 fmol/μg DNA.

The study was designed and conducted according to ICH-Good Clinical Practice guidelines and was approved by the Karolinska Institute Regional Ethics Committee.

Statistical methods

Time to event was calculated as time from date of randomisation to date of disease recurrence, contralateral breast cancer, other cancer or death without a reported recurrence, whichever came first. Time for alive and event-free patients, was calculated from the date of randomisation to the common end-date for follow-up, December 31 2005. Kaplan-Meier technique was used to estimate failure probability, and the log rank test was used to test for difference in time to event between treatment groups. Hazard rate ratios and 95% confidence intervals were estimated using proportional hazards regression. When assessing the main treatment effect of tamoxifen or goserelin, the other treatment was used as control. Test for interactions was performed by inclusion of product terms in the regression models. The analyses were on the basis of intention to treat. No analyses were done on the basis of treatment received. We also analysed the effect of treatment on time to first recurrence according to level of ER content, i.e. ER negative (< 0.05 fmol/μg DNA), intermediate ER (0.05-0.59 fmol/μg DNA), and high ER (≥ 0.60 fmol/μg DNA). The cut-off for the two ER positive groups was made at a level, which created equally sized groups. Data on ER content was available on 793 patients (86%). Interaction between treatments and ER content were further investigated graphically by use of Subpopulation Treatment Effect Pattern Plots (STEPP) [START_REF] Bonetti | A graphical method to assess treatment-covariate interactions using the cox model on subsets of the data[END_REF]. The STEPP analysis was performed using the program stepp_tail, implemented in the statistical software Stata [START_REF] Royston | A new approach to modelling interactions between treatment and continuous covariates in clinical trials by using fractional polynomials[END_REF].

Results

A total of 927 women in Stockholm were recruited to the study from 31 May 1990 to 8 January 1997. Of the 927 recruited women, 234 were randomised to the control (C) arm, 231 were randomised to receive goserelin (G), 231 to tamoxifen (T) and 231 to goserelin plus tamoxifen (GT) therapy. Patient characteristics were well balanced between the treatment groups as seen in table 1. Nodal status was known for all women and hormone receptor status reported for 86% of the women included in the analysis. One woman was incorrectly randomised twice and one woman was diagnosed with recurrent disease at the date of randomisation. The remaining 925 women were included in the analysis.

The common end of follow-up was January 1, 2006. After a median follow up time of 12.3 years, 166 women presented with loco-regional recurrence as a first event, 159 had a distant metastases, 54 had contra-lateral breast cancer, 50 women were diagnosed with other cancers and there were 6 deaths as first event. The overall number of deaths from breast cancer was 225 and there were 26 non-breast cancer deaths. The total number of first events was 435.

There were 128 (55%) first events in the C group, 98 (42%) in G treated, 101 (44%) among T treated and 108 (47%) in the GT group.

Three main sets of analyses were carried out, first the overall effect of endocrine therapy versus no endocrine therapy, secondly the effect of goserelin with or without tamoxifen and finally the effect of tamoxifen with or without goserelin.

In the first set of analysis, the control group was used as a reference. In comparison to the controls, endocrine treatment with either goserelin alone, combined with tamoxifen or tamoxifen alone, reduced the risk of first recurrence by 32 % (CI, 0.52 to 0.89), 24 % (CI, 0.59 to 0.98) and 27 % (CI, 0.56 to 0.94) respectively (P = 0.021) (Figure 2). The second analysis showed that goserelin treatment reduced the risk of first recurrence by 16% (CI, 0.68 to 1.02). There was no additional beneficial effect of goserelin with tamoxifen, but in the absence of tamoxifen, first recurrence was reduced by 32% (CI, 0.53 to 0.89). In the third set of analysis, we examined the effect of tamoxifen. The main effect of tamoxifen was not statistically significant (HR: 0.89, CI, 0.74 to 1.08), but when examined separately for those not treated with goserelin, there was a 27% benefit for tamoxifen (CI, 0.56 to 0.95). A test for interaction between goserelin and tamoxifen was statistically significant (P = 0.025) (Table 2, Figure 3).

When we examined the treatment effect for different levels of ER content, there was an overall effect of goserelin in the high ER group (HR: 0.71, CI, 0.50 to 0.99, P = 0.044) and a trend towards greater risk reduction with increasing levels of ER content (Figure 4). The greatest risk reduction from goserelin treatment in the group with high ER was observed among those not receiving tamoxifen (HR: 0.52, CI, 0.32 to 0.84, P = 0.007).

The main effect of tamoxifen in the high ER group was not significant (HR: 0.85, CI, 0.61 to 1.18) (Figure 4). Neither was there a significant risk reduction for tamoxifen alone (HR: 0.68, CI, 0.44 to 1.05, P = 0.081). For the intermediate ER group, there was neither a main effect of goserelin (HR: 0.80, CI, 0.57 to 1.14), nor tamoxifen (HR: 0.97, CI, 0.68 to 1.38). Finally, among those classified as ER negative, there was no main effect of goserelin (HR:1.25, CI, 0.85 to 1.82), tamoxifen (HR: 0.85, CI, 0.59 to 1.24) or combined goserelin and tamoxifen (HR: 1.03, CI, 0.63 to 1.67). In the intermediate and ER negative groups, the treatment effect of goserelin and tamoxifen, was not modified by each other (Figure 4).

Discussion

In previously published and recently updated ZIPP trial analysis, some inconsistencies in the study design were noted, i.e. randomisation to tamoxifen had been optional in some centres [START_REF] Baum | Adjuvant goserelin in pre-menopausal patients with early breast cancer: Results from the zipp study[END_REF][START_REF] Hackshaw | Long-term effectiveness of adjuvant goserelin in premenopausal women with early breast cancer[END_REF]. Subsequently, some women received tamoxifen electively. The Stockholm and GIVIO centres alone, maintained the 2 x 2 factorial randomisation throughout the trial. In addition, the Stockholm cohort was well defined according to hormone receptor content, nodal status and whether chemotherapy and/or radiotherapy was given or not.

The ZIPP trial was included in the meta-analysis of the Early Breast Cancer Overview group, which concluded that LHRH agonists alone are effective in the adjuvant setting in hormone receptor positive breast cancer, both in terms of recurrence-free and overall survival. The optimal timing and duration of use however, remains unclear and there is lack of data from randomised trials where LHRH agonists are tested against chemotherapy, with or without tamoxifen in both arms [START_REF] Cuzick | Use of luteinising-hormone-releasing hormone agonists as adjuvant treatment in premenopausal patients with hormone-receptor-positive breast cancer: A metaanalysis of individual patient data from randomised adjuvant trials[END_REF]. Our results from the present Stockholm sub-cohort, are in concordance with the previously published results of the conjoint ZIPP trial data, where goserelin effect on recurrence is considerable and the most marked effect is seen among those not receiving concomitant tamoxifen. The pooled trial results also showed a survival benefit not possible for us to examine here because of a small sample size. Despite the limited sample size, the strength of this report is the strict randomised design and execution of the study, as recently recommended by the Cochrane review group. This design allows a formal test of interaction which is not possible for the overall trial data of 2706 patients. Furthermore, the single laboratory analyses of ER content with a quantitative cytosol method, allowed an analysis of the effect of different receptor levels on outcome. This formal test of interaction between goserelin and tamoxifen in a randomised cohort confirms the indication made in the Hackshaw report, that tamoxifen provides no additional benefit among women treated with goserelin [START_REF] Hackshaw | Long-term effectiveness of adjuvant goserelin in premenopausal women with early breast cancer[END_REF]. The same is true for the opposite, where goserelin provides no additional benefit for tamoxifen treated. Tamoxifen is effective in extending the time to first recurrence, but only among those not receiving goserelin. The lack of additional benefit of combined endocrine therapy has previously been demonstrated in the postmenopausal adjuvant as well as the metastatic setting [START_REF] Baum | Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early breast cancer: First results of the ATAC randomised trial[END_REF][START_REF] Bergh | First results from FACTan open-label, randomized phase III study investigating loading dose of fulvestrant combined with anastrozole versus anastrozole at first relapse in hormone receptor positive breast cancer[END_REF]. As our study is not powered for survival analysis, the ongoing Suppression of Ovarian Function Trial (SOFT), should answer the question of the effect of combination endocrine therapy in the premenopausal setting, more decisively. In our strictly randomised cohort, the analysis of estrogen receptor levels confirms the lack of effect on ER negative women, in line with previous reports [18]. An intriguing finding is however, that the effect of goserelin increases with higher ER levels, whereas the same does not apply for tamoxifen. This may be a finding of chance, but the data indicates a clear difference, which may be based on biological grounds. Goserelin, which has stronger estrogen suppressor effect compared to tamoxifen, is potentially more effective when ER content is high. In this group, tamoxifen may not have sufficient effect to counteract the highly estrogenic milieu of premenopausal women. The STEPP plots demonstrating this effect, are however based on small subsets of patients, and the data will therefore have to be interpreted conservatively. Among postmenopausal women, earlier studies have shown a significant trend in reduction of recurrence rate with higher ER levels among tamoxifen treated, supporting the significance of stronger ER content in endocrine treatment [START_REF] Fisher | Influence of tumor estrogen and progesterone receptor levels on the response to tamoxifen and chemotherapy in primary breast cancer[END_REF][START_REF] Rutqvist | The stockholm trial on adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor level and treatment effect[END_REF][START_REF] Viale | Prognostic and predictive value of centrally reviewed expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and tamoxifen adjuvant therapy for postmenopausal early breast cancer: Big 1-98[END_REF][START_REF] Khoshnoud | Immunohistochemistry compared to cytosol assays for determination of estrogen receptor and prediction of the long-term effect of adjuvant tamoxifen[END_REF].

Quality of life aspects and the long-term side effects are important factors to be taken into consideration when assessing improvements in adjuvant therapy. The overview has shown that goserelin is an option for women where chemotherapy is contraindicated or strongly opposed to cytotoxic drugs [START_REF] Cuzick | Use of luteinising-hormone-releasing hormone agonists as adjuvant treatment in premenopausal patients with hormone-receptor-positive breast cancer: A metaanalysis of individual patient data from randomised adjuvant trials[END_REF]. Premature menopause is highly probable after chemotherapy and desire for pregnancy is not uncommon after completed treatment. Moreover, some women may be reluctant to the adverse effects of permanent ovarian failure, such as infertility and accelerated bone loss. These factors have been studied extensively in the Stockholm cohort and reported in several publications [START_REF] Sverrisdottir | Adjuvant goserelin and ovarian preservation in chemotherapy treated patients with early breast cancer: Results from a randomized trial[END_REF][START_REF] Sverrisdóttir | Bone mineral density among premenopausal women with early breast cancer in a randomized trial of adjuvant endocrine therapy[END_REF][START_REF] Berglund | Effect of endocrine treatment on sexuality in premenopausal breast cancer patients: A prospective randomized study[END_REF][START_REF] Nystedt | Side effects of adjuvant endocrine treatment in premenopausal breast cancer patients: A prospective randomized study[END_REF]. Our study on ovarian function showed that amenorrhea among goserelin and chemotherapy treated, was more often reversible compared to those not receiving goserelin [START_REF] Sverrisdottir | Adjuvant goserelin and ovarian preservation in chemotherapy treated patients with early breast cancer: Results from a randomized trial[END_REF]. We did not observe this effect among women who received tamoxifen in addition to goserelin. However, there was lack of power for analysis of survival effect from different endocrine treatment in the subset of women that developed chemotherapy induced amenorrhea. Results from studies examining the possible protective effect of goserelin on ovarian function are nevertheless conflicting and results from ongoing trials are awaited. Our study on bone mineral density (BMD) showed a substantial but reversible decrease in BMD after 2 years of goserelin therapy whereas the addition of tamoxifen to goserelin resulted in less changes in BMD [START_REF] Sverrisdóttir | Bone mineral density among premenopausal women with early breast cancer in a randomized trial of adjuvant endocrine therapy[END_REF]. The possible role of aromatase inhibitor (AI) treatment in combination with an LHRH agonist is not clear but the addition of AI's is likely to exaggerate the bone effects further. At present, there is limited data on this approach in the adjuvant setting, but AI's have shown a marked effect on bone metabolism.

The Austrian Breast and Colorectal Cancer Study Group (ABCSG)-12 trial showed that zoledronic acid not only counteracts effectively the demineralising effects from goserelin, but improves disease-free survival as well [START_REF] Gnant | Endocrine therapy plus zoledronic acid in premenopausal breast cancer[END_REF]. Accordingly, bone loss is manageable and should not restrict the use of goserelin. Further studies on the Stockholm cohort showed that sexual dysfunction as well as physical effects from goserelin diminished with time, whereas chemotherapy related symptoms were on-going at follow-up at 3 years [START_REF] Berglund | Effect of endocrine treatment on sexuality in premenopausal breast cancer patients: A prospective randomized study[END_REF][START_REF] Nystedt | Side effects of adjuvant endocrine treatment in premenopausal breast cancer patients: A prospective randomized study[END_REF] A limitation of the Stockholm study may be the 2 years of 40 mg/day tamoxifen, which was standard at the time, instead of 5 years of 20 mg/day treatment as currently recommended. In contrast to tamoxifen, 2 years of goserelin is still within the recommended treatment period, although the optimal duration of treatment is not yet determined. Another limitation is the concurrent use of chemotherapy in the trial, which may not be optimal based on the risk of endocrine therapy interaction with chemotherapy, and sequential treatment currently being recommended as a standard. However, this concern is primarily based on the report by Albain et al, and the study lacked power to reveal significant statistical difference, although the hazard ratio indicates that concurrent therapy should not be given [START_REF] Albain | Adjuvant chemotherapy and timing of tamoxifen in postmenopausal patients with endocrineresponsive, node-positive breast cancer: A phase 3, open-label, randomised controlled trial[END_REF].

In conclusion, goserelin is effective in reducing risk of recurrence and improving survival in endocrine responsive premenopausal breast cancer. These effects are not enhanced further by the addition of tamoxifen, and there is a significant interaction between goserelin and tamoxifen. Quality of life is affected and side effects are considerable from goserelin, but they are manageable and moreover, reversible. This study indicates that there may be an additional subgroup of women besides the very young, i.e. those with strongly ER positive tumors, who benefit more from goserelin treatment, whereas the effect of tamoxifen does not seem to be modified by ER content. A significant interaction indicates that the effect of goserelin depends on whether tamoxifen is given or not and the effect of tamoxifen depends on whether goserelin is given or not. Our data supports, that there is no additional benefit from combination endocrine therapy in the premenopausal setting. Within the limitations of the exploratory approach and limited power as a stand-alone trial and, our results should be viewed mainly as hypothesis generating, awaiting data from ongoing trials such as SOFT.

Legend Table 2. Effects of goserelin and tamoxifen on time to first recurrence 1

Test for interaction between goserelin and tamoxifen p=0.025.

Abbreviations: CI, confidence interval. 1 First of: disease recurrence, contralateral breast cancer, other cancer or death without a reported recurrence. 2 Comparison of goserelin given versus goserelin not given (reference), controlling for tamoxifen. 3 Comparison of tamoxifen given versus tamoxifen not given (reference), controlling for goserelin.

Legend

Figure 1 .Figure 2 .Figure 4 .

 124 Figure 1. Study design and flow diagram

Figure 1 .Figure 2 .

 12 Figure 1. Study design and flow diagram

Table 2 .

 2 Effects of goserelin and tamoxifen on time to first recurrence1

	Analyses

>0.05 fmol/ g DNA.

<0.05 fmol/ g DNA.

Likelihood-ratio test.

Acknowlegdements

This trial was supported by the Swedish Cancer Society and King Gustav V Jubilee Fund. Jonas Bergh's research group was supported as well by The Swedish Research Fund, Stockholm County Council Research Funds (ALF/FoU), The Stockholm Cancer Society, The Swedish Breast Cancer Association (BRO), Karolinska Instituet, Stockholm County Council Research Strategy Initiative and Märit and Hans Rausing's Initiative Against Breast Cancer. We would like to thank the following contributing physicians participating in the trial in Stockholm: J. Einhorn, L.Perbeck, B. Cedermark, U.Ringborg, N. Wilking, L. Skoog, M. Iiristo, G. Svane, I. Fredriksson, P. Nikolaidis, J. Frisell, K. Sandelin, P. Gunnarsson, E. Göransson, (Karolinska University Hospital), U. Glas, A. Somell, F. Celebioglu, L (Södersjukhuset Hospital). R. Fernstad, L. Löfgren (St. Görans Hospital), M-L. Hjalmar, K. Dahlberg (Danderyd Hospital), E. Af Trampe, (Sophiahemmet). L. Lenner (Visby Hospital), C. Wadström, M. Al Haider (Södertälje Hospital), T. Teve (Sabbatsberg Hospital), J.

Askegren (Huddinge University Hospital), as well as all former and present members of the Stockholm Breast Cancer Group. Finally we like to thank the nursing staff at the breast units at Karolinska-, Danderyd-, Huddinge-and Söder Hospitals, contributing to the logistics of the trial, coordinating patient visits and examinations.

Legend

The curves show hazard ratios on a logarithmic scale for treatment ER interactions. Circles represents the treatment effect (plotted at the category mean) in the ER-categories: <0.05, 0.05-0.59 and ≥0.60 fmol/μg/DNA.