Activated HER-receptors in predicting outcome of ER-positive breast cancer patients treated with adjuvant endocrine therapy

The Breast 21 (2012) 662e668

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Original article

Activated HER-receptors in predicting outcome of ER-positive breast cancer patients treated with adjuvant endocrine therapy Mathilde S. Larsen a, b, *, Karsten Bjerre c, Anne E. Lykkesfeldt b, Anita Giobbie-Hurder d, Anne-Vibeke Lænkholm e, f, Katrine L. Henriksen b, g, Bent Ejlertsen c, Birgitte B. Rasmussen a a

Department of Pathology, Herlev Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark Department of Breast Cancer Research, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen Ø, Denmark Danish Breast Cancer Cooperative Group, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark d IBCSG Statistical Center, Dana-Farber Cancer Institute, 450 Brookline Ave., CLSB 11007, Boston, MA, USA e Department of Pathology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 March 2012 Received in revised form 28 June 2012 Accepted 4 July 2012

The four human epidermal growth factor receptors (HER1-4) are involved in growth stimulation and may play a role in endocrine resistance. The receptors form dimers, leading to activation by mutual phosphorylation. Our purpose was to explore the role of the activated receptors (pHER1, pHER2, pHER3) in endocrine treated breast cancer in terms of co-expression and association with disease-free survival (DFS) in 1062 patients with ER-positive tumors. Furthermore, HER2 amplification was evaluated. We found positive associations between the phosphorylated receptors. pHER1 and pHER3 were co-expressed with one or two of the other activated receptors in 85% and 89% of tumors, respectively, whereas pHER2 was co-expressed with the other activated receptors in 54% of tumors. Except for HER2, which was associated with poor prognosis, none of the remaining markers were associated with DFS. However, frequent co-expression indicates a role of the other HER-family members in activation of HER2. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Breast cancer ER-positive Endocrine resistance HER-receptors Phosphorylated HER

Introduction Endocrine therapy is the mainstay of adjuvant therapy for patients with estrogen receptor (ER)-positive breast cancer.1 Endocrine therapy with antiestrogens or aromatase inhibitors targets estrogen action and thereby prevents the growth stimulation mediated by the activated ER.2,3 However, tumor resistance to endocrine therapy is inevitable in many of these patients. The human epidermal growth factor receptor (HER)-family comprises four homologous transmembrane receptors (HER1, also

* Corresponding author. Department of Pathology, Herlev Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark. Tel.: þ45 38689248; fax: þ45 38683711. E-mail addresses: [email protected] (M.S. Larsen), kb@ dbcg.dk (K. Bjerre), [email protected] (A.E. Lykkesfeldt), [email protected] (A. Giobbie-Hurder), [email protected] (A.-V. Lænkholm), k.weischenfeldt@ gmail.com (K.L. Henriksen), [email protected] (B. Ejlertsen), birgitte.bruun.rasmussen@ regionh.dk (B.B. Rasmussen). f Present address: Department of Pathology, Slagelse Hospital, Ingemannsvej 18, 4200 Slagelse, Denmark. g Present address: Signal Transduction in Cancer and Metabolism Group, German Cancer Research Center (DKFZ), Im Neunheimerfeld 580, D-69120 Heidelberg, Germany. 0960-9776/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.breast.2012.07.005

known as EGFR, HER2, HER3 and HER4), which form a complex system involved in growth regulation.4 Upon ligand binding, the HER-receptors form dimers, resulting in autophosphorylation, activation of the tyrosine kinase activity and downstream signaling pathways.5 Recent evidence suggests that growth factor receptor signaling contributes to endocrine resistance.6e8 Both the ER- and the HER-receptor pathway are capable of driving growth of breast cancer, and there is evidence that the two signaling pathways are intertwined.9 While membrane ER can activate HER2 signaling, the kinase cascade downstream of HER2 can phosphorylate and activate ER and its co-regulatory proteins.10 The importance of molecular crosstalk in endocrine resistance is indicated by the association between increased expression of HER1 and/or HER2 in breast cancer tumors and a poor response to tamoxifen treatment.11e14 An association between positive HER1 status and ER-negative status has been reported in many studies, but the prognostic significance of HER1 status remains unclear.15 Approximately 15% of all breast cancers are HER2-positive,16 and the proportion is about 10% in ER-positive breast cancers.17 HER2-positive status is reported to be an independent predictor of poor prognosis.18,19 Furthermore, high expression of HER2 has been associated with

M.S. Larsen et al. / The Breast 21 (2012) 662e668

resistance to endocrine therapy.11,14,17,20,21 HER3 is expressed in 20e70% of breast cancers22,23 but the prognostic value of HER3 remains unclear23e27 although HER3 overexpression in patients with early relapse on tamoxifen has been reported.28 HER4 signaling has been described to promote differentiation and growth inhibition of breast cancer cells.29 In addition, HER4 is related to a favorable prognosis in breast cancer25,30,31 and loss of HER4 expression may represent a marker for resistance to tamoxifen.32 The HER-receptors are activated by mutual interaction and, therefore, a more comprehensive profile of all the HER-receptors and their activated forms could be more informative than the expression of individual HER-family members. As phosphorylation is a hallmark of active signaling, the presence of the activated form of the receptors, as detected by antibodies directed at the phosphorylated form, might play a potential role for clinical outcome in ER-positive breast cancer patients. Little work has been conducted on the phosphorylated HER-receptors in breast cancer. Thor et al33 found prognostic value of phosphorylated HER2 (pHER2) in a subgroup of node-positive patients, and in a report by Cicenas et al.,34 pHER2 was significantly associated with poor survival in both HER2-negative and HER2-positive patients. In a pilot study by Frogne et al.6 we evaluated the importance of expression of the activated forms of HER1, HER2 and HER3 by immunohistochemical (IHC) analysis of primary breast tumors from patients treated with adjuvant tamoxifen. We found that phosphorylated HER1 (pHER1), pHER2 and phosphorylated HER3 (pHER3) were positively associated with poor DFS in univariate analysis. Furthermore, strong expression of pHER2 in more than 10% of the tumor cells was associated with both poor DFS and poor OS when adjusting for the current standard prognostic parameters. The present study was designed to use IHC to determine both the total level of each of the four HER-receptors and the level of the activated HER1, HER2 and HER3 in order to evaluate the role of the individual receptors and the activated forms, as well as the impact of concomitant expression of activated HER-receptors on response to adjuvant endocrine therapy. Patients and methods Patients Archival paraffin tissue blocks of primary breast cancer samples from Danish patients participating in the randomized, doubleblinded, clinical trial BIG 1-98, were used for this study. BIG 1-98 was a phase 3 trial, investigating the effect of adjuvant tamoxifen and letrozole treatment in postmenopausal women with hormone receptor-positive breast cancer. Treatment arms were: tamoxifen or letrozole for 5 years, 2 years of tamoxifen or letrozole followed by 3 years of letrozole or tamoxifen.35 In total, 1396 Danish patients were randomized in BIG 1-98. Primary tumor samples from 1323 patients were available for tissue microarray (TMA) preparation with two 2-mm cores from each

663

tumor.36,37 IHC staining was performed on 1303 TMA samples available for the study. Thirty patients with negative or unknown ER status were excluded from the analysis; 211 patients were excluded because a complete set of the investigated markers was unattainable due to insufficient tumor tissue in either of the stainings. The remaining 1062 patients were included in the analysis. The study has been approved by The Danish National Committee on Biomedical Ethics in Denmark in 1997 with the current biomarker study as an addendum approved in 2004. Immunohistochemistry and fluorescence in situ hybridization (FISH) Sections were cut from the TMA blocks. Table 1 shows details about IHC antibodies, dilutions, retrieval methods and visualization systems used. The IHC analyses of HER1, pHER1, pHER2, HER3 and pHER3 were performed on the fully automated instruments: PT Link, Pre-treatment Module (Dako), and Autostainer Link 48 instrument (Dako). HER2 staining with antibody included in “HercepTest for the TechMate Instrument” (Dako) was performed according to the manual. Tumors with IHC HercepTest score of 2þ were further analyzed by FISH as recommended by Herceptest Guidelines using Histology FISH accessory kit, K5599 from Dako. HER4 immunostaining was carried out on the automated immunostainer, Tech-mate Horizon (Dako). With each batch of slides, specimens or cell lines known to exhibit positive reaction for the different HER/pHER antibodies were stained simultaneously: skin tissue (HER1, pHER1), carcinoma of the lung (HER1, pHER1), HER2amplified breast carcinoma (HER2, pHER2), normal colon tissue (HER3), MCF-7 cells stimulated with Heregulin (Recombinant Human HRG1-b1, R&D Systems) (pHER3), and control MCF-7 cells grown under standard conditions (HER4). Evaluation of immunohistochemistry The evaluation of all markers was performed blindly without reference to the patient history. Only staining of the membrane of tumor cells was evaluated. HER2 and HER4 were evaluated according to the HercepTest guidelines. For tumors with a HER2 score of 2þ, FISH-analysis was performed; a HER2/CEN ratio of 2.0 or grater was considered to be HER2 amplified. Furthermore, HER2 was also evaluated together with HER1, pHER1, pHER2, HER3 and pHER3 using an extended HercepTest scoring system, where category 0 was subdivided in two categories: 00 (no reaction), 01 (positive reaction in <10%). The alternative scoring system was applied to explore the clinical relevance of very low expression of the receptors. In subsequent analyses, HER1, pHER1, pHER2, HER3, pHER3 and HER4 were classified as positive if the score was 1þ or more, whereas analyses of HER2 implied two classifications: (1) positive if the score was 1þ (as the rest of the receptors), or (2) amplified if the score was 3þ or 2þ with FISH amplification (as the conventional HER2 assessment used in the clinic).

Table 1 Antibodies and methods used for immunohistochemical staining. IHC HER1 pHER1 HER2 pHER2 HER3 pHER3 HER4

Phospho-site Tyr 1173 Tyr 1221/1222 Tyr 1289

Antibody/Assay

Dilution

M7298, Dako #4407, Cell Signaling HercepTestÔ for the TechMate #2243, Cell Signaling M7297, Dako #4791, Cell Signaling RB9045, Neomarkers

1:50 EnvisionÔ FLEX Kit, Dako 1:100 EnvisionÔ FLEX Kit, Dako Instrument, Dako 1:100 EnvisionÔ FLEX Kit, Dako 1:100 EnvisionÔ FLEX Kit, Dako 1:200 EnvisionÔ FLEX Kit, Dako 1:300 Pronase E, Sigma 15 min, 37  C

Antigen retrieval

Visualization system

ChemMate EnVisionþDetection Kit, Dako

664

M.S. Larsen et al. / The Breast 21 (2012) 662e668

Clinical data and endpoint Clinical data and follow-up data were obtained from the International Breast Cancer Study Group (IBCSG) Statistical Center. DFS was defined as the time from random assignment to the earliest of invasive recurrence in local, regional, or distant sites, new contralateral invasive breast cancer, new secondary non-breast malignancy, or death from any cause. Observations of follow-up were censored at the date of last patient contact before the data retrieval date of July 2008. Statistics The threshold for HER1, pHER1, HER2, pHER2, HER3, pHER3 and HER4 was set at score 1þ to define a positive tumor.6 The coexpression of total and phosphorylated HER-receptors was assessed by Fisher’s exact test after this dichotomization. The association between HER-family receptor scores and standard clinical and pathological prognostic markers was evaluated by the Wilcoxon rank-sum test. The included variables were: tumor size, number of positive lymph nodes, tumor grade, and age at randomization, see Table 2. Follow-up time for DFS was quantified in terms of a KaplaneMeier (KM) estimate of median potential follow-up. DFS according to HER-receptor scores was estimated by the KM method and statistical significance was estimated by the log-rank test. The association between DFS and HER-receptor scores was evaluated in univariate Cox proportional hazards models, stratified by randomization option and allocated treatment. The score category with most observations was used as reference level. If the

Table 2 Characteristics of Danish participants of the BIG 1-98 study. Patients in the study population had ER-positive disease and had a complete set of observations of HER1, pHER1, HER2, pHER2, HER3, pHER3 and HER4. Characteristic

Study population N (%)

All 1062 Year of surgery 1998e99 250 2000e01 475 2002e03 337 Age at randomization, y 45e54 124 55e64 577 65e75 361 Postmenopausal category Postmenopausal 1012 Not postmenopausal 5 HRT stopped 45 Tumor size, mm 0e20 496 21e50 535a 50þ 31 Histological type Inv Ductal 902a Inv Lobular 30 Other 130 Tumor grade I 227 II 551 III 152 Unknown 132 Number of nodes found positive 0 365 1e3 467a 4e9 147 10 83 a

Excluded

Total

N (%)

N (%)

(100.0)

334 (100.0)

1396 (100.0)

(23.5) (44.7) (31.7)

87 (26.0) 135 (40.4) 112 (33.5)

337 (24.1) 610 (43.7) 449 (32.2)

(11.7) (54.3) (34.0)

48 (14.4) 188 (56.3) 98 (29.3)

172 (12.3) 765 (54.8) 459 (32.9)

(95.3) (0.5) (4.2)

322 (96.4) 1 (0.3) 11 (3.3)

1334 (95.6) 6 (0.4) 56 (4.0)

(46.7) (50.4) (2.9)

188 (56.3) 134 (40.1) 12 (3.6)

685 (49.1) 668 (47.9) 43 (3.1)

(84.9) (2.8) (12.2)

267 (79.9) 8 (2.4) 59 (17.7)

1167 (83.6) 38 (2.7) 189 (13.5)

(21.4) (51.9) (14.3) (12.4)

83 166 30 55

(24.9) (49.7) (9.0) (16.5)

310 717 182 187

(22.2) (51.4) (13.0) (13.4)

(34.4) (48.6) (13.8) (7.8)

140 113 51 30

(41.9) (33.8) (15.3) (9.0)

505 580 198 113

(36.2) (41.5) (14.2) (8.1)

Including one observation with missing value.

highest HER-receptor score category had fewer than 10 observations, the category was merged with the second-highest score category. The potential prognostic impact of co-expression of phosphorylated HER-receptors was evaluated for a derived variable expressing the number of positive receptors at the 10% level. Statistical significance was defined as P < 0.05. Results The patient characteristics of the study population are presented in Table 2. Relative to the study population, the excluded patients had a higher frequency of node-negative, low grade, and small tumors (0e20 mm). Table 3 shows the distribution of the scores. For HER1, pHER1, pHER2 and pHER3, no staining was found in the majority of the tumors. The frequencies of positive tumors at the 1þ threshold (10% positive tumor cells) were 3.9% (HER1), 13.0% (pHER1), 56.6% (HER2), 20.7% (pHER2), 45.3% (HER3), 7.2% (pHER3), and 42.6% (HER4). Of the pHER1, pHER2 and pHER3 positive tumors 3%, 6% and 10.5% respectively were scored as 3þ (Fig. 1). Notably, more tumors were scored as pHER1-positive than as HER1-positive. When conventional HER2 assessment including FISH was used, 9% of the tumors were HER2 amplified. Among tumors eligible for HER1 staining but excluded from the study as ER-negative, 45% (10/22) were HER1-positive, a notably higher frequency than among ER-positive tumors. The total receptors HER1, HER2, HER3, and HER4 were significantly associated with standard prognostic factors in two instances. HER2 was positively associated with tumor grade (P < 0.0001) and HER3 was positively associated with the number of positive lymph nodes (P ¼ 0.01). The activated receptors pHER1, pHER2, and pHER3 were all negatively associated with tumor size (P ¼ 0.015, P ¼ 0.0005, P ¼ 0.004) and pHER1 and pHER3 were negatively associated with the number of positive lymph nodes (P ¼ 0.03, P ¼ 0.01). In addition, pHER1 was negatively associated with age at randomization (P ¼ 0.008) and pHER2 was positively associated with tumor grade (P ¼ 0.002). The pair-wise co-expression of HER-receptors is shown in Table 4. The association between the total HERs and their phosphorylated forms was positive for HER2 and pHER2 (P < 0.0001) and for HER3 and pHER3 (P < 0.0001), whereas there was no association between HER1 and pHER1. Although a phosphorylated form should theoretically co-occur with a total form this was not always the case: Among the 461 tumors scored as HER2-negative, 68 were pHER2-positive (14.8%) and, in comparison, among the 601 tumors scored as HER2-positive, 152 were pHER2-positive (25.3%). Among the HER3-negative tumors, 4.1% were pHER3-positive, and among the HER3-positive tumors, 10.8% were pHER3-positive. Among the HER1-negative tumors, 12.7% were pHER1-positive, and among the HER1-positive tumors, 19.5% were pHER1-positive. Table 3 Distribution of basic scores. HER4 scores according the IHC HercepTest scoring system and HER1, pHER1, HER2, pHER2, HER3, and pHER3 scores according to the extended HercepTest scoring system (n ¼ 1062). Score

HER1

pHER1

HER2

pHER2

HER3

pHER3

HER4

00 01 1þ 2þ 3þ

889 132 30 8 3

834 90 103 31 4

114 347 451 86 64

745 97 150 57 13

469 112 446 35 0

919 67 51 17 8

610b 259 146 47

Positivea (%)

(3.9)

(13.0)

(56.6)

(20.7)

(45.3)

(7.2)

(42.6)

a

10% positive cells regardless of intensity. Score 0 of the IHC HercepTest scoring system is equivalent of score 00 and 01 combined. b

M.S. Larsen et al. / The Breast 21 (2012) 662e668

665

Fig. 1. Positive immunohistochemical staining of pHER1 (A), pHER2 (B) and pHER3 (C), all scored as 3þ.

There were statistically significant, positive relationships between the co-expression of phosphorylated receptors pHER1 and pHER2 (P < 0.0001), pHER1 and pHER3 (P < 0.0001), and pHER2 and pHER3 (P < 0.0001). HER4 was positively correlated with pHER1, HER2, pHER2 and HER3. HER2 amplification assessed by conventional HercepTest and subsequent FISH was significantly correlated with HER2, pHER2, HER3, pHER3 and HER4, but not with HER1 or pHER1 (Table 4). In total, 24% of the tumors had positive expression of at least one of the three phosphorylated receptors (12% expressed one, 7% two, and 5% three phosphorylated receptors). Compared to this, 74% of the tumors expressed at least one of the total receptors HER1, HER2 or HER3 (43% expressed one, 31% expressed two, and <1% expressed three total receptors). Table 5 shows the frequency with which the activated receptors were expressed alone compared to how frequently they were co-expressed with one or two of the others. Of the 138 tumors classified as pHER1-positive, 46% were also positive for either pHER2 or pHER3, and 39% were positive for

both pHER2 and pHER3. In total, 85% of the pHER1-positive tumors were positive for at least one of the other phosphorylated HERreceptors. Likewise, pHER3-positive tumors expressed either pHER1 or pHER2 simultaneously in 18% of the cases, and both pHER1 and pHER2 in 71% of the cases, resulting in 89% of the cases being simultaneously expressed with at least one of the other activated receptors. In contrast, pHER2 was only expressed in combination with one or two other activated receptors in 55% of the cases (Table 5). Neither total HER1, HER3, HER4 nor any of the phosphorylated receptors expressed alone or simultaneously showed any association to DFS. However, as expected, there was a strong correlation between HER2 and DFS both when assessed according to the extended scoring system (P ¼ 0.002, Fig. 2) and when assessed by the conventional HercepTest guidelines including FISH evaluation (P ¼ 0.0002). Hazard ratios and P-values of univariate Cox regression models with each of the total or phosphorylated receptors are shown in Table 6.

Table 4 Frequencies and co-expression of HER-receptors. HER1 Neg pHER1 Neg 891 Pos 130 a P-value HER2 Neg 436 Pos 585 P-value pHER2 Neg 808 Pos 213 P-value HER3 Neg 557 Pos 464 P-value pHER3 Neg 946 Pos 75 P-value HER4 Neg 587 Pos 434 P-value HER2 IHC þ FISH Normal 924 Amplified 97 P-value All a

1021

Pos

pHER1

HER2

Neg

Neg

Pos

Pos

pHER2

HER3

Neg

Neg

Pos

Pos

pHER3

HER4

Neg

Neg

Pos

All

33 8 0.23 25 16 0.024 ()

421 503

40 98 0.00022 (þ)

34 7 0.70

816 108

26 112 <0.0001 (þ)

393 68

449 152 <0.0001 (þ)

24 17 0.64

535 389

46 92 <0.0001 (þ)

288 173

293 308 <0.0001 (þ)

497 345

84 136 <0.0001 (þ)

40 1 0.36

908 16

78 60 <0.0001 (þ)

439 22

547 54 0.0082 (þ)

828 14

158 62 <0.0001 (þ)

557 24

429 52 <0.0001 (þ)

23 18 0.87

555 369

55 83 <0.0001 (þ)

284 177

326 275 0.017 (þ)

523 319

87 133 <0.0001 (þ)

369 212

241 240 <0.0001 (þ)

572 414

38 38 0.19

38 3 1.00

839 85

123 15 0.53

461 0

501 100 <0.0001 (þ)

783 59

179 41 <0.0001 (þ)

541 40

421 60 0.002 (þ)

902 84

60 16 0.002 (þ)

566 44

396 56 0.005 (þ)

41

924

138

461

601

842

220

581

481

986

76

610

452

Fishers exact test. Associations were considered significant when P < 0.05.

%

Pos 924 138

(87.0) (13.0)

461 601

(43.4) (56.6)

842 220

(79.3) (20.7)

581 481

(54.7) (45.3)

986 76

(92.8) (7.2)

610 452

(57.4) (42.6)

962 100

(90.6) (9.4)

1062

666

M.S. Larsen et al. / The Breast 21 (2012) 662e668

Table 5 Frequency of tumors positive for activated HER1, HER2, and HER3 alone or in coexpression with one or two of the other activated HER-receptors. Receptor

Total

Expressed alone (%)

Co-expressed with one (%)

Co-expressed with two (%)

pHER1 pHER2 pHER3

138 220 76

20 (14.5) 100 (45.5) 8 (10.5)

64 (46.4) 66 (30.0) 14 (18.4)

54 (39.1) 54 (24.5) 54 (71.1)

Discussion In the light of the potential role of all HER-receptors, and especially the activated forms of the receptors, as growthmediating factors in endocrine resistant breast cancer, we evaluated the IHC expression of HER1, HER2, HER3, HER4, pHER1, pHER2 and pHER3 in ER-positive tumors from patients treated with adjuvant endocrine therapy and tested the impact of expression of these receptors on clinical outcome. Due to lack of an applicable commercially available antibody, pHER4 staining could not be included. Our pilot study6 showed that all three activated HER-receptors were associated with poor DFS. Although we have used the same antibodies and IHC evaluation techniques, with the exception of an extension with score 01, we could not confirm an association of pHER1, pHER2 or pHER3 with poor DFS in the current study. The frequency of pHER1, pHER2 and pHER3 were 13%, 21% and 7%, which are significantly lower than the frequencies of 18%, 40% and 15% reported in the pilot study, respectively.6 It has been reported that some phosphoproteins are labile and are affected by the amount of time from excision to fixation, e.g. the phosphorylated forms of the signaling molecules Akt and Erk.38 Although specimens from breast cancer tissue are handled very uniformly throughout Denmark, including recommendations for fixation time,39 the tissue used in the pilot study was from one hospital, and special precautions were taken to ensure that the sections were fixed rapidly. Therefore preservation of phosphorylated HER-receptors may have been more optimal in the pilot

Fig. 2. KaplaneMeier curves of association between DFS the level of HER2 according to the extended HercepTest (n ¼ 1064).

Table 6 Association between DFS and HER-family receptors: HER2 IHCþFISH, HER1, pHER1, HER2, pHER2, HER3, pHER3, and HER4.a Risk factor

Level

n

HR

(95% CI)

Pb

HER2 IHCþFISH

Normal Amplified 00 01 1þ 2þ(3þ) 00 01 1þ 2þ(3þ) 00 01 1þ 2þ 3þ 00 01 1þ 2þ 3þ 00 01 1þ 2þ(3þ) 00 01 1þ 2þ(3þ) 0 1þ 2þ 3þ

962 100 889 132 30 11 834 90 103 35 114 347 451 86 64 745 97 150 57 13 469 112 446 35 919 67 51 25 610 259 146 47

1.00 1.91 1.00 1.04 1.88 1.27 1.00 1.03 1.20 1.39 1.19 0.89 1.00 1.11 2.10 1.00 1.07 1.34 1.17 0.94 1.00 1.26 1.23 1.34 1.00 0.86 1.05 1.02 1.00 1.02 1.21 0.81

referent (1.37e2.68) referent (0.74e1.47) (1.05e3.38) (0.47e3.44) referent (0.69e1.55) (0.82e1.74) (0.78e2.49) (0.82e1.72) (0.67e1.17) referent (0.72e1.72) (1.39e3.18) referent (0.71e1.62) (0.97e1.84) (0.71e1.92) (0.30e2.95) referent (0.87e1.85) (0.96e1.59) (0.70e2.56) referent (0.52e1.43) (0.61e1.79) (0.48e2.17) referent (0.77e1.35) (0.87e1.68) (0.44e1.50)

0.0002

HER1

pHER1

HER2

pHER2

HER3

pHER3

HER4

a b

0.19

0.58

0.0024

0.50

0.33

0.94

0.58

Univariate analysis stratified for randomization option and allocated treatment. Wald’s test.

material. Another important difference between the two studies is the study population. The samples used in the pilot study6 were from patients for whom both paraffin blocks and fresh frozen tumor were available, and thus may contain a greater proportion of large tumors. Furthermore, the study was a retrospective cohort, and the median duration of tamoxifen treatment was only 1.8 years. We used samples from a large, randomized controlled study (BIG 198) where treatment length with tamoxifen and/or letrozole was 5 years as it is recommended today. Finally, the median time of follow up in the pilot study6 was 12.4 years compared with 6 years in this study. Thus, the patient material is very different between the two studies and this may explain the different results regarding clinical outcome and frequency of markers. We found a percentage of HER2-amplified cases of 7%, which is similar to the one found in BIG 1-98 patient cohort as a whole, as well as in an identical patient population in the ATAC trial.14,17 It would be relevant to repeat the analysis when longer follow-up is obtained. Strong HER4 expression has been associated with good prognosis.6,23 In contrast, we did not find an association with DFS. We have scored HER4 staining according to the HercepTest guideline, which only evaluates the membrane staining. Witton et al.23 also evaluated the HER4 staining of the membrane only, although the HercepTest guidelines were not used, and found strong expression to be associated with increased survival. The pilot study6 classified HER4 to be positive if staining was observed in membrane, cytoplasm, and/or nucleus in more than 10% of the tumor cells. The different approaches to evaluation, and the fact that the follow-up time in both published studies was longer than in the current study, may explain the different results. Our local assessment of HER2 status revealed 9% HER2amplified tumors, which is concordant with 7% HER2-amplified

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tumors found in the central assessment of 3650 tumors from the BIG 1-98 international study.14 Also in agreement with the central evaluation, we found significantly shorter DFS for the HER2amplified tumors. Besides the conventional assessment of HER2 as either amplified or normal, we also used an extended scoring system, which was used to evaluate the other HER-markers, as well. The idea was to explore if reaction in less than 10% of tumor cells had an effect on the prognosis in this patient group. The analysis of the association between all levels of HER2 and DFS showed that only tumors with score 3þ were significantly different from the other groups. However, we observed interesting results concerning the coexpression of the phosphorylated receptors. Activated HER2 was co-expressed with other activated HER-receptors in 55% of the cases, whereas pHER1 and pHER3, were expressed with one or two other activated HER-receptors in 85% and 89% of the cases, respectively. As HER3 has no kinase activity, it must form heterodimers to elicit downstream signaling.40 Therefore, our finding of co-expression of pHER3 with other activated HER-receptors could be expected. We found that HER2 amplification status, assessed by conventional HercepTest and subsequent FISH, correlated with positive expression of pHER3 but not with pHER1. It has been stated that HER2 is the preferred heterodimerization partner of all other receptors of the HER-family, and the preferred dimerization partner of HER2 is HER3, which may explain the findings of HER3 playing a critical role in HER2 mediated promotion of breast cancer cell growth and proliferation.41,42 We observed a higher frequency of pHER3 but not of pHER1 expression in HER2-amplified tumors compared to HER2-normal tumors. This may indicate that HER3 is linked to HER2 activation, and even though only HER2 amplification is a marker of prognosis, HER3 could theoretically be a relevant therapeutic target. Surprisingly, tumors scored positive for activated HER-receptor were not always positive for total receptor. In particular, only 6% of the pHER1-positive tumors were recorded as HER1-positive. As seen in Table 3, this may partly be ascribed to a greater sensitivity of the antibody detecting pHER1, as only 3.9% of the tumors were HER1-positive whereas 13.0% were pHER1-positive. The threshold between positive and negative was set at a score of 1þ for all total and phosphorylated receptors. The possibility exists that this threshold is not biologically meaningful for all 7 markers evaluated in this study. Conclusion We could not confirm a prognostic value of HER1, pHER1, pHER2, HER3, pHER3 or HER4, but HER2 amplification was a strong indicator of poor prognosis for patients receiving endocrine therapy. Our finding of strong correlations between the expression of the three activated HER-receptors indicates a potential role of HER1 and HER3 in the activation of HER2. Therefore treatment targeting other HER-receptors, especially HER3 may be important and should be considered. Ethical approval Ethical approval was obtained for the BIG 1-98 study. Funding sources We wish to thank “Breast Friends”, “Dansk Kraeftforsknings Fond”, “Danish Agency for Science Technology and Innovation” (grant nos 09-063068 and 09-066017) and “ A Race Against Breast Cancer ” for financial support. Partial funding was provided by the US National Institutes of Health (grant no CA-75362).

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