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Originally published as JCO Early Release 10.1200/JCO.2007.13.5939 on January 7 2008 © 2008 American Society of Clinical Oncology. Short Preoperative Treatment With Erlotinib Inhibits Tumor Cell Proliferation in Hormone Receptor–Positive Breast Cancers
From the Departments of Medicine, Pathology, Biostatistics, Surgery, and Cancer Biology, Breast Cancer Research Program, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Medicine, Nashville, TN; Ventana Medical Systems, Inc, Tucson, AZ; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC; University of Alabama at Birmingham Cancer Center, Birmingham, AL; and Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA Corresponding author: Carlos L. Arteaga, MD, Division of Oncology, Vanderbilt University Medical Center, 2220 Pierce Ave, 777 PRB, Nashville, TN 37232-6307; e-mail: carlos.arteaga{at}vanderbilt.edu
Purpose To administer the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib to patients with operable untreated breast cancer during the immediate preoperative period and to measure an antiproliferative and/or a proapoptotic effect in the post-therapy specimen and determine a biomarker profile associated with evidence of erlotinib-mediated cellular activity.
Patients and Methods Newly diagnosed patients with stages I to IIIA invasive breast cancer were treated with erlotinib 150 mg/d orally for 6 to 14 days until the day before surgery. Erlotinib plasma levels were measured by tandem mass spectrometry the day of surgery. Drug-induced changes in tumor cell proliferation and apoptosis were assessed by Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling analysis, respectively, in biopsies from the pretherapy and surgical specimens. Biopsies were also evaluated for P-EGFR, P-HER-2, P-MAPK, P-Akt, P-S6, and S118 P-ER
Results In drug-sensitive PC9 xenografts, 5 days of treatment with erlotinib were enough to induce a maximal inhibition of cell proliferation and induction of apoptosis. Forty-one patients completed preoperative treatment with erlotinib. Grade Conclusion A presurgical approach to evaluate cellular responses to new drugs is feasible in breast cancer. EGFR inhibitors are worthy of testing against ER-positive breast cancers but are unlikely to have clinical activity against HER-2–positive or triple-negative breast cancers.
In breast cancer, there are many novel molecular-targeted therapies in preclinical and clinical development. For the majority, if not all, of these therapies, there is not a clear biomarker profile in tumors that can allow for the selection or exclusion of patients into phase II efficacy trials with these drugs or with combinations that include them. Some data suggest that short-term, tissue-based pharmacodynamic trials may provide information that can be later used for patient selection. For example, administration of antiestrogens for a period of 1 to 3 weeks has been shown to induce a significant antiproliferative effect in estrogen receptor (ER) –positive breast cancers.1-3 These studies have evaluated proliferation in the tumor by measuring the percentage of cells that stain with an antibody against the nuclear antigen Ki67.4 Interestingly, in all of these trials, there was no effect in the ER-negative cancers. In other neoadjuvant trials, short-term end points have correlated with clinical outcome. For example, treatment-induced tumor cell apoptosis, as measured by cleaved caspase-3 immunohistochemistry (IHC) 1 week after administration of the human epidermal growth factor receptor 2 (HER-2) antibody trastuzumab, correlates with clinical response of HER-2–overexpressing breast cancers.5 The neoadjuvant Immediate Preoperative Anastrozole, Tamoxifen, or Combined With Tamoxifen trial compared anastrozole, tamoxifen, and the combination of the two drugs. Drug-induced inhibition of tumor cell proliferation at 2 weeks, as measured by Ki67, was better in patients treated with anastrozole versus patients in the other two arms.6 This result parallels the result of the large Arimidex, Tamoxifen, Alone or in Combination trial, in which relapse-free survival was greater in patients treated with adjuvant anastrozole compared with tamoxifen or the combination.7 Furthermore, this presurgical approach allows access to tumor tissues in 100% of enrolled patients, facilitating the unbiased discovery of biomarkers that can be linked to evidence of cellular and/or clinical activity.
We present here the results of a short-term presurgical study with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib in women with untreated operable breast cancer. The results can be summarized as follows. First, short-term treatment with erlotinib inhibited tumor cell proliferation (Ki67), P-EGFR, and P-HER-2. Second, the inhibition of proliferation occurred mainly in ER-positive but not in HER-2–positive or triple-negative cancers. Third, treatment was associated with a marked reduction in levels of P-MAPK, P-Akt, P-S6, and S118 P-ER
Clinical Trial This was a multicenter trial of erlotinib in patients with untreated operable breast cancer. After signing informed consent, patients with stages I to IIIA (T1-2, N0-2, M0) breast cancer who were considered not to require neoadjuvant chemotherapy underwent a core biopsy and started erlotinib 150 mg/d orally for 6 to 10 days until approximately 24 hours before surgery. Pretherapy and surgical specimen biopsies were promptly frozen or immersed in formalin. ER and progesterone receptors (PR) were measured using standard IHC methods. HER-2 was graded as per the Dako Herceptest (Dako, Carpinteria, CA). HER-2 gene amplification was assessed by fluorescent in situ hybridization using the Vysis method (Abbott Molecular Inc, Des Plaines, IL). HER-2–positive tumors were those scoring 3+ by IHC or with  two copies of the HER-2 gene by fluorescent in situ hybridization.
Erlotinib Plasma Levels
IHC
Statistical Analysis
Xenograft Studies
Cell Lines, Drugs, and Chemicals
Immunoblot and Immunoprecipitation
Erlotinib Inhibits Proliferation and Induces Apoptosis in EGFR-Dependent Xenografts We first examined the optimal timing for detecting the cellular activity of erlotinib in tumors established in nude mice. PC9 cells contain a del746-750 in exon 19 of the EGFR that results in hypersensitivity to erlotinib10,11; A549 cells harbor a K-Ras mutation and are erlotinib resistant.13 Subcutaneous PC9 and A549 xenografts measuring 600 ± 130 µL and 800 ± 145 µL, respectively, were randomly assigned to erlotinib 100 mg/kg/d orally or to vehicle. Control and treated tumors were harvested after 5, 10, and 14 days of treatment. Proliferation and apoptosis in situ were measured by BrdU incorporation and TUNEL, respectively. PC9 tumors were eliminated by therapy, whereas the A549 tumors did not respond (Fig 1). On day 5, tumor sections from PC9-treated xenografts already exhibited a 77% reduction in BrdU incorporation and an 88% increase in TUNEL-positive cells. Similar changes were observed on days 10 and 14 (erlotinib v vehicle, P < .05; Fig 2), suggesting that drug-induced cellular activity at 5 days already predicts the clinical activity observed after more prolonged therapy. Conversely, A549 tumors did not show changes in proliferation at any of the time points. Interestingly, the proportion of TUNEL-positive cells on days 5 and 14 was lower in treated A549 xenografts than in controls (Fig 2).
Clinical Trial, Toxicity, and Erlotinib Levels Fifty-two patients were enrolled. Table 1 lists demographics and tumor characteristics. Only five cancers were EGFR-positive by IHC. Of 23 hormone receptor–positive cancers, 20 were ER positive/PR positive, and three were ER positive/PR negative. Four patients were not eligible, one patient withdrew before starting treatment, and six patients withdrew while on erlotinib as a result of toxicity. Forty-one patients completed treatment ranging from 6 to 10 days. Toxicity is listed in Table 1. Rash (grade 1 in 25% of patients and grade 2 in 35%) was the most common adverse effect. Two patients stopped treatment as a result of skin toxicity; this resulted in no delay in surgery and was promptly reversible on discontinuation of erlotinib. Blood was collected within 24 hours of the last dose of erlotinib and was available in 30 patients. Erlotinib plasma levels were 8.8 ± 7.4 µmol/L.
Erlotinib Inhibits EGFR and HER-2 Phosphorylation in Primary Breast Tumors Five (one HER-2 positive, two triple negatives, and two ER positives) and nine (one HER-2 positive, four triple negatives, and four ER positives) cancers exhibited detectable P-EGFR and P-HER-2 in the pretherapy specimen, respectively. In four of five P-EGFR–positive and eight of nine P-HER-2–positive post-therapy cores, there was a marked reduction in P-EGFR and P-HER-2 (Fig 3); the overall inhibition of phosphorylation was 83% and 89%, respectively (Fig 3). Of note, the highest P-HER-2 score was detected in an HER-2–overexpressing cancer, but the other eight P-HER-2–positive tumors scored anywhere from 0 to 2+ by IHC.
The effect of erlotinib on activated HER-2 could be interpreted as direct or indirect via blockade of the EGFR tyrosine kinase. To answer this, we used Rat1 cells expressing hemagglutinin-tagged EGFR or HER-2 chimeras. These chimeric receptors contain FK506-binding protein in their carboxyl terminus and the low-affinity nerve growth factor receptor in the ectodomain to prevent activation by autocrine ligands.14 Addition of the bivalent FK506-binding protein ligand AP1510 resulted in phosphorylation of chimeric EGFR and HER-2 as measured by phosphotyrosine immunoblot of hemagglutinin precipitates. Preincubation with  1 µmol/L erlotinib inhibited ligand-induced EGFR phosphorylation, whereas 3 µmol/L inhibited P-HER-2 (Fig 4). We next confirmed the effect of erlotinib on endogenous HER-2 in the HER-2–overexpressing BT-474 and MDA-453 breast cancer cells. The MDA-453 cells lack EGFR expression.15 In both cell lines, 1 to 3 µmol/L of erlotinib suppressed basal HER-2 phosphorylation as measured by phosphotyrosine immunoblot of cell lysates precipitated with a HER-2 antibody (Fig 4).
Erlotinib Inhibits Cell Proliferation in Primary Breast Cancers In 34 patients with paired pre- and post-therapy cores, there was an overall reduction in cell proliferation (P = .004; Fig 5A). The baseline Ki67 index was lower in ER-positive cancers compared with HER-2–positive and triple-negative cancers (10.9%, 27.5%, and 43.9% Ki67-positive cells, respectively; P = .004). The inhibition of proliferation was only significant in the ER-positive tumors (10.9% pretreatment v 3.2% post-treatment; P = .0006). No significant change was observed in HER-2–positive tumors (27.5% pretreatment v 16.4% post-treatment; P = .17) and triple-negative tumors (43.9% pretreatment v 38.9% post-treatment; P = .57; Fig 5D). There was no induction of apoptosis by TUNEL analysis.
Erlotinib Inhibits ErbB Signaling in Primary Breast Tumors and Breast Cancer Cell Lines Changes in Akt, MAPK, and ribosomal protein S6 phosphorylation were evaluated as readouts of the effect of erlotinib on signaling pathways downstream of ErbB receptors. Erlotinib treatment induced significant changes in S473 P-Akt (n = 30), S235/236 P-S6 (n = 31), and P-MAPK (n = 31; Fig 6). When analyzed by tumor type, these changes were statistically significant only in ER-positive tumors (P-Akt, pretreatment v post-treatment, P = .001; P-MAPK, P = .05; P-S6, P = .006).
We next treated BT-474 and MDA-453 cells with increasing concentrations of erlotinib for 3 hours in serum-free conditions and examined effects on Akt, S6, and MAPK using phospho-specific antibodies. The erlotinib-sensitive BT-474 cells (inhibitory concentration at 50% < 1 µmol/L) showed a clear reduction in S473 P-Akt, S235/235 P-S6, and P-MAPK, whereas the erlotinib-resistant MDA-453 cells (inhibitory concentration at 50% > 5 µmol/L) exhibited a minor inhibition of phosphorylation only at the higher dose (Fig 7).
Erlotinib Inhibits S118 P-ER  and Cell Proliferation in ER-Positive Breast CancersThe inhibition of ErbB receptor signaling in situ and the effect on tumor cell proliferation in ER-positive cancers in response to erlotinib prompted us to examine a possible mechanism that would link these two observations. ErbB receptor signaling has been shown to post-translationally modify the ER and coactivators and induce ER transcriptional activity and is associated with relapse after adjuvant endocrine therapy.16,17 MAPK phosphorylates ER in S118, and mutations in this site markedly impair ER function. Furthermore, S118 P-ER correlates with P-MAPK,18 suggesting that phosphorylation at this site is a marker of cross-talk between ErbB receptors and ER. Therefore, we examined phosphorylation of ER at S118 before and after treatment with erlotinib. Analysis of 14 paired samples from hormone receptor–positive tumors showed an overall significant decrease (P = .021; Figs 8A, 8B, and 8C) and a significant correlation between S118 P- ER and P-MAPK after treatment (r = 0.52, P = .04).
A complete cell cycle response (CCCR), as defined by a 1% Ki67 index after neoadjuvant therapy with the aromatase inhibitor letrozole, has been shown to predict a good outcome after adjuvant hormonal therapy.19 Furthermore, the Ki67 value 2 weeks after neoadjuvant hormonal therapy has been shown to predict relapse-free survival in postmenopausal patients with ER-positive tumors.20 These data suggested to us that a 1% Ki67 index in the posterlotinib specimen could be construed as a rigorous marker of cellular response. Thus, we examined whether evidence of a CCCR in the surgical specimen was different according to tumor type. A CCCR was observed in 13 (59%) of 22 ER-positive, four (44%) of nine HER-2–positive, and zero of nine triple-negative post-treatment surgical specimens (Fig 8D). Of note, three of four HER-2–positive specimens that exhibited a Ki67 1% in the post-treatment surgical biopsy were ER positive. Thus, 16 of 17 tumors achieving a CCCR were hormone receptor positive, including all three ER-positive/PR-negative tumors.
We report here the results of a presurgical trial with erlotinib in patients with untreated invasive breast cancer. Data on the clinical activity of EGFR antagonists in breast cancer are mixed.21-26 In these trials, no biomarker was used to select patients for enrollment. The purpose of this study was two-fold. First, we aimed to examine the feasibility of this approach with a drug that has not yet been approved for the treatment of breast cancer. Second, we aimed to identify a biomarker profile associated with breast cancers in which short treatment with the EGFR TKI reduces proliferation and/or induces apoptosis. We propose that, in turn, this biomarker profile can be used for patient selection in phase II trials with EGFR antagonists. Patients were treated for 6 to 10 days before surgery, and erlotinib action was assessed in the resected cancer specimen. Several arguments suggest that this duration of therapy is adequate to assess the full cellular effect of erlotinib on the tumor. In highly responsive PC9 xenografts, a drug-induced effect on tumor cell proliferation and apoptosis was already maximal after 5 days of treatment (Fig 2). Furthermore, previous pharmacokinetic studies indicate that, after 7 days of erlotinib at the dose of 150 mg/d, a minimum steady-state level in plasma ranging from 0.8 to 6.7 µmol/L is achieved.27,28 In all patients tested, the plasma levels of erlotinib on the day of surgery were consistent with these reported levels. A significant inhibition of cell proliferation was observed in several EGFR-negative cancers. We cannot rule out the possibility of this being secondary to an off-target effect of the small-molecule EGFR inhibitor. Nonetheless, in four of five cancers that were P-EGFR–positive at baseline, there was a reduction in phosphorylation in the erlotinib-treated specimen, which is suggestive of EGFR specificity. Drug activity in cancers with undetectable receptor levels is not unprecedented because the EGFR antibody cetuximab has been shown to induce tumor regression in patients with colon cancers that are EGFR negative by IHC.29 Moreover, several P-EGFR–positive tumors in our cohort were negative when stained with total EGFR antibodies, underscoring the weakness of the latter reagents in detecting low levels of EGFR expression.
Activated HER-2 was also inhibited by short-term treatment with erlotinib. Although a direct inhibitory effect of erlotinib against the HER-2 tyrosine kinase is difficult to prove in humans, data presented in Figures 3 and 4 suggest that the effect may be direct. First, at pharmacologically achievable concentrations, erlotinib inhibited the phosphorylation of ligand-activated HER-2 chimeric receptors. Second, Despite drug-induced inhibition of P-HER-2, there was no inhibition of cell proliferation in HER-2–overexpressing tumor. This is consistent with the report by Sergina et al33 that proposes that, in HER-2–overexpressing breast cancer cells, the transient inhibition of HER-2 phosphorylation induced by erlotinib is followed by feedback upregulation of active HER-3 and P-Akt. Indeed, inhibition of P-Akt did not occur in erlotinib-resistant MDA-453 cells (Fig 4) or in the HER-2–positive tumors. Unfortunately, in our hands, commercially available P-HER-3 antibodies were inadequate for quantification of active HER-3 in formalin-fixed specimens. Erlotinib did not inhibit proliferation in triple-negative breast cancers. This result is relevant because of the reported overexpression of EGFR in this subtype of breast cancer and the proposal that the receptor is a therapeutic target in triple-negative tumors.34 Current trials with EGFR antibodies in patients with this type of breast cancer will shed light on this question. Given the data shown in Figure 5, we believe that EGFR inhibitors are unlikely to have clinical activity in triple-negative breast cancers.
The inhibition of proliferation and postreceptor signaling pathways was only detectable in ER-positive tumors. These data are consistent with at least three reports showing predominant activity of gefitinib in ER-positive breast cancers.23-25 At a first glance, this result is counterintuitive. However, the marked inhibition of P-MAPK by erlotinib and the known ability of this kinase to phosphorylate ER In summary, this presurgical study supports the feasibility of testing novel therapies during the preapproval process to investigate a tumor profile of potential use in subsequent clinical studies that address drug efficacy. This approach requires additional examples and experience. We speculate that this approach may expedite the drug development process by potentially informing the exclusion of nonresponsive patients who will dilute the net signal of clinical activity of a drug or a combination. In the case of erlotinib, these patients would be those with HER-2–positive and the triple-negative cancers. In this study, short treatment with erlotinib inhibited active EGFR and HER-2, ErbB receptor signaling, and tumor cell proliferation in situ. The inhibition of P-MAPK was pronounced, suggesting, first, that, in breast cancer, this signal transducer is mainly under regulation by the ErbB receptor network and, second, that changes in P-MAPK are a good surrogate of ErbB signaling activation and inactivation. This inhibition of cell proliferation and signaling was limited to hormone receptor–positive tumors, suggesting that EGFR antagonists are worthy of testing in hormone-dependent tumors in combination with antiestrogens.
Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Ian E. Krop, Genentech (C); Carlos L. Arteaga, Sunesis (C), Bristol-Myers Squibb Co (C), AstraZeneca (C), GlaxoSmithKline (C), InNexus (C), Monogram (C), OSI Pharmaceuticals (C) Stock Ownership: None Honoraria: None Research Funding: Ian E. Krop, Genentech; Carlos L. Arteaga, Genentech Expert Testimony: None Other Remuneration: None
Conception and design: Marta Guix, Gary Pestano, Yu Shyr, Senthil Muthuswamy, Mark C. Kelley, Carlos L. Arteaga Financial support: Carlos L. Arteaga Administrative support: Bobbye M. Wieman, Kerek E. Frierson, Carlos L. Arteaga Provision of study materials or patients: Nara de Matos Granja, Ingrid Meszoely, Violeta Sanchez, Ana M. Grau, Ingrid A. Mayer, Gary Pestano, Benjamin Calvo, Helen Krontiras, Ian E. Krop, Mark C. Kelley, Carlos L. Arteaga Collection and assembly of data: Marta Guix, Nara de Matos Granja, Theresa B. Adkins, Kerek E. Frierson, Violeta Sanchez, Melinda E. Sanders, Gary Pestano, Mark C. Kelley, Carlos L. Arteaga Data analysis andinterpretation: Marta Guix, Nara de Matos Granja, Melinda E. Sanders, Gary Pestano, Yu Shyr, Senthil Muthuswamy, Mark C. Kelley, Carlos L. Arteaga Manuscript writing: Marta Guix, Nara de Matos Granja, Melinda E. Sanders, Gary Pestano, Ian E. Krop, Mark C. Kelley, Carlos L. Arteaga Final approval of manuscript: Marta Guix, Nara de Matos Granja, Theresa B. Adkins, Bobbye M. Wieman, Kerek E. Frierson, Violeta Sanchez, Melinda E. Sanders, Ana M. Grau, Ingrid A. Mayer, Gary Pestano, Yu Shyr, Senthil Muthuswamy, Benjamin Calvo, Helen Krontiras, Ian E. Krop, Mark C. Kelley, Carlos L. Arteaga
Immunohistochemistry Immunohistochemistry was performed on 4-µm paraffin-embedded tumor sections using the following eight antibodies: Ki67 (MIB-1), S473 P-Akt, S235/236 P-S6, P- MAPK, S118 P-ER , EGFR (clone H11), Y1068 P-EGFR, and Y1221/1222 P-HER-2. The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling assay was performed using the Apop-Tag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Billerica, MA) according to the manufacturer's instructions. For all reactions, slides were deparaffinized in xylenes and graded alcohols. Peroxidase and protein blocks, when performed, were achieved with 3% H2O2 for 20 minutes and Dako protein block reagent (Dako, Carpinteria, CA) for 10 minutes, respectively. Staining with Y1068 P-EGFR and Y1221/1222 P-HER-2 antibodies was achieved with the automated Discovery XT staining platform (Ventana Medical Systems, Inc, Tucson, AZ), and primary antibodies were detected using the UltraMap DAB anti-Rb detection kit (Ventana). Finally, slides were counterstained with hematoxylin, dehydrated with graded alcohols and xylenes, and mounted. Ki67, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling, P-MAPK, and P-ER were scored by recording the percentage of positive nuclei. S235/236 P-S6 and P- MAPK staining was scored according to the staining intensity using the following histoscore: P-S6 score = 1x (percent cells with weak staining) + 2x (percent cells with moderate staining) + 3x (percent cells with strong staining). We developed the following scoring system for P-Akt, which measures both the percentage of stained nuclei and the intensity of cytoplasmic staining, combining them into one score: percent positive nuclei + 1/3 (cytoplasmic intensity x 100). The cytoplasmic staining intensity for P-Akt was graded as follows: 0 = none; 1 = weak; 2 = moderate; 3 = strong. Y1068 P-EGFR and Y1221/1222 P-HER-2 staining was quantified using the same histoscore as S235/236 P-S6 and P- MAPK. All stains were scored by two pathologists (N.M.G. and M.E.S.).
Cell Lines, Drugs, and Chemicals
Immunoblot and Immunoprecipitation
published online ahead of print at www.jco.org on January 7, 2008. Supported in part by National Institutes of Health Grant No. R01 CA80195 (C.L.A.), Breast Cancer Specialized Program of Research Excellence (SPORE) Grant No. P50 CA98131, Avon-National Cancer Institute Grant No. CA098131-03S1 (C.L.A.), and Vanderbilt-Ingram Comprehensive Cancer Center Support Grant No. P30 CA68485. M.E.S. and I.A.M. are recipients of Vanderbilt Physician-Scientist Development Awards. A grant from Genentech also partially supported the clinical trial. Presented in part at the 29th Annual San Antonio Breast Cancer Symposium, December 14-17, 2006, San Antonio, TX. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Perez-Torres M, Guix M, Gonzalez A, et al: Epidermal growth factor receptor (EGFR) antibody down-regulates mutant receptors and inhibits tumors expressing EGFR mutations. J Biol Chem 281:40183-40192, 2006 11. Koizumi F, Shimoyama T, Taguchi F, et al: Establishment of a human non-small cell lung cancer cell line resistant to gefitinib. Int J Cancer 116:36-44, 2005[CrossRef][Medline] 12. Wang SE, Shin I, Wu FY, et al: HER2/Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by transforming growth factor beta. Cancer Res 66:9591-9600, 2006 13. Yauch RL, Januario T, Eberhard DA, et al: Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 11:8686-8698, 2005 14. Muthuswamy SK, Gilman M, Brugge JS: Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers. Mol Cell Biol 19:6845-6857, 1999 15. Arteaga CL, Hurd SD, Dugger TC, et al: Epidermal growth factor receptors in human breast carcinoma cells: A potential selective target for transforming growth factor alpha-Pseudomonas exotoxin 40 fusion protein. Cancer Res 54:4703-4709, 1994 16. Bjornstrom L, Sjoberg M: Mechanisms of estrogen receptor signaling: Convergence of genomic and nongenomic actions on target genes. Mol Endocrinol 19:833-842, 2005 17. Ali S, Coombes RC: Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2:101-112, 2002[CrossRef][Medline] 18. Sarwar N, Kim JS, Jiang J, et al: Phosphorylation of ERalpha at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERalpha phosphorylation in breast cancer progression. Endocr Relat Cancer 13:851-861, 2006 19. Ellis MJ, Tao Y, Bhatnagar AS, et al: Cell cycle complete response after neoadjuvant letrozole predicts superior relapse-free and overall survival: Long-term follow-up of the letrozole P024 study. J Clin Oncol 25:20s, 2007 (suppl 18S; abstr 570)[CrossRef] 20. Dowsett M, Smith IE, Ebbs SR, et al: Prognostic value of Ki67 expression after short-term presurgical endocrine therapy for primary breast cancer. J Natl Cancer Inst 99:167-170, 2007 21. Baselga J, Albanell J, Ruiz A, et al: Phase II and tumor pharmacodynamic study of gefitinib in patients with advanced breast cancer. J Clin Oncol 23:5323-5333, 2005 22. Winer EP, Cobleigh M, Dickler M, et al: Phase II multicenter study to evaluate the efficacy and safety of Tarceva (erlotinib, OSI- 774) in women with previously treated locally advanced or metastatic breast cancer. Breast Cancer Res Treat 76:445A, 2002 (abstr) 23. Agrawal A, Gutteridge E, Cheung KL, et al: Efficacy and tolerability of gefitinib in oestrogen receptor negative and tamoxifen resistant oestrogen receptor positive locally advanced or metastatic breast cancer. Breast Cancer Res Treat 24:S61, 2005 (suppl)[CrossRef] 24. Polychronis A, Sinnett HD, Hadjiminas D, et al: Preoperative gefitinib versus gefitinib and anastrozole in postmenopausal patients with oestrogen-receptor positive and epidermal-growth-factor-receptor-positive primary breast cancer: A double-blind placebo-controlled phase II randomised trial. Lancet Oncol 6:383-391, 2005[CrossRef][Medline] 25. Ciardiello F, Troiani T, Caputo F, et al: Phase II study of gefitinib in combination with docetaxel as first-line therapy in metastatic breast cancer. Br J Cancer 94:1604-1609, 2006[Medline] 26. Tan AR, Yang X, Hewitt SM, et al: Evaluation of biologic end points and pharmacokinetics in patients with metastatic breast cancer after treatment with erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor. J Clin Oncol 22:3080-3090, 2004 27. Hidalgo M, Siu LL, Nemunaitis J, et al: Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 19:3267-3279, 2001 28. Lu JF, Eppler SM, Wolf J, et al: Clinical pharmacokinetics of erlotinib in patients with solid tumors and exposure-safety relationship in patients with non-small cell lung cancer. Clin Pharmacol Ther 80:136-145, 2006[CrossRef][Medline] 29. Chung KY, Shia J, Kemeny NE, et al: Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol 23:1803-1810, 2005 30. Moulder SL, Yakes FM, Muthuswamy SK, et al: Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res 61:8887-8895, 2001 31. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al: Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353:123-132, 2005 32. Thatcher N, Chang A, Parikh P, et al: Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: Results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 366:1527-1537, 2005[CrossRef][Medline] 33. Sergina NV, Rausch M, Wang D, et al: Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature 445:437-441, 2007[CrossRef][Medline] 34. Livasy CA, Perou CM, Karaca G, et al: Identification of a basal-like subtype of breast ductal carcinoma in situ. Hum Pathol 38:197-204, 2007[CrossRef][Medline] Submitted July 24, 2007; accepted October 30, 2007.
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
