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Originally published as JCO Early Release 10.1200/JCO.2005.04.3034 on June 5 2006 © 2006 American Society of Clinical Oncology. Estrogen-Independent Proliferation Is Present in Estrogen-Receptor HER2-Positive Primary Breast Cancer After Neoadjuvant Letrozole
From the Siteman Comprehensive Cancer Center, Washington University School of Medicine, St Louis, MO; Duke University Medical Center, Durham, NC; Edinburgh Breast Unit, Edinburgh University, Edinburgh, Scotland; Royal Marsden Hospital, London, United Kingdom; and Novartis Pharma AG, Basel, Switzerland. Address reprint requests to Matthew J. Ellis, MB, PhD, FRCP, Washington University School of Medicine, Campus Box 8056, 660 S Euclid Ave, St Louis, MO 63110; e-mail: mellis{at}wustl.edu
PURPOSE: To investigate the impact of human epidermal growth factor receptor (HER) 1 and HER2 gene amplification on endocrine therapy responsiveness, a fluorescence in situ hybridization (FISH) study was conducted on tumor samples from 305 postmenopausal patients with stage II and III estrogen receptor (ER) –positive (ER  10%) breast cancers treated on two independent neoadjuvant endocrine therapy trials. PATIENTS AND METHODS: FISH analysis focused on HER1 and/or HER2 immunohistochemistry (IHC) -positive patients and a random selection of HER1/2 IHC-negative patients. HER2 FISH status was correlated with response and changes in the proliferation marker Ki67. RESULTS: HER1 was rarely amplified (< 1%), and HER2 amplification was observed in 9.2% of patients. Letrozole response by clinical measurement (71% HER2 FISH positive v 71% HER2 FISH negative), mammogram (44% HER2 FISH positive v 47% HER2 FISH negative), or ultrasound (47% HER2 FISH positive v 54% HER2 FISH negative) was not impaired by HER2 FISH-positive status. In contrast, HER2 FISH-positive tumors showed higher histologic grade (P = .009), higher pretreatment Ki67 (P = .005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = .0001). Similar observations regarding Ki67 were made in a smaller cohort of tamoxifen-treated tumors. CONCLUSION: Neoadjuvant letrozole is clinically effective in ER-positive HER2 FISH-positive tumors, indicating sensitivity to short-term estrogen deprivation. However, continued proliferation despite ongoing letrozole or tamoxifen treatment in the majority of ER-positive HER2 FISH-positive samples (88%) could imply therapeutic resistance that may manifest later in the clinical course of the disease. Discordance between clinical and biomarker findings in this study serves to emphasize the need for surrogate end point validation in neoadjuvant endocrine trials through correlation with information on long-term outcomes.
Most studies1 have concluded that estrogen receptor (ER) –positive breast cancers exhibiting human epidermal growth factor receptor (HER) 2 protein overexpression have diminished responsiveness to tamoxifen. The exceptions have tended to occur in the setting of adjuvant studies where chemotherapy use may obscure the interaction.2 In contrast, a neoadjuvant study that compared tamoxifen with letrozole indicated that preoperative regression of ER-positive tumors in response to 4 months of letrozole was not impeded by overexpression of HER1 and/or HER2.3 A similar conclusion was reached in a preoperative study of anastrozole versus tamoxifen,4 but the size of the ER-positive, HER2-positive subsets in both these studies was too small for definitive conclusions. Small sample size is a consistent problem with the literature because ER-positive, HER2-positive tumors are uncommon, with only 10% of ER-positive disease showing evidence of HER2 gene amplification.5 Nonetheless, studies that focus on the correct endocrine approach for ER-positive HER2-positive disease are important because half of HER2 fluorescence in situ hybridization (FISH) –positive primary breast cancers exhibit some degree of hormone receptor expression. Since our initial publication,3 FISH testing has replaced immunohistochemistry (IHC) as the gold standard for HER2 assessment. Therefore, we re-examined our tumor bank using HER1 and HER2 FISH probes. Treatment-associated changes in Ki67 staining are also presented because this proliferation biomarker provides useful additional information on endocrine responsiveness in the preoperative setting.6,7 To increase our sample size, we combined clinical information and tumor samples from two independent studies of preoperative endocrine therapy.
Study Population and Tumor Bank The P024 protocol was a double-blind randomized trial that compared 4 months of preoperative letrozole with tamoxifen in postmenopausal women with stage II and III breast cancers who were ineligible for breast conservation. The clinical findings and tumor bank characteristics have been described previously.3,6,8 The P024 letrozole cohort was expanded with an additional 106 samples from a consecutive series of patients treated with preoperative letrozole in the Edinburgh Breast Unit.9 Eligibility criteria and tumor assessments for patients treated on the Edinburgh study were similar to those used in the letrozole P024 trial. However, some of the tumors treated in the Edinburgh cohort were smaller than 3 cm, and the Edinburgh patients were treated for a shorter period (typically approximately 12 weeks rather than 16 weeks). All studies were conducted with approval from the institutional review boards or ethics committees of the institutions involved in either enrolling patients or in sample analysis.
Biomarker Analysis: IHC
Biomarker Analysis: FISH
Biostatistical Analysis
Definition of Cell Cycle Complete Response
Clinical and Biomarker Characteristics of ER-Positive, HER2 Gene–Amplified Tumors and HER1 FISH Analysis HER2 gene amplification was detected in 9.2% of the samples (28 of 305 samples). The baseline clinical and biomarker characteristics of the HER2 FISH-positive versus HER2 FISH-negative population are outlined in Table 1. The clinical characteristics of patients with ER-positive, HER2 FISH-positive disease were not statistically different from patients with ER-positive, HER2-negative disease. In terms of the biomarker status, HER2 FISH-positive tumors exhibited higher pretreatment Ki67 levels than HER2 FISH-negative tumors (Mann-Whitney U test, P = < .005) and higher histologic grade (P = .0088). No statistically significant differences in clinical or biomarker status could be detected between the patients treated with tamoxifen and patients treated with letrozole, except that the tamoxifen-treated patients were slightly younger (Table 2). HER1 FISH analysis was conducted on all patients from the P024 study (nine cases in total) who had HER1 based on IHC 2 or 3+ staining. Only one case with 3+ staining exhibited HER1 gene amplification. Interestingly, this case exhibited gross amplification, with HER1 gene copies too numerous to count.
Effect of HER2 Gene Amplification on the Clinical Efficacy of Neoadjuvant Endocrine Therapy HER2 FISH analysis of specimens from the tamoxifen arm of the P024 study identified nine HER2 FISH-positive tumors, and 90 were designated HER2 FISH negative. Tamoxifen-treated tumors harboring HER2 gene amplification exhibited lower response rates than HER2 FISH-negative tumors by two of the three criteria examined (Table 3). These differences were not statistically significant, and the sample size provided limited statistical power to address the interaction. To more adequately examine the impact of HER2 FISH status on the effectiveness of letrozole, additional samples from a single-arm neoadjuvant letrozole study were analyzed. The combined 202-sample letrozole data set included 17 HER2 FISH-positive tumors and 185 HER2 FISH-negative tumors. The letrozole response data, outlined in Table 4, indicated that the presence of HER2 gene amplification did not substantially reduce the clinical effectiveness of neoadjuvant letrozole treatment, although the relatively small number of HER2 FISH-positive tumors does not rule out a limited effect. The 95% CI for the letrozole clinical response in HER2 FISH-positive tumors was 71% ± 23%. Thus, despite the sample size, a clinical response rate to letrozole of less than 48% in ER-positive, HER2 FISH-positive disease can be excluded by these data. An exploratory analysis was conducted to compare the efficacy of tamoxifen and letrozole within subsets defined by HER2 FISH status (Tables 5 and 6). These results should be interpreted with caution because the additional letrozole samples were not drawn from the original P024 double-blind randomized trial. Nonetheless, these findings do not contradict our earlier conclusions that letrozole is clinically more effective neoadjuvant therapy than tamoxifen. This advantage seems to be preserved in the HER2 FISH-positive subset.3,8
HER2 Gene Amplification and the Antiproliferative Effects of Letrozole An examination of the raw Ki67 data revealed that most HER2 FISH-negative tumors showed a dramatic decline in Ki67 on treatment (Fig 1A). The geometric mean baseline Ki67 level in the HER2 FISH-negative samples was 6.25 (95% CI, 5.16% to 7.58%); this level decreased to 0.68% after treatment (95% CI, 0.53% to 0.87%). This decrease was significant (Wilcoxon signed rank test, P = .0001; Fig 1C). In contrast, an examination of the HER2 FISH-positive group showed that the impact of letrozole was blunted, with few samples decreasing close to 0% (Fig 1B). At baseline, the geometric mean Ki67 score in the HER2 FISH-positive group was 14.73% (95% CI, 9.67% to 22.44%), and after treatment, the geometric mean decreased to 8.1% (95% CI, 4.16% to 15.75%). In contrast to the HER2 FISH-negative samples, this decrease was not statistically significant (Fig 1D). The Mann-Whitney U test, which was applied to determine whether the paired Ki67 values in the HER2 FISH-positive group were different from the values in the HER2 FISH-negative cohort, was significant (P = .0001).
HER2 Gene Amplification and the Antiproliferative Effects of Tamoxifen The 87 tamoxifen-treated HER2 FISH-negative tumors with paired Ki67 data showed marked and statistically significant decreases with treatment (Fig 2A). The baseline geometric mean Ki67 level was 5.79% (95% CI, 4.43% to 7.56%); this level decreased to 1.29% (95% CI, 0.88% to 1.88%) on treatment (Wilcoxon signed rank test, P = .0001; Fig 2C). In contrast, the Ki67 score in the tamoxifen-treated HER2 FISH-positive group did not show a statistically significant decrease; the baseline geometric mean was 6.9% (95% CI, 1.81% to 26.4%), and it was 6.5% (95% CI, 1.7% to 24.78%) in the post-treatment samples (P = .65; Fig 2B). A statistical comparison between the Ki67 changes in the HER2 FISH-positive samples and the HER2 FISH-negative samples did not reach significance (P = .0925; Fig 2D) presumably because of the small sample size; however, overall, the pattern of the results was similar to the letrozole result, which is consistent with prior conclusions that HER2 gene amplification reduces the antiproliferative effects of tamoxifen as well as letrozole.12
HER2 Gene Amplification Prevents Cell Cycle CR Table 7 examines the relationship between cell cycle CR and HER2 gene amplification in tumors treated with letrozole. HER2 FISH-positive tumors only occasionally exhibited a cell cycle CR (12%), underscoring the failure of letrozole to induce complete cell cycle arrest in most of the ER-positive HER2-positive tumors treated. In contrast, the majority (60%) of the HER2 FISH-negative letrozole-treated tumors met the definition of a cell cycle CR at the time of surgery. This difference was statistically significant (P = .0001). A second exploratory analysis was conducted that restricted the comparison to samples in which the baseline Ki67 was at least 5% in both HER2 FISH-negative tumors and HER2 FISH-positive tumors in an attempt to adjust for the fact that HER2 FISH-positive tumors had higher levels of Ki67 at baseline than HER2 FISH-negative samples (Table 1). The relationship between cell cycle CR and HER2 status remained significant in this subset (P = .0012; data not shown), indicating that the low rate of cell cycle CR in HER2 gene–amplified tumors was not simply explained by the higher baseline Ki67 level. The relationship between cell cycle CR and HER2 status on the tamoxifen arm was similar, with only one (11%) of nine HER2 FISH-positive tumors exhibiting a cell cycle CR compared with 37 (42%) of 87 HER2 FISH-negative tumors. In Table 8, a combined analysis is presented, drawing on the entire data set, regardless of whether the tumor was treated with letrozole or tamoxifen. The high level of correlation between the lack of cell cycle CR and the presence of a positive HER2 FISH test (P = .0001) is consistent with the conclusion that the presence of HER2 gene amplification generates resistance at the level of cell cycle progression regardless of which endocrine therapy is used.
This study was designed to investigate the relationship between HER2 gene amplification and the efficacy of neoadjuvant endocrine therapy. By combining two sample sets, we confirmed that the clinical efficacy of letrozole in the neoadjuvant setting was not obviously compromised by the presence HER2 gene amplification. This finding might be most helpful when considering a preoperative protocol for a postmenopausal patient with ER-positive HER2 FISH-positive disease for whom a more toxic preoperative regimen is not suitable because of extreme age, frailty, or the presence of cardiac disease. The combination of letrozole and trastuzumab for the neoadjuvant treatment of postmenopausal women with ER-positive HER2 FISH-positive disease is an appealing research question, although the relative scarcity of these tumors will make study accrual a challenge. The finding that ER-positive HER2 FISH-positive tumors and ER-positive HER2 FISH-negative tumors respond similarly to letrozole at the clinical level is a striking contrast to the consistent finding that ER-positive HER2 FISH-positive tumors are less sensitive to the cell cycle–inhibitory effects of letrozole (as well as other endocrine therapies). Dowsett et al12 have reported similar Ki67 results after a much briefer exposure to a selective estrogen receptor modulator or an aromatase inhibitor (several weeks only). Together, these results suggest that the disparity between the data on tumor regression and Ki67 changes in the present study may be a primary influence of HER2 gene amplification on the endocrine responsiveness of the initial tumor cell population rather than an acquired property of a subpopulation of estrogen-independent tumor cells that selectively emerge after several months of therapy. The discordance between the data on tumor response and Ki67 changes raises several provocative questions. The first is a biologic issue. How can major responses to estrogen deprivation occur in ER-positive HER2-positive tumor despite poor, transient, or absent cytostatic effects? Second, which end point, Ki67 changes or tumor regression (or some combination of the two end points), will best predict the likelihood that the patient will do well on long-term adjuvant endocrine treatment? Estrogen deprivation–induced tumor regression must be a complicated event, presumably involving not just the induction of cell cycle arrest, but also the involvement of other processes such as apoptosis, normalization of the tumor vasculature,13 and other changes in the composition of the tumor mass. Certainly, there are considerable data that tumor angiogenesis can be estrogen dependent, perhaps through direct regulation of vascular endothelial growth factor,14,15 and letrozole therapy can reduce tumor gadolinium contrast accumulation on breast magnetic resonance imaging, implying an effect of estrogen deprivation on the tumor vasculature.11 With respect to apoptosis, this has been quite difficult to assess in neoadjuvant endocrine studies because cells staining for apoptosis markers are rare at baseline and not obviously modulated by endocrine therapy.16 We have presented preliminary data on tumor grade and have uncovered potential differences in the morphologic response to tamoxifen and letrozole. Specifically, letrozole seems to reduce nuclear grade to a greater extent than tamoxifen.17 Whether these changes contribute to relative differences in treatment-induced decrease in tumor size is unclear. The data presented in this article serve to emphasize a need to critically evaluate neoadjuvant endocrine therapy end points in relation to relapse risk. The few studies that have addressed this issue do support the notion that a neoadjuvant response, whether defined clinically or on the basis of Ki67, does portend a better prognosis.18,19 Only when the relationships between short-term neoadjuvant outcomes and the risk of relapse are better understood will we be able to incorporate information on the short-term success of neoadjuvant endocrine therapy into planning other aspects of the adjuvant treatment plan, such as the need to administer chemotherapy or trastuzumab. Data on the influence of HER2 status on the relative benefits of tamoxifen and letrozole as adjuvant therapy in the Breast International Group (BIG) 1-98 trial are, therefore, of considerable interest. A recent preliminary report indicated that HER2-positive status was associated with a significantly higher relapse rate in the BIG 1-98 trial, regardless of whether letrozole or tamoxifen was used.20 This conclusion, if consistent in further follow-up, supports the validity of the Ki67-based predictive models presented in this article. An answer to the question of whether letrozole maintains a therapeutic advantage in the adjuvant setting despite HER2 FISH-positive status (suggested by the clinical response data) also awaits longer follow-up of the BIG 1-98 trial population. This investigation supports the notion that well-designed neoadjuvant endocrine therapy studies could be useful to identify novel molecular explanations for endocrine therapy resistance. The data outlined in Table 7 suggest that the majority of cases of endocrine resistance detected at the level of Ki67 remain unexplained because 73 (40%) of 184 ER-positive HER2-negative tumors failed to achieve a cell cycle CR and, therefore, display some degree of letrozole resistant-cell cycling. The combination of candidate gene sequencing, array-based comparative genomic hybridization, and gene expression profiling should allow the identification of these additional causes of endocrine therapy resistance and ultimately foster additional targeted approaches to the problem of endocrine therapy failure.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. 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.
Dollar Amount Codes (A) < $10,000 (B) $10,000-$99,900 (C)
We recognize the contribution of our patients and clinical research staff who made these studies possible.
Supported by Novartis Pharma AG, Basel, Switzerland (M.J.E., J.M.D., and W.R.M.) and by National Institutes of Health Grants No. CA0961402 and P30CA9184205 (M.J.E.). Presented in part at the 41st Annual Meeting of American Society of Clinical Oncology, Orlando, FL, May 13-17, 2005. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Hu JC, Mokbel K: Does c-erbB2/HER2 overexpression predict adjuvant tamoxifen failure in patients with early breast cancer? Eur J Surg Oncol 27:335-337, 2001[CrossRef][Medline] 2. Berry DA, Muss HB, Thor AD, et al: HER-2/neu and p53 expression versus tamoxifen resistance in estrogen receptor-positive, node-positive breast cancer. J Clin Oncol 18:3471-3479, 2000 3. Ellis MJ, Coop A, Singh B, et al: Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: Evidence from a phase III randomized trial. J Clin Oncol 19:3808-3816, 2001 4. Smith IE, Dowsett M, Ebbs SR, et al: Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: The Immediate Preoperative Anastrozole, Tamoxifen, or Combined With Tamoxifen (IMPACT) multicenter double-blind randomized trial. J Clin Oncol 23:5108-5116, 2005 5. Lal P, Tan LK, Chen B: Correlation of HER-2 status with estrogen and progesterone receptors and histologic features in 3,655 invasive breast carcinomas. Am J Clin Pathol 123:541-546, 2005[CrossRef][Medline] 6. Ellis MJ, Coop A, Singh B, et al: Letrozole inhibits tumor proliferation more effectively than tamoxifen independent of HER1/2 expression status. Cancer Res 63:6523-6531, 2003 7. Dowsett M, Smith IE, Ebbs SR: Short-term changes in Ki-67 during neoadjuvant treatment of primary breast cancer with anastrozole or tamoxifen alone or combined correlate with recurrence-free survival. Clin Cancer Res 11:951s-958s, 2005 8. Eiermann W, Paepke S, Appfelstaedt J, et al: Preoperative treatment of postmenopausal breast cancer patients with letrozole: A randomized double-blind multicenter study. Ann Oncol 12:1527-1532, 2001 9. Dixon JM: Role of endocrine therapy in the neoadjuvant surgical setting. Ann Surg Oncol 11:18S-23S, 2004[Medline] 10. Going JJ, Keith WN, Neilson L, et al: Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett's mucosa. Gut 50:373-377, 2002 11. Tao Y, Klause A, Vickers A, et al: Clinical and biomarker endpoint analysis in neoadjuvant endocrine therapy trials. J Steroid Biochem Mol Biol 95:91-95, 2005[Medline] 12. Dowsett M, Harper-Wynne C, Boeddinghaus I, et al: HER-2 amplification impedes the antiproliferative effects of hormone therapy in estrogen receptor-positive primary breast cancer. Cancer Res 61:8452-8458, 2001 13. Jain RK: Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy. Science 307:58-62, 2005 14. Ruohola JK, Valve EM, Karkkainen MJ, et al: Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells. Mol Cell Endocrinol 149:29-40, 1999[CrossRef][Medline] 15. Heer K, Kumar H, Read JR, et al: Serum vascular endothelial growth factor in breast cancer: Its relation with cancer type and estrogen receptor status. Clin Cancer Res 7:3491-3494, 2001 16. Dowsett M, Ebbs SR, Dixon JM, et al: Biomarker changes during neoadjuvant anastrozole, tamoxifen, or the combination: Influence of hormonal status and HER-2 in breast cancer—A study from the IMPACT trialists. J Clin Oncol 23:2477-2492, 2005 17. Turner J, Bentey R, Tao Y, et al: Changes in tumor grade with neoadjuvant endocrine therapy: Morphological evidence for fundamental differences in the anti-tumor effects of letrozole and tamoxifen. Breast Cancer Res Treat 88:S37, 2004 (abstr 407) 18. Horobin JM, Preece PE, Dewar JA, et al: Long-term follow-up of elderly patients with locoregional breast cancer treated with tamoxifen only. Br J Surg 78:213-217, 1991[Medline] 19. Miller WR, Anderson TJ, White S, et al: Growth factor signaling in clinical breast cancer and its impact on response to conventional therapies: The Edinburgh experience. Endocr Relat Cancer 12:S119-S123, 2005 (suppl 1) 20. Viale G, Regan M, Dell'Orto P, et al: Central review of ER, PgR and HER-2 in BIG 1-98 evaluating letrozole vs. tamoxifen as adjuvant endocrine therapy for postmenopausal women with receptor-positive breast cancer. Presented at the 28th San Antonio Breast Cancer Symposium, San Antonio, TX, December 8-11, 2005 (abstr 44) Submitted September 19, 2005; accepted February 3, 2006.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 tests were used to define associations between HER2 gene amplification status and clinical, mammography, ultrasound, and cell cycle responses. The nonparametric Mann-Whitney U test was applied to compare differences in Ki67 changes between HER2-positive and HER2-negative tumors. The differences between baseline and post-treatment Ki67 values within each HER2 group (amplified or not amplified) were assessed by the Wilcoxon signed rank test. The 95% CI of the geometric Ki67 mean was calculated to show the size of effects in pair-wise comparisons. All statistical analyses were performed using SAS 9.1.2 (SAS Institute Inc, Cary, NC). 
