Annals of Oncology Advance Access originally published online on September 5, 2007
Annals of Oncology 2008 19(2):315-320; doi:10.1093/annonc/mdm429
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breast cancer |
Expression patterns and predictive value of phosphorylated AKT in early-stage breast cancer
1 Department of Breast Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
2 Department of Medicine and Translational Research Unit UPRES EA 03535
3 Department of Biostatistics, Laboratoire d'Immunologie des tumeurs humaines, Institut Gustave Roussy, PR1 and IFR54, Villejuif, France
4 Department of Pathology, Institut Gustave Roussy, Laboratoire d'Immunologie des tumeurs humaines, Institut Gustave Roussy, PR1 and IFR54, Villejuif, France
5 Department of Institut National de la Santé et de la Recherche Médicale U753, Laboratoire d'Immunologie des tumeurs humaines, Institut Gustave Roussy, PR1 and IFR54, Villejuif, France
* Correspondence to: Dr F. J. Esteva, Department of Breast Medical Oncology, Unit 1354, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Tel: +1-713-792-2817; Fax: +1-713-563-0739; E-mail: festeva{at}mdanderson.org
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Abstract |
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Background: AKT phosphorylation is a critical step in the activation of growth factor receptors and can mediate tumor resistance to anthracyclines. We evaluated the expression patterns and predictive value of phosphorylated AKT (pAKT) in breast cancer tissues.
Patients and methods: pAKT expression was assessed by immunohistochemistry in 823 tumors from patients with early breast cancer enrolled in two randomized trials. The distribution of pAKT expression was correlated with HER2 and epidermal growth factor receptor (EGFR) expression. The predictive value of pAKT for the efficacy of adjuvant chemotherapy was determined by test for interaction.
Results: pAKT, EGFR, and HER2 were expressed in 119 of 781 (15%), 118 of 758 (16%), and 99 of 775 (13%) assessable tumors. Staining was positive for pAKT in 28 of 99 (28%) and 90 of 676 (13%) HER2+ and HER2– tumors (P < 0.001). pAKT was expressed in 15 of 94 (16%) and 75 of 563 (13%) HER2–/EGFR+ and HER2–/EGFR– tumors, respectively (P = 0.49). A positive staining for pAKT did not correlate with prognosis (P = 0.94), and did not predict the resistance to anthracyclines (test for interaction, P = 0.70).
Conclusions: AKT phosphorylation is associated with HER2 expression but not EGFR expression in patients with early breast cancer. pAKT is not predictive for the efficacy of anthracycline-based adjuvant chemotherapy.
Key words: AKT, breast cancer, EGFR, HER2
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introduction |
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AKT phosphorylation is a major molecular event occurring after activation of growth factor receptors and has been proposed as a surrogate marker for HER2 and epidermal growth factor receptor (EGFR) activation [1, 2]. Phosphorylated AKT (pAKT) induces mammalian target of rapamycin (mTOR) activation and, subsequently, cell proliferation, survival, and motility [3]. Several drugs, including rapamycin analogues, have been developed recently in order to specifically inhibit the PI3K/AKT/mTOR pathway through mTOR inhibition [3]. In breast cancer, AKT phosphorylation has been more specifically associated with in vitro resistance to tamoxifen [4] and doxorubicin [5–7]. Additional studies have reported that AKT/mTOR pathway inhibition could sensitize breast cancer cells to doxorubicin [8, 9]. Altogether, these data give rise to the hypotheses that AKT activation could be associated with resistance to tamoxifen or anthracycline-based chemotherapy in patients with breast cancer.
A variety of EGFR and HER2 inhibitors have been tested in patients with breast cancer. While HER2 inhibitors have been successful in the metastatic (e.g. trastuzumab, lapatinib) and adjuvant (trastuzumab) settings [10, 11], inhibition of the EGFR tyrosine kinase using gefitinib [12] or erlotinib [13] resulted in low response rates (<5%) in patients with metastatic breast cancer. These data gave rise to the hypothesis that while HER2 is an important therapeutic target in breast cancer, inhibiting EGFR activation may not be sufficient to alter the progression of breast cancer.
Although activation of the AKT pathway appears to be a potentially major event in the survival of breast cancer cells, few studies have investigated its prognostic and predictive value in tissue from breast cancer patients. Six retrospective studies [14–19] investigated the correlation between pAKT staining and clinical characteristics, outcome, or both in breast cancer. These studies reported that AKT activation was associated with HER2 expression and a higher rate of relapse after tamoxifen therapy [14, 15]. However, the results were not concordant among the studies, which was probably due to the lack of statistical power since these studies included between 99 and 402 patients.
In the present study, we carried out pAKT immunostaining in 823 patients enrolled in two prospective randomized trials that compared adjuvant anthracycline-based chemotherapy to no chemotherapy. The predefined aims of this study were to evaluate the frequency of AKT activation in HER2- and EGFR-positive human breast cancer; to determine whether AKT pathway activation correlates with the immunophenotypical patterns based on HER2, EGFR, estrogen receptor (ER), and cytokeratin (CK) 5/6 expressions; and to determine the predictive value of AKT activation for the efficacy of anthracycline-based chemotherapy in the adjuvant setting.
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patients
Primary breast cancer tissue was obtained from 823 patients who participated in two French multicenter randomized trials. The inclusion criteria and results of these trials have been reported elsewhere [20], and these two trials were approved by the Institutional Review boards of Institut Gustave Roussy (IGR). All patients gave written informed consent before registration. The primary objective of these trials was to determine the efficacy of adjuvant anthracycline-based chemotherapy compared with no chemotherapy in pre- and postmenopausal patients with early-stage breast cancer. A total of 1146 patients were enrolled in the two clinical trials from 1989 to 1995. Nine hundred and thirty-five patients (83% of the overall population) were treated at the IGR. Of the 935 patients treated at IGR, 688 were postmenopausal and presented either with histologically confirmed disease in axillary lymph nodes or with no cancer in the nodes and histologic grade II or III primary cancer. The remaining 247 patients were premenopausal and presented with negative axillary nodes and histologic grade II or III primary disease. Histologic grade was defined according to a modified Scarff–Bloom–Richardson classification scheme [21]. ER expression was determined by ligand-binding assay (cut-off 10 fmol/mg protein). The primary tumor sample was not found for 112 out of 935 patients included at the IGR. The patients corresponding to the 112 non-retrieved tumors were a bit younger than those corresponding the 823 retrieved tumors (mean age 54 versus 56, P = 0.02), less postmenopausal (63% versus 75%, P = 0.01), and less progesterone receptor-positive (ligand-binding assay, 54% versus 76%, P < 0.001). There was no evidence for a selection bias based on the stage of the disease, tumor grade, or ER positivity (ligand-binding assay).
Patients were randomly assigned to receive six cycles of adjuvant anthracycline-based chemotherapy (5-fluorouracil 500 mg/m2, epirubicin 50 mg/m2 or doxorubicin 50 mg/m2, and cyclophosphamide 500 mg/m2 administered i.v. on day 1 of a 28-day cycle) or no chemotherapy. All postmenopausal patients received adjuvant tamoxifen for at least 2 years, and they were allowed to participate in a French trial comparing 2 years of tamoxifen with long-term treatment. After they completed treatment, patients underwent clinical examination every 6 months for the first 5 years and once per year thereafter; mammography was also done annually.
tissue array and immunostaining
EGFR, HER2, and pAKT expression was assessed using immunohistochemistry on a tissue array containing primary breast cancer tissue from 823 (88%) of 935 patients treated at the IGR [22]. The tissue array contained three spots of each primary tumor.
For pAKT staining, each slide was deparaffinized and rehydrated using xylene, serial dilutions of ethanol (100%, 95%, 80%, and 70%), and distilled water. The slides were twice washed for 15 min with Tris-buffered saline (TBS) Tween-20 (TBST; DAKO, Carpinteria, CA). Antigen retrieval was completed by treating the slides in antigen retrieval solution (DAKO) in a steamer for 40 min. The slides were then cooled down at room temperature for 10 min and twice washed for 5 min with TBS Tween-20 buffer. Then the slides were placed in 3% hydrogen peroxide in phosphate-buffered saline (PBS) (DAKO) (pH 7.4) for 10 min in the dark. The slides were washed under tap water to completely remove hydrogen peroxide, and then they were twice washed for 5 min with TBS Tween-20 buffer. The slides were incubated in PBS for 15 min at room temperature in a humidified chamber and then incubated with pAKT antibody (1 : 100 dilution with 1% bovine serum albumin in PBS solution, pH 7.4; Cell Signaling Technologies, Beverly, MA) in a humidified chamber at room temperature for 2 h. After washing twice with TBS Tween buffer for 10 min each, the slides were incubated with the secondary antibody, labeled streptavidin biotin blocking system (LSAB; DAKO), at room temperature in a humidified chamber for 20 min, and then they were washed twice with TBS Tween buffer for 10 min each. The slides were incubated with LSAB at room temperature in a humidified chamber for 20 min, washed twice with TBS Tween buffer for 10 min each, incubated with diamino benzidine (DAB) solution for 1 min at room temperature, rinsed with water to remove residue DAB, counterstained with Mayer’s hematoxylin (DAKO) for 3 min, and dehydrated using serial dilutions of ethanol (70%, 80%, 95%, and 100%) and xylene. Staining was considered positive for pAKT when 
10% of the tumor cells presented cytoplasmic or membrane staining regardless of the intensity.
Each slide was also stained with anti-HER2 (DO485; DAKO), anti-ER (clone 6F11; Novocastra, Burlingame, CA), anti-cytokeratin 5/6 (clone D5/16B4; Zymed, San Francisco, CA), and anti-EGFR (clone 3C6; Ventana Medical Systems, Tucson, AZ) antibodies according to the manufacturers’ recommendations. Staining was considered positive for HER2 when an intense complete membrane staining was observed in >10% of the tumor cells. Staining was considered positive for ER when >10% of the tumor cells were stained. Stainings were considered positive for CK 5/6 and EGFR when staining was observed in at least one cell, as previously reported [23]. When a discrepancy was observed between the three spots in the tissue array, a decision rule was implemented. For ER and HER2, the definitive score was the one observed in two of the three spots. For EGFR and CK 5/6, staining was considered positive if it was observed in at least one spot. The breast cancers were divided into four immunophenotypical patterns as follows. The luminal tumors subgroup included all ER-positive tumors. HER2+/ER– tumors were defined by basal-like tumors were defined as HER2-negative/ER-negative/EGFR-positive and/or CK 5/6-positive tumors, as previously defined by Nielsen et al. [23]. HER2-negative/ER-negative/non-basal-like tumors were defined as lacking HER2, ER, CK 5/6, and EGFR stainings. We then compared levels and patterns of pAKT expression between these immunophenotypical patterns.
statistical analysis
Chi-square tests were used to compare the pAKT expression between tissue specimens according to HER2 and EGFR expression and according to immunophenotypical patterns. Disease-free survival (DFS) time was defined as the time between the date of randomization and the date of the last follow-up or the date of the first event: locoregional recurrence, distant metastasis, diagnosis of a second primary tumor, or cancer-unrelated death. Overall survival (OS) time was defined as the time between the date of randomization and the date of last follow-up or death from any cause. We used a Cox model [24] stratified by trial and adjusted for the following clinical prognostic variables: histologic grade (I, II, or III), lymph node status (positive or negative), and age (Table 1) to investigate the prognostic effect of pAKT. The predictive value of pAKT staining for the efficacy of adjuvant chemotherapy was studied by testing the interaction between the relevant variables and the attributed treatment (chemotherapy or no chemotherapy) in the same Cox model (Wald test). Two-sided P values of P < 0.01 were considered statistically significant. All analyses were carried out using SAS software, version 8.2 (SAS Institute Inc., Cary, NC).
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pAKT staining patterns
pAKT staining was assessable in 781 patients. This accounts for 95% of the 823 available tumor blocks, 83% of the patients included in the two trials at the IGR, and 68% of the overall population of the two trials. Staining for pAKT was positive in 119 cases (15%) (Table 1). Representative stains are shown in Figure 1.
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Correlations between pAKT stainings and HER2 and EGFR expression are reported in Table 2. HER2 and EGFR expression were assessable in 775 and 758 patients, respectively, for whom pAKT immunohistochemical expression data were available. HER2 and EGFR were expressed in 99 (13%) and 118 (16%) assessable tumors, respectively. EGFR expression was observed in a significantly higher proportion of HER2-positive tumors (25%) than HER2-negative tumors (14%) (P < 0.01). A statistically significant difference in positive staining for pAKT was observed between HER2-positive and HER2-negative tumors (28% versus 13%; P < 0.0001). No statistically significant difference was found between EGFR-positive and EGFR-negative tumors regarding pAKT staining (19% versus 15%; P = 0.20).
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In order to assess whether pAKT expression was increased in EGFR+ tumors, independently to HER2 staining, we evaluated pAKT expression according to EGFR staining in the subset of patients with HER2-negative tumors. We found a positive staining for pAKT in 16% of the EGFR-positive/HER2-negative cases and 13% of the EGFR-negative/HER2-negative cases (P = 0.49).
Because EGFR has been shown to contribute to HER2 activation through heterodimerization, we evaluated whether dual EGFR-HER2 expression could be associated with an increased rate of pAKT staining. A positive staining for pAKT was observed in 33% of the EGFR-positive/HER2-positive cases as compared with 26% of the EGFR-negative/HER2-positive cases (P = 0.49).
The tumors’ immunophenotypical patterns could be determined in 763 patients (Table 2). pAKT expression was observed in 16 (26%) HER2-positive/ER-negative tumors, 15 (16%) basal-like tumors, 12 (11%) HER2-negative/ER-negative/non-basal-like tumors, and 74 (15%) luminal tumors (P = 0.07).
prognostic and predictive value of pAKT expression
After a median follow-up of 10 years, pAKT expression was not associated with DFS [hazard ratio (HR) = 0.99; 95% confidence interval (CI) 0.72–1.36; P = 0.94] or OS (HR = 1.39; 95% CI 0.97–2.00; P = 0.08). Since some drugs have been specifically designed to inhibit pAKT-mTOR pathway, and considering that pAKT was more frequently expressed in HER2-overexpressing tumors, we analyzed the prognostic value of pAKT according to HER2 expression. Results are reported in Table 3. These data indicated a trend for a worse prognosis in pAKT+/HER2+ tumors as compared with the three other subgroups. The HR for death was indeed 2.07 (95% CI 1.17–3.64), 1.14 (95% CI 0.68–1.9), and 1.03 (95% CI 0.65–1.64) in patients with HER2+/pAKT+, HER2+/pAKT–, and HER2–/pAKT+ tumors as compared with those with HER2–/pAKT– tumors. Nevertheless, the prognostic value of the HER2/pAKT-based classification was not statistically significant (P = 0.18 and 0.10 for DFS and OS, respectively).
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Since patients were randomly assigned to receive adjuvant anthracycline-based chemotherapy or no chemotherapy, and considering that AKT activation has been reported to mediate anthracycline resistance in vitro [5–7], we analyzed the predictive value of pAKT expression for the efficacy of anthracycline-based chemotherapy. pAKT expression was not predictive for the efficacy of adjuvant anthracycline-based chemotherapy in terms of either OS (test for interaction, P = 0.73) or DFS (test for interaction, P = 0.95). The DFS curves according to pAKT staining and adjuvant chemotherapy are reported in Figure 2.
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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In the present study, pAKT expression was demonstrated in 15% of the breast cancer tissue samples. Our study also demonstrates that HER2 expression is associated with a higher rate of AKT activation, while EGFR expression is not associated with an increased frequency of AKT activation in breast carcinoma. In addition, we report that pAKT expression is neither prognostic nor predictive for the efficacy of adjuvant anthracycline-based chemotherapy.
Although it is now clear that HER2 positivity is an early oncogenic event in breast carcinoma [25], few biomarker studies have addressed the activation of the AKT pathway in HER2-positive breast cancer. Tokunaga et al. [14] reported a close correlation between HER2 and pAKT expression in a population of 252 primary breast carcinomas. This finding was confirmed by Kirkegaard et al. [15], who reported a higher frequency of pAKT expression in HER2-positive tumors than in HER2-negative tumors in 402 patients with ER-positive tumors. Our study's findings are concordant with these. These data confirm that the AKT pathway is activated in a significant proportion of HER2-positive breast cancers, indicating that this latter transmembrane receptor is activated in a subset of cases.
PTEN is a phosphatase that blocks activation of the PI3K/AKT pathway. It has been reported that PTEN down-regulation results in AKT phosphorylation and is associated with resistance to trastuzumab in patients with breast cancer [26]. This observation led to the development of inhibitors of the PI3K/AKT/mTOR pathways, like rapamycin analogs, as a therapeutic approach against HER2-overexpressing breast cancer in order to overcome resistance to trastuzumab. Our data, by showing that HER2 expression is associated with a higher percentage of pAKT staining, together with the finding that the HER2+/pAKT+ tumors exhibit a trend for worse prognosis, support the use of rapamycin analogs in patients with HER2+ tumors. The use of pAKT staining as predictive biomarker for the efficacy of mTOR inhibitors in this setting still needs to be determined. A recent report [27] has shown that PI3KCA gene harbor mutations in 26% of breast cancers. This activating mutation is correlated with both ER and HER2 expressions. Further studies will aim at determining whether PI3KCA mutations correlate with AKT phosphorylation and whether mTOR inhibitors should be tested in this setting.
Although AKT was found to be activated in a significant proportion of HER2-positive breast cancers, we did not find a correlation between EGFR expression and pAKT expression. This finding would indicate that EGFR is not activated in a significant proportion of breast carcinomas. Our finding is consistent with the apparent lack of efficacy of EGFR inhibitors in breast cancer. Indeed, few objective responses were observed in phase II trials of erlotinib [12] or gefitinib [13], the latter of which was shown to be unable to modulate pAKT expression or Ki-67 in vivo. Taken together, the findings that EGFR expression is not associated with AKT pathway activation and that EGFR inhibitors do not lead to significant tumor response rates indicate that EGFR by itself is not a relevant therapeutic target in breast carcinoma.
It must be pointed out that, as previously reported [14], no correlation was observed between pAKT staining and ER expression in our study. Nevertheless, this data should be taken with caution since some authors indicate that only extranuclear ER interacts with AKT signaling [28]. Finally, we evaluated the predictive value of pAKT expression in patients receiving adjuvant anthracycline-based chemotherapy. Several studies have reported that AKT activation can mediate anthracycline resistance [5–7]. However, pAKT expression was not predictive for anthracyline efficacy in the present study. Our data therefore do not support the use of AKT and possibly mTOR inhibitors to modulate anthracycline resistance in breast carcinoma. Nevertheless, some investigators have reported that doxorubicin induces a secondary AKT activation that could lead to either antiapoptotic signaling or secondary tamoxifen resistance. This latter consideration indicates that while there is no rationale to modulate pAKT before anthracycline exposure, rapamycin analogues might be used to modulate anthracycline-induced AKT activation.
In summary, our data indicate that the AKT pathway is frequently activated in HER2-positive breast cancer and that AKT/mTOR inhibitors should be evaluated in this setting. However, EGFR expression does not appear to be associated with substantial AKT activation, which may explain the limited clinical efficacy of EGFR inhibitors in unselected patients with breast cancer.
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funding |
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Fondation de France, Fondation Lilly, American Society of Clinical Oncology to F.A.; European Society of Medical Oncology to R.C; Beast Cancer Research Foundation.
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We thank A. Todd for editing.
Received for publication June 19, 2007. Revision received July 19, 2007. Accepted for publication July 23, 2007.
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