Annals of Oncology 19: 315–320, 2008
Published online 5 September 2007
Expression patterns and predictive value of
phosphorylated AKT in early-stage breast cancer
F. Andre1,2, R. Nahta1, R. Conforti2, T. Boulet3, M. Aziz4, L. X. H. Yuan1, F. Meslin5,
M. Spielmann2, G. Tomasic3, L. Pusztai1, G. N. Hortobagyi1, S. Michiels4, S. Delaloge2&
F. J. Esteva1*
1Department of Breast Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA;2Department of Medicine and Translational
Research Unit UPRES EA 03535, Departments of;3Biostatistics and,4Pathology, Institut Gustave Roussy;5Institut National de la Sante ´ et de la Recherche Me ´dicale
U753, Laboratoire d’Immunologie des tumeurs humaines, Institut Gustave Roussy, PR1 and IFR54, Villejuif, France
Received 19 June 2007; revised 19 July 2007; accepted 23 July 2007
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 HER22 tumors
(P < 0.001). pAKT was expressed in 15 of 94 (16%) and 75 of 563 (13%) HER22/EGFR+ and HER22/EGFR2 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
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 . Several drugs,
including rapamycin analogues, have been developed recently
in order to specifically inhibit the PI3K/AKT/mTOR pathway
through mTOR inhibition . In breast cancer, AKT
phosphorylation has been more specifically associated with in
vitro resistance to tamoxifen  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  or erlotinib
 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
*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;
ª 2007 European Society for Medical Oncology. For Permissions, please email: email@example.com
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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.
patients and methods
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 , 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
. 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/m2or 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 .
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 . 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+/ER2 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. . 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
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  stratified by trial and adjusted
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316 | Andre et al.Volume 19|No. 2|February 2008
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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).
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
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).
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
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+/pAKT2,
and HER22/pAKT+ tumors as compared with those with
HER22/pAKT2 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).
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.
Table 1. Patient characteristics
(n = 781)
(n = 662)
(n = 119)
Axillary node involvement
ER expression (fmol/mg)
P = 0.45
P = 0.67
P < 0.001
P = 0.14
P = 0.64
pAKT, phosphorylated AKT; ER, estrogen receptor.
Annals of Oncology
Volume 19|No. 2|February 2008 doi:10.1093/annonc/mdm429 | 317
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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
Although it is now clear that HER2 positivity is an early
oncogenic event in breast carcinoma , few biomarker
studies have addressed the activation of the AKT pathway in
HER2-positive breast cancer. Tokunaga et al.  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. , 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,
Figure 1. Illustrative staining for phosphorylated AKT expression.
Table 2. pAKT staining according to HER2 and EGFR expressions and
(n = 662)
(n = 119)
HER2 expression (n = 775)
HER2+ (n = 99)
HER22 (n = 676)
EGFR expression (n = 758)
EGFR+ (n = 118)
EGFR2 (n = 640)
EGFR expression in HER22 tumors (n = 657)
EGFR+/HER22 (n = 94)
EGFR2/HER22 (n = 563)488 (87%)
EGFR expression in HER2+ tumors (n = 97)
EGFR+/HER2+ (n = 24)
EGFR+/HER2+ (n = 73)
Molecular subclasses (n = 763)
HER2+/ER2 (n = 61)
Basal like (n = 93)
(n = 106)
Luminal like (n = 503)
79 (84%) 15 (16%)
429 (85%) 74 (15%)
pAKT, phosphorylated AKT; EGFR, epidermal growth factor receptor; ER,
Figure 2. Disease-free survival according to phosphorylated AKT
expression in (A) patients who did not receive adjuvant chemotherapy and
(B) patients treated by anthracycline-based adjuvant chemotherapy.
Table 3. Prognostic value of a combined pAKT/HER2 classification
ratio for relapse or
death (95% CI)
ratio for death
HER2+/pAKT+ (n = 28)
HER2+/pAKT2 (n = 71)
HER22/pAKT+ (n = 90)
HER22/pAKT2 (n = 586)
pAKT, phosphorylated AKT; CI, confidence interval.
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318 | Andre et al.Volume 19|No. 2|February 2008
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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 .
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  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  or gefitinib , 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 , 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 . 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
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.
We thank A. Todd for editing.
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