ARTICLE
Auteur(s) : A Barbier1, J Domont2,
N Magné3,4, J-L Goldmard5, C
Genestie1, C Hannoun6, J-C
Vaillant6, A Bellanger7, D
Khayat4, F Capron1, J-P
Spano4
1Département d’anatomopathologie, groupe hospitalier
Pitié-Salpêtrière, Paris, France
2Département d’oncologie médicale, institut
Gustave-Roussy, Villejuif, France
3Département de radiothérapie, institut Gustave-Roussy,
Villejuif, France
4Département d’oncologie médicale, groupe hospitalier
Pitié-Salpêtrière, Paris, France
5Département de statistiques médicales, groupe
hospitalier Pitié-Salpêtrière, Paris, France
6Département de chirurgie viscérale, groupe hospitalier
Pitié-Salpêtrière, Paris, France
7Département de pharmacie hospitalière, groupe
hospitalier Pitié-Salpêtrière, Paris, France
Article reçu le 22 Septembre 2009, accepté le 24 Novembre
2009
Introduction
During the past few years, the systemic treatment of colorectal
cancer (CRC) has become a rapidly evolving field. For more than
40 years, 5-fluorouracil (5-FU) was the standard of care for
patients with metastatic CRC (mCRC). The addition of effective
newer cytotoxic agents, such as irinotecan and oxaliplatin to
5-FU-based therapies, the introduction of oral fluoropyrimidines
and the recent development of targeted agents have prolonged the
overall median survival time from one to two years [1-3]. Recently,
three targeted agents were approved in the treatment of mCRC: the
anti-vascular endothelial growth factor (VEGF) monoclonal antibody
(mAb), bevacizumab in combination with first-line 5-FU-based
chemotherapy regimens [4] and the human epidermal growth factor
receptor (EGFR)-targeted mAbs, cetuximab as monotherapy or in
combination with irinotecan as second-line therapy in refractory
cancer [5, 6] and panitumumab after progression with 5-FU,
oxaliplatin and irinotecan [7].
Based on the importance of the EGFR axis in tumorigenesis and
tumor progression, EGFR expression has been investigated as a
possible prognosis indicator in CRC. EGFR expression was found in
up to 82% of CRC [5, 6, 8]. In a review of 200 studies
involving more than 20,000 patients and 10 cancer types,
Nicholson et al. showed that increased EGFR expression was
associated with reduced recurrence-free or overall survival rates
in 52% of studies (13/25) with EGFR status considered as a modest
prognostic indicator in CRC [9]. With the advent of EGFR targeted
therapies, immunohistochemical screening strategies for EGFR
expression were developed in clinical trials to select patients
with EGFR-expressing tumors. However, the clinical data do not
support a relationship between EGFR expression as assessed by
immunohistochemistry (IHC) and response to EGFR-targeted mAbs
[10-12].
Several technical and biological factors could be advocated to
explain this lack of correlation. Recently, Francoual et al.
pointed out the existence of a heterogeneous population of EGFR in
CRC tumors with both one class (78% of tumors with physiologically
relevant high-affinity binding sites) and two classes of binding
sites (22% of tumors with a mixed presence of low- and
high-affinity binding sites) and suggested that IHC could not be
sensitive enough to quantify EGFR as high-affinity EGF binding
sites [13]. From a biological point of view, the EGFR signaling
pathway is complex and other molecular mechanisms such as
activating EGFR mutations, increased ligand expression, alteration
of downstream signaling pathways and cross-talk among different erB
receptor family members are critically involved in the action of
anti-EGFR mAbs, and therefore more predictive of treatment response
than the total level of the receptor per se [14]. Currently, some
biomarkers have been identified in CRC tumor samples as potential
predictors of response to cetuximab or panitumumab therapy, i.e.
activated EGFR [15], EGFR amplification [16], absence of KRAS
mutations [17-21], PTEN (phosphatase protein homologue to tension)
expression [22], low VEGF receptor expression [23], nuclear
factor-κB tumor expression [24] or epiregulin and amphiregulin
expression [20].
Another explanation suggested for the apparent discordance
between EGFR status and clinical response to anti-EGFR mAb therapy
in mCRC is a possible difference in EGFR status between primary
tumor, which was usually assessed in the clinical trials, and
related metastatic sites [25]. Scartozzi et al. showed that
36% of primary tumors expressing EGFR showed a loss of expression
in the corresponding metastatic sites [25]. However, conflicting
data were reported thereafter [26-28]. Moreover, very few data are
available for other biological markers from major downstream
signaling pathways that could emerge as new molecular predictive
factors to anti-EGFR therapies [29-31].
The aim of the present study was to analyze and compare the
expression of a panel of molecular markers, namely EGFR,
phospho-EGFR (pEGFR), VEGF, PTEN, phospho-AKT (pAKT) and p21,
assessed by IHC on tissue microarrays of primary colorectal tumor
samples and/or liver metastases. In a subgroup of patients treated
with cetuximab, the predictive value of these biomarkers on the
response to treatment was also evaluated.
Patients and methods
Patients
Forty-six consecutive patients, who underwent surgical resection of
the primary colon tumor and/or the liver metastases and treated at
the Oncology Department of the Pitié Salpétrière Hospital (Paris,
France), were selected from a pathological database of colorectal
cancer cases. Eighteen patients were treated with cetuximab at an
initial dose of 400 mg/m2 intravenously followed by
weekly doses of 250 mg/m2 combined with
irinotecan-based chemotherapy. Tumor response was evaluated by
computerized tomodensitometry according to the Response Evaluation
Criteria in Solid Tumors [32] and classified as complete (CR),
partial response (PR), stable disease (SD) or progressive disease
(PD). This retrospective study was approved by independent local
ethics committee. The study was conducted in accordance with the
Declaration of Helsinki (1996). All patients provided written
informed consent.
Tumor specimens and tissue microarray
Sixty-three paraffin-embedded specimens from primary CRC (designed
as « T ») (29 samples) and liver metastases (« M »)
(34 samples), resected before treatment, were available. For
14 cases, samples « T » and « M » from the same patient were
obtained. All resected samples were received fresh, then
immediately fixed in 10% pH neutral formalin for 48 hours and
embedded in paraffin before processing. Paraffin-embedded tissue
blocks containing viable tumor were selected for each case. Tissue
microarrays (TMA) included two (« M ») or three (« T »)
1-mm-core-biopsies from each block, cut using a manual
tissue-arraying instrument (Manual Tissue Array; Alphelys; Beecher
Instruments Inc) as follows: (a) for « T » one sample from tumor,
one sample from infiltrative forehead, one sample from colic safe
tissue and (b) for « M » one sample from tumor, one sample from
liver safe tissue.
Antibodies and immunohistochemical techniques
Tissue was stained with antibodies to the following markers: EGFR
(2-18C9, Dakocytomation, diluted at 1:200, pH 8); pEGFR
((Tyr1068)1H12, Cell signaling technology, 1:200, pH 8); VEGF
(sc-7269, Santa cruz biotechnology, 1:100, pH 6); PTEN (138G6, Cell
signaling technology, 1:50, pH 6); pAKT ((Thr308) 244F9, Cell
signaling technology, 1:100, pH 9); p21 (Ras) (DCS-60.2,
Interchim, NeoMarkers, 1:20, pH 9). Antigen retrieval was conducted
by treatment with high temperature
(Tris/EDTA-bain-marie-dakocytomation wash buffer) and final
detection involved standard staining methods
(avidin-biotin-peroxidase).
Immunohistochemistry scoring
Semi-quantitative evaluation of immunohistochemical staining was
carried out by two independent pathologists (A. Bardier and C.
Genestie), who were blinded regarding the clinicopathological data,
through defining of the percentage of positive cells and the
staining intensity. Grading of immunolabeling was performed using
the immunoreactive score (IRS). The IRS score was obtained by
multiplying the two parameters and ranged from 0 to 12.
Statistical analysis
Since marker expression distributions are not Gaussian and sample
sizes are small, statistical analyses were performed using non
parametric tests. Descriptive statistics used median and
inter-quartile interval. The correlation between tumor and
metastasis biomarker expressions has been tested using the Spearman
rank correlation coefficient test (each patient independently).
Relationships between biomarker expressions and therapeutic
response to cetuximab have been studied by considering
independently marker expressions of tumor and metastasis, even when
measured from the same subject. Furthermore, marker expressions
have been recoded as binary variables, with positive (IRS >0) or
negative (IRS = 0) values. The proportion of positive marker
expressions has been compared between responder and non responder
patients using the Fisher’s exact test. All the tests were
two-sided, and used a significant threshold of p = 0.05. Analyses
were performed using the SAS V8 statistical package.
Results
Clinical and pathological features
At the time of primary diagnosis, the patients (N = 46)
(26 male, 20 female) had a median age of 64 years
(range, 28 to 79 years). Clinical characteristics are
summarized in table 1. Metastatic sites
were mainly located at liver and lung. The majority of patients
(78.3%) had metastasis in a single site. Prior systemic therapy
consisted in first line (95.7% of patients), second line (69.6%)
and third line chemotherapy (43.5%). Eighteen patients (39.1%) were
treated with cetuximab and 9 patients (19.6%) with
bevacizumab. Under cetuximab plus irinotecan-based chemotherapy,
9 patients had PR, 4 had SD and 5 progressed,
whereas no patient showed CR.
Table 1 Characteristics of mCRC patients (N = 46).
|
Characteristic
|
|
N
|
%
|
|
Age, years
|
Median
|
64
|
|
Range
|
28-79
|
|
Sex
|
Male
|
26
|
56.5
|
|
Metastatic sites,
|
Liver
|
37
|
80.4
|
|
Lung
|
6
|
13.0
|
|
Peritoneal
|
2
|
4.3
|
|
Other
|
9
|
19.6
|
|
Missing data
|
2
|
4.3
|
|
Number of metastatic sites
|
1
|
36
|
78.3
|
|
2
|
5
|
10.9
|
|
> 2
|
2
|
4.3
|
|
Missing data
|
2
|
4.3
|
|
Systemic therapy
|
Adjuvant chemotherapy
|
17
|
37.0
|
|
First line
|
44
|
95.7
|
|
Second line
|
32
|
69.6
|
|
Third line
|
20
|
43.5
|
|
Line with cetuximab
|
18
|
39.1
|
|
Line with bevacizumab
|
9
|
19.6
|
|
Missing data
|
2
|
4.3
|
Biomarkers expressions in primary tumors and liver
metastases
Among the 63 tumor samples, 60 were analyzable (T =
28 and M = 32). Overall, 15 cases were EGFR positive
(25.4%; 1 missing data - MD), 22 cases were pEGFR
positive (37.9%; 2 MD), 23 were VEGF positive (38.3%),
44 were pVEGF positive (47.6%; 1 MD), 36 were PTEN
positive (61.0%; 1 MD), 42 cases were pAKT positive
(70.0%) and 29 cases were p21 positive (50.9%;
3 MD). The biomarkers expressions assessed as IRS in primary
tumor and liver metastasis samples are presented in table 2. A significant correlation was
observed between primary tumors and metastases for pAKT (p = 0.037)
and pEGFR (p = 0.0002) status.
Table 2 Marker expression in primary tumors and
metastases.
|
Biomarker
|
Tumor (N = 28)
|
Metastases (N = 32)
|
|
|
IRS = 0
|
IRS > 0
|
IRS = 0
|
IRS >0
|
p
|
|
n
|
n
|
Median IRS [min-max]
|
n
|
n
|
Median IRS [min-max]
|
|
EGFR
|
23 (85.2)
|
4 (14.8)
|
2 .5 [1-12]
|
21 (65.6)
|
11 (34.3)
|
3.5 [2-12]
|
0.074
|
|
pEGFR
|
17 (65.4)
|
9 (34.6)
|
4 (2-12]
|
19 (59.4)
|
13 (40.6)
|
6 [2-12]
|
0.0002
|
|
VEGF
|
19 (67.9)
|
9 (32.1)
|
8 [3-12]
|
18 (56.3)
|
14 (43.8)
|
4 [1-12]
|
0.600
|
|
pVEGF
|
8 (29.6)
|
19 (70.4)
|
8 [3-12]
|
7 (21.9)
|
25 (78.1)
|
4 [1-12]
|
0.174
|
|
PTEN
|
11 (40.7)
|
16 (59.3)
|
6 [1-12]
|
12 (37.5)
|
20 (62.5)
|
4 [1-12]
|
0.700
|
|
pAKT
|
8 (28.6)
|
20 (71.4)
|
8 [4-12]
|
10 (31.3)
|
22 (68.7)
|
8 [2-12]
|
0.037
|
|
p21
|
15 (62.5)
|
10 (37.5)
|
2 [2-6]
|
13 (40.6)
|
19 (59.4)
|
2 [1-12]
|
0.300
|
Biomarkers expressions and therapeutic response
to cetuximab
Among the subgroup of 18 patients treated by cetuximab-based
therapy, only p21 status appeared as significant predictive
factor of response (p = 0.036) (table
3).
Table 3 Marker expression and therapeutic response to
cetuximab.
|
Biomarker
|
Responders (PR or SD) (n = 13)
|
Non responders (n = 5)
|
pa
|
|
n (%)
|
n (%)
|
|
EGFR
|
2 (16.7)b
|
1 (20.0)
|
1.000
|
|
pEGFR
|
7 (58.3)b
|
2 (40.0)
|
0.620
|
|
VEGF
|
7 (53.8)
|
0
|
0.101
|
|
pVEGF
|
11 (91.7)
|
3 (60.0)
|
0.191
|
|
PTEN
|
11 (84.6)
|
2 (40.0)
|
0.099
|
|
pAKT
|
9 (69.2)
|
3 (60.0)
|
1.000
|
|
p21
|
8 (61.5)
|
0
|
0.036
|
Discussion
The present study is, to our knowledge, the first to evaluate the
expression of a panel of molecular markers, namely EGFR, pEGFR,
VEGF, pVEGF, PTEN, pAKT and p21, in primary CRC and the related
distant metastases in a significant number of patients.
A significant correlation between primary tumors and liver
metastases was observed only for pEGFR and pAKT. No correlation was
found for the other biomarkers. Most of the published data compared
the EGFR status between primary and related metastatic sites.
Scartozzi et al. retrospectively evaluated EGFR
immunohistochemistry from primary tumors and related metastatic
sites in 99 CRC patients [25]. EGFR was found to
be positive in 53% of primary tumors; in 36% of these primary
tumors expressing EGFR the corresponding metastatic site was found
negative. In other recent studies using IHC analysis, EGFR
reactivity was similar in the primary tumor and the related
metastases [27, 28]. Bibeau et al. studied EGFR expression
using IHC in primary CRC tumors (n = 32) and their related
metastases (n = 53) on tissue sections and TMA generated from the
same paraffin blocks [26]. On tissue section, a concordant
EGFR-positive status was showed in 78% of cases. On TMA, 65% of the
primary CRC, 66% of the metastases and 43% of the matched primary
CRC metastases were EGFR-positive; no concordant EGFR status was
found. Our results obtained on TMA are similar to those reported by
Bibeau et al. with no significant correlation for EGFR status
between primary and metastatic tumors. Bibeau et al. showed
that results obtained on TMA were systemically lower than those
observed on the whole tissue sections and explained the discordant
results between the two technologies by the cases containing rare
stained cells (i.e., <10%) or small invasive clusters, which may
be not selected by TMA. This can explain the fact that EGFR
positivity reached only 15% and 34% in primary and metastatic CRC,
respectively in the present study. The interpretation of the IHC
analysis, which differs from one study to another, could also
explain the differences observed in EGFR positivity.
However, a significant correlation between primary CRC and
related liver metastases was found for the activated form of EGFR
and the downstream effector protein pAKT. Activated EGFR stimulates
a number of different signal transduction pathways, including the
phosphatidyl inositol 3-kinase (PI-3K) and the downstream
protein-serine/threonine kinase Akt pathway [33]. Akt transduces
signals that trigger a cascade of responses from cell growth and
proliferation to survival and motility [14]. A greater rate of
positivity for pEGFR than for EGFR was evidenced in both primary
and metastatic tumor samples with approximately 40% of positive
cases for pEGFR. Phospho-AKT was strongly expressed in both types
of tumor cells (70% of cases). Scartozzi et al. also found a
strong expression of pAKT in 98 cases of paired primary CRC
tumors (74% positive cases) and liver metastases (73%) [34].
Phospho-AKT in primary CRC changed from positive to negative in 16%
paired metastases and from negative to positive in 13% related
metastatic sites. Their findings suggest, in opposition to our
results, a lack of correlation between primary CRC tumors and
corresponding metastases for pAKT status. They also shown that Akt
and MAPK (mitogen-activated protein kinase) could be independent of
EGFR status both in primary and metastatic sites, thus suggesting
that EGFR downstream signaling pathway can be overactivated even in
the absence of EGFR expression.
PTEN was deleted in 39% of cases with no correlation between
primary and metastatic tumor samples. PTEN is a lipid phosphatase
and tumor suppressor protein that regulates the PI-3K/Akt signaling
pathway. With the loss of PTEN function, the major substrate for
PTEN, phosphatidylinositol 3,4,5-triphosphate, which is a second
messenger of PI-3K, accumulates in the cell membrane, when it binds
and activates Akt [14]. Thus, the loss of PTEN function results in
overactivation of the Akt pathway, increasing its cellular
antiapoptotic functions. PTEN expression was decreased in
approximately 40% of colorectal cancers, often with associated PTEN
mutation or deletion [35]. The cyclin-dependent kinase inhibitor
p21 was deleted in 62.5% of primary tumor cells and 41% of
metastatic cells with no correlation between both types of cells.
The expression of p21 gene is tightly controlled by the tumor
suppressor protein p53, through which this protein mediates the
p53-dependent cell cycle G1 phase arrest in response to a
variety of stress stimuli.
An increasing body of evidence suggests that EGFR-mediated
pathways are intimately involved in tumor angiogenesis through
up-regulation of VEGF and other mediators of angiogenesis. It has
been reported that VEGF is strongly related to liver metastases of
CRC and its expression levels are useful not only as a predictive
marker for distant metastases but also as a prognostic marker
[36-38]. Takahashi et al. reported that protein expressions of
VEGF and its receptor, KDR, were higher in metastatic than in
non-metastatic neoplasms in CRC by using IHC staining [36]. They
also found that VEGF expression and vessel count were correlated
with time to recurrence [39]. Similar results were obtained in the
present study with a stronger expression of VEGF and pVEGF in
metastases compared to primary tumors. However, Kuramochi
et al. observed no difference between VEGF mRNA levels of
31 primary CRC tumors and corresponding liver metastases [40].
Berney et al. found that VEGF protein expression evaluated by
IHC was significantly reduced in the metastatic liver tumors
compared with primary CRC tumors [41]. No consensus can be drawn
from these studies and additional experiments are needed to
evaluate the VEGF status in primary and metastatic CRC.
Among the subgroup of patients treated with cetuximab, only
p21 status was a significant factor of response. Huether
et al. showed that cetuximab inhibited growth of
p53 wild-type HepG2 hepatocellular cancer cells in a
time- and dose-dependent manner [42]. Cetuximab treatment resulted
in arresting the cell cycle in the
G1/G0-phase due to an increase of expression
of the cyclin-dependent kinase inhibitors p21Waf1/CIP1
and p27Kip1 and a decrease in cyclin D1 expression.
Two markers, VEGF and PTEN had p-values around 10% and could be
considered as potential candidates for further studies, since the
small sample sizes (13 responders and 5 non-responders)
give this study a very low power. In our study, 85% of responder
patients expressed PTEN versus 40% in non responders. PTEN loss of
expression was shown to predict cetuximab efficacy in mCRC in
several recent studies [43, 44]. Response to cetuximab was
associated with high expression of VEGF in the present study with
54% of responders expressing VEGF versus 0% in non responders.
In summary, this study showed that biomarkers status may change
between primary and corresponding metastatic sites in CRC. No
correlation was found for EGFR, VEGF, pVEGF, PTEN and
p21 between primary CRC and related liver metastases. Similar
expression was shown for pEGFR and pAKT only. These results may
have implications for the identification of patients who are likely
to respond to anti-EGFR treatment.
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