Androgen-independent prostate cancer (AIPC) is defined as biochemical and/or clinical
disease progression during androgen deprivation with castration levels of serum testosterone.
Patients are in need of palliation, and novel strategies focus on the epidermal growth
factor receptor (EGFR) family and related intracellular pathways.
Based on current knowledge, the EGFR family comprises four members; c-erbB-1 (EGFR),
c-erbB-2 (HER2/neu), c-erbB-3 (HER3) and c-erbB-4 (HER4) (Olayioye et al, 2000;Suo
and Nesland, 2002). All members of this family have an extracellular ligand-binding
domain, a single transmembrane domain and a cytoplasmatic tyrosine kinase domain,
which for c-erbB-3 is nonfunctioning. Binding of ligands to the extracellular domains
leads to the formation of homo- and heterodimers. This starts a complex signalling
cascade finally resulting in cellular proliferation, prevention of apoptosis and promotion
of tumour cell mobility, adhesion and invasion (Barton et al, 2001).
Previous observations have demonstrated increased levels of c-erbB-1 immunoreactivity
in hormone-independent human prostate cancer cell lines (MacDonald and Habib, 1992;
Sherwood et al, 1998). In tissue from patients with metastatic AIPC, the c-erbB-1
expression has been reported as high as about 90–100% (Scher et al, 1995; Di Lorenzo
et al, 2002). For c-erbB-2 expression, there have been reports of divergent rates
in primary prostate carcinoma (Mellon et al, 1992; Kuhn et al, 1993; Fossa et al,
2002). In xenografts, an upregulation of c-erbB-2 has been found when the tumour becomes
androgen-independent (Craft et al, 1999). In tumours from patients with AIPC, c-erbB-2
expression is reported from relatively uncommon (Reese, 2001; Savinainen et al, 2002)
up to about 50–80% (Signoretti et al, 2000; Osman et al, 2001; Di Lorenzo et al, 2002).
To the best of our knowledge, data are lacking from a larger series that compare the
expression of all EGFRs for the same patient before and after the development of AIPC.
The primary aim of the present study is to fill this gap and, secondarily, to provide
information about the prognostic significance of EGFR expression in AIPC patients.
MATERIALS AND METHODS
From the files of the Department of Pathology, The Norwegian Radium Hospital (NRH),
we identified AIPC patients with an adenocarcinoma-positive biopsy from the prostate
before the start of androgen deprivation (pretreatment (PRTR) biopsy) and after the
development of symptomatic AIPC (AIPC biopsy). The latter histological material was
obtained by palliative surgery due to local problems (transurethral resections of
the prostate (TUR-P)/transvesical prostatectomy (TV)), or represented a diagnostic
biopsy in order to differentiate between a growing prostate cancer and rectal cancer
(transrectal/transperineal core biopsies). Patients displaying primary androgen-independence,
defined as clinical progression within 3 months after the start of castration therapy,
were excluded from the present study. Any concomitant or prior medical and/or surgical
treatment was allowed, whereas radiotherapy (RT) to the prostate prior to the AIPC
biopsy was a criterion for exclusion. In all, 10 specimens of archival formalin-fixed
normal prostate tissue represented a clinical control group.
Clinical information
The following information was extracted from the medical records:
Initial diagnosis and treatment: Date, serum prostate-specific antigen (PSA) level,
extent of the disease categorised as local, regional or distant metastases according
to the registration routines of the Cancer Registry of Norway (www.kreftregisteret.
no), date of start and type of androgen deprivation.
Clinical course and diagnosis of AIPC: Date and type of clinical progression (local
vs distant), date and status of last observation (death or 15 October 2001). For all
PRTR and AIPC biopsies, we recorded the date and type of biopsy together with the
Gleason score, the latter differentiating between Gleason score 7a (grade 3+4) and
Gleason score 7b (grade 4+3) (Lilleby et al, 2001).
Immunohistochemistry
For each eligible patient, the archival paraffin blocks of the PRTR biopsy and of
the first available biopsy after diagnosis of AIPC were collected. Serial paraffin
sections (5 μm thick) were cut from the blocks with the most representative amount
of tumour tissue, as determined in haematoxylin and eosin-stained slides. The newly
cut sections were mounted on silane-coated slides and dried for 1 h in 56°C followed
by 37°C overnight. The sections were deparaffinised, rehydrated and incubated with
0.3% H2O2 in methanol for 30 min to block endogenous peroxidase. The pretreatment
conditions and the primary antibodies against PSA and the EGFRs are presented in Table
1
Table 1
Primary antibodies and conditions
Antibody against
Source
Cat. no.
Dilution
Pretreatment
PSA
DAKO, CA, USA
M0750
1 : 20
Microwave 2 × 5 mina
c-erbB-1
DAKO, CA, USA
M3563
1 : 100b
Pronase 10 min
c-erbB-2
Novocastra Laboratories Ltd, Newcastle, UK
NCL-CB11
1 : 200
Microwave 2 × 5 min
c-erbB-3
SantaCruz Biotechnology Inc., CA, USA
SC-415
1 : 50
None
c-erbB-4
SantaCruz Biotechnology Inc., CA, USA
SC-283
1 : 50
Microwave 2 × 5 min
a
In 10 mmol/l citrate buffer (pH 6.0).
b
Applied overnight.
. Automatic immunostaining with the biotin–streptavidin amplified (B-SA) system (Optimax®
Automated Cell Staining System Plus, BioGenex, San Raman, CA, USA) was applied. All
series included both a positive control (known positive case) and a negative control
in which a nonreacting immunoglobulin of the same subclass had substituted the primary
antibody. All controls were satisfactory.
Scoring procedure
The results of the immunostaining were reviewed by an experienced pathologist (JMN)
and scored semiquantitatively as follows: no staining: −(minus); scattered cells to
less than 1% tumour cells positive: +; 1 to <10% tumour cells positive: ++; 10–50%
tumour cells positive: +++ and >50% cells positive: ++++. Scores of −, + and ++ were
grouped together as ‘immunohistochemistry (IHC)-negative’ as opposed to ‘IHC-positive’
findings in the examination of the clinical impact. For the EGFRs, only moderate to
strong cell membrane-specific immunostaining was taken into account.
Statistics
Statistical analyses were performed using the computer-based program SPSS (Statistical
Package for Social Sciences) version 10.0. Individual changes of immunostaining after
diagnosis of AIPC were assessed by the McNemar test for related samples. The overall
survival was estimated by the Kaplan–Meier method from the date of the AIPC biopsy
to the last observation/death applying the logrank test to assess statistical significance.
P-values <0.05 were regarded statistically significant.
RESULTS
Patient characteristics
A total of 106 prostate cancer patients (diagnosed 1970–1997) fulfilled the eligibility
criteria. The median age at initial diagnosis was 70 years (range: 50–86) (Table 2
Table 2
Demographics at diagnosis of prostate cancer
Age (years)
70 (50–86)a
Extent of the disease
Loco-regional
81 (76%)b
Distant metastases
24 (23%)
Unknown
1 (1%)
Primary hormone treatment
Orchiectomy
56 (53%)
LHRH analogues
33 (31%)
Combined LHRH analogues/antiandrogens
15 (14%)
Oestrogens
2 (2%)
Initial diagnosis to diagnosis of AIPC (months)
32 (7–167)
a
Median (range).
b
Number of patients (%).
). In total, 23% of the patients had distant metastases at the time of initial diagnosis.
Only five of 34 patients with initially available serum PSA values displayed levels
less than 10 μg/l. Androgen deprivation was started within median 2 months after initial
diagnosis (range 0–109). After a median time of 32 months (range 7–167) AIPC was diagnosed
in all patients. Distant metastases were recorded for 43 patients after the development
of AIPC. Of the 105 PRTR biopsies evaluable for Gleason score, 79% were ⩾7b (Table
3
Table 3
Histological specimens
PRTR biopsy
AIPC biopsy
P
a
Type of biopsy
TUR-P
26 (25%)
99 (93%)
TV prostatectomy
7 (7%)
—
Transrectal/transperineal core biopsy
73 (69%)
7 (7%)
Gleason score
⩽7a (grade 3+4)
21 (20%)
—
⩾7b (grade 4+3)
84 (79%)
106 (100%)
Not evaluable
1 (1%)
—
IHC positivity
PSA
88 (83%)
73 (69%)
0.006
c-erbB-1
24 (23%)
46 (43%)
0.001
c-erbB-2
35 (33%)
25 (24%)
0.174
c-erbB-3
15 (14%)
22 (21%)
0.21
c-erbB-4
25 (24%)
31 (29%)
0.43
a
McNemar test for related samples.
). All AIPC biopsies were Gleason score ⩾7b.
Immunostaining
The percentage of PSA immunoreactive specimens decreased from 83% (88 of 106 patients)
to 69% (73 patients) as the patients developed AIPC (Table 3). A total of 21 patients
lost PSA positivity as they developed AIPC. Before androgen deprivation, c-erbB-1
positivity was demonstrated in 24 patients (23%) and in 46 patients (43%) after the
development of AIPC (P=0.001). The comparable figures for c-erbB-2 were 33% and 24%,
respectively. c-erbB-1 and c-erbB-2 IHC positivity after the development of AIPC is
illustrated by Figure 1A–B
Figure 1
Immunohistochemical detection after the development of AIPC of (A) c-erbB-1 (magnification
× 40) and (B) c-erbB-2 (magnification × 20).
.
The EGFRs remained negative in 48–71% of the patients during the development of AIPC
(Figure 2
Figure 2
Intrapatient change (PRTR → AIPC) of IHC stainability (106 patients). ▪ Negative →
Positive; ▪ Positive → Positive; □ Negative → Negative;
Positive → Negative. Θ Number of patients.
). Of 82 patients whose PRTR biopsies were c-erbB-1-negative, 31 displayed c-erbB-1
positivity in their AIPC biopsy (P=0.001). However, nine of 24 patients with c-erbB-1
positivity before hormone treatment lost this immunoreactivity after the development
of AIPC. The direction of intrapatient changes for c-erbB-2, c-erbB-3 and c-erbB-4
was less pronounced. Overall, the positivity of EGFRs was not significantly related
to Gleason score or PSA immunoreactivity (data not shown). During the development
of AIPC, the number of c-erbB-1-positive specimens was doubled in those patients who
lost PSA positivity, whereas the number of c-erbB-2 positive specimens decreased from
eight to three (Table 4
Table 4
Growth factor receptors in 21 patients with a PSA-positive PRTR biopsy changing to
PSA negativity at the development of AIPC
PRTR biopsy
AIPC biopsy
P
a
IHC positivity
c-erbB-1
6 (29%)
11 (52%)
0.180
c-erbB-2
8 (38%)
3 (14%)
0.125
c-erbB-3
5 (24%)
5 (24%)
1.00
c-erbB-4
7 (33%)
7 (33%)
1.00
a
McNemar test for related samples.
). For the 10 specimens of normal prostate tissue, four displayed c-erbB-1 positivity,
whereas none were positive for c-erbB-2, c-erbB-3 or c-erbB-4.
Survival
At the time of the last observation, 91 patients (86%) were dead. The median overall
survival time from the date of the AIPC biopsy was 20 months (95% CI: 15–26 months).
The immunoreactivity of c-erbB-2 determined decreased survival (P=0.029) (Figure 3A
Figure 3
(A) C-erbB-2 stainability of the AIPC biopsy and overall survival. — c-erbB-2 negative
(81), - - - - c-erbB-2 positive (25). (B) C-erbB-4 stainability of the AIPC biopsy
and overall survival. — c-erbB-4 negative (75),
- - - - c-erbB-4 positive (31).
), whereas the positivity of c-erbB-4 tended to indicate a better 2-year survival
than c-erbB-4 negativity (P=0.054) (Figure 3B) (Table 5
Table 5
EGFR family expression and survival from the date of AIPC biopsy
Immunoreactivity
Number of patients (%)
2-year survival
P
a
c-erbB-1
Negative
60 (57%)
0.526
Positive
46 (43%)
0.362
0.118
c-erbB-2
Negative
81 (76%)
0.498
Positive
25 (24%)
0.320
0.029
c-erbB-3
Negative
84 (79%)
0.463
Positive
22 (21%)
0.409
0.266
c-erbB-4
Negative
75 (71%)
0.416
Positive
31 (29%)
0.539
0.054
a
Logrank test for trend.
). The coexpression of c-erbB-1 and c-erbB-2 in the AIPC biopsy (17 patients) did
not represent a statistically significant poor prognosis factor (P=0.064).
DISCUSSION
In this descriptive study, 43% of the prostate cancer specimens obtained from growing
pelvic tumours of AIPC patients expressed the c-erbB-1 protein and 24% expressed c-erbB-2,
the latter being a poor prognosis factor. During the development of AIPC, intrapatient
changes occurred in two opposite directions for each of the four EGFRs: (1) negativity
of the PRTR biopsies changed to positivity of the paired AIPC biopsies and (2) positivity
of the PRTR biopsies changed to negativity of the paired AIPC biopsies. The development
of AIPC was significantly associated with the increase of c-erbB-1 expression, whereas
no changes for the other growth factor receptors reached the level of statistical
significance. The Gleason score and PSA immunoreactivity were not associated with
growth factor receptor expression before or after the development of AIPC. In normal
prostate tissue, four out of 10 specimens displayed c-erbB-1 positivity, whereas none
were positive for c-erbB-2, c-erbB-3 or c-erbB-4.
Previous observations of the expression of c-erbB-1 and c-erbB-2 in prostate cancer
tissue and cell lines have demonstrated highly divergent rates. Our results display
figures far below the highest reported values of c-erbB-1 and c-erbB-2 expression,
but for c-erbB-2 our figures are higher than the results from Savinainen et al (2002).
The following explanations are offered: Firstly, the cancer cell population in biopsies
from human AIPC is much more heterogeneous than that from laboratory cell lines. Secondly,
there may be differences in the pattern of EGFRs between AIPC tissue from the prostatic
tumour, as used in the present study, and that observed in metastatic cancer tissue
as examined by Scher et al (1995). Tissue from metastases may express higher levels
of growth factor receptors than the primary tumour. Thirdly, several methodological
differences as to IHC may lead to variability of the results. In our study, only moderate
or strong membrane staining was considered a positive finding, whereas both Scher
et al (1995) and Di Lorenzo et al (2002) included weak membrane staining of c-erbB-1
in their cohort of positive specimens. The use of different antibodies represents
another cause of variability of the results. Finally, uncontrollable variations as
the duration of formalin fixation of the archival specimens may additionally influence
the immunoreactivity of EGFRs.
The results of the present study are in agreement with the published observations:
expression of c-erbB-1 increases significantly, as the tumour becomes androgen independent.
Xie et al (1995) reported that c-erbB-1-mediated signals are associated with the invasiveness
of DU-145 human prostate carcinoma cells. However, we were unable to confirm the findings
of Di Lorenzo et al (2002), that c-erbB-1 expression increased with increasing Gleason
score. Unlike the series of Di Lorenzo et al, 80% of the untreated cases of the present
study were already Gleason score ⩾7b. Di Lorenzo et al also found a positive correlation
between serum PSA and c-erbB-1, which was not confirmed in our study applying IHC-detected
PSA. Lee et al (2003) indicated that decreased PSA secretion in androgen-independent
LNCaP C-81 cells is associated with a low expression of c-erbB-2, similar to our observation
of a trend of reduced c-erbB-2 expression in biopsies that became PSA negative.
Even though high levels of c-erbB-1 can be demonstrated in human prostate cancer,
especially after the development of AIPC, its clinical role is not yet clear. This
receptor's function is further obscured as it has become clear that internalisation
has to take place for its activation (Kim et al, 2003). Thus, the protein expression
of c-erbB-1 per se is only one of several conditions for this receptor's functionality.
The role of c-erbB-2 in prostate cancer is also controversial, contrary to this receptor's
clinical importance for advanced breast cancer (Cobleigh et al, 1999; Slamon et al,
2001). As seen in the present study, immunoreactivity for this receptor has been reported
in 9% (Mark et al, 1999) up to 30% in untreated patients (20%, Osman et al, 2001;
25%, Signoretti et al, 2000; 29%, Xie et al, 1995; and 30%, Fossa et al, 2002). Savinainen
et al (2002) did not find c-erbB-2 immunoreactivity in any of 54 specimens from untreated
patients, nor in 20 lymph node metastases or 50 hormone-refractory tumours. The percentage
of c-erbB-2-positive cases among untreated prostate cancer patients thus seems generally
lower than in breast cancer. During the development of AIPC, several authors agree
that c-erbB-2 positivity increases (Xie et al, 1995; Signoretti et al, 2000; Osman
et al, 2001), contrary to our results that showed reversal to c-erbB-2 negativity
in 27 out of 35 AIPC patients. The c-erbB-2 immunoreactivity may, however, still be
important in the clinical management of a subgroup of these patients, and Moasser
et al (2001) found that cell lines expressing c-erbB-2 are particularly sensitive
to ZD1839. Furthermore, c-erbB-2 and c-erbB-3 frequently act together creating heterodimers.
Recently, a naturally occurring inhibitor of c-erbB-3 has been detected (Lee et al,
2001), and at least in some tumour cell lines, ZD1839 also inhibits c-erbB-3 (Moasser
et al, 2001).
Both c-erbB-1 and c-erbB-2 immunoreactivity have been shown to be associated with
an unfavourable prognosis in a homogenous series of hormonally untreated cancer patients,
in particular if these two receptors are combined (Di Lorenzo et al, 2002). Owing
to the clinical heterogeneity of our untreated cases, we did not perform a survival
analysis based on the PRTR biopsies. However, we found a significant association between
c-erbB-2 positivity of AIPC biopsies and a poor prognosis, whereas coexpression of
c-erbB-1 and c-erbB-2 in AIPC patients was not statistically significantly associated
with an unfavourable prognosis. The finding of a slightly better survival in AIPC
patients with c-erbB-4 immunoreactivity needs further confirmation. Previously, the
association between c-erbB-4 positivity and a better clinical outcome has been demonstrated
for advanced breast cancer patients (Suo and Nesland, 2002).
The strength of the present study is its large number of patients and the possibility
to analyse intrapatient changes of expression of EGFRs during the development of AIPC.
Our investigations of EGFRs are, however, limited to IHC only. The correlation between
IHC-detected c-erbB-2 expression and comparable results of FISH analyses has been
debated (Press et al, 2002). Analysis by IHC labels the gene product and has been
sufficiently accurate for screening purposes, defining positive cases by moderate
or strong membrane-specific staining in >10% of the tumour cells. On the other hand,
the FISH technique demonstrates gene amplification, and is currently applied in breast
cancer patients with nonconclusive c-erbB-2 IHC results and possible therapeutic consequences.
It may be discussed whether the pattern of expression of EGFRs in our AIPC patients,
all with local problems, is representative for AIPC patients in general. As we have
previously indicated, the clinical course and survival of AIPC patients with local
problems seem to differ from that of AIPC patients with symptomatic bone metastases
(Hernes et al, 2000,2003).
In summary, we find that during the development of AIPC intrapatient changes occur
in two opposite directions for each of the four EGFRs: negativity change to positivity,
and vice versa, statistically significant only for the increase of c-erbB-1 expression
(P=0.001). c-erbB-2 is a poor prognosis factor in AIPC patients with local problems.
These findings support ongoing attempts to develop new treatment strategies for subgroups
of prostate cancer patients with the inhibition of EGFRs and their signalling pathways.
The sole immunohistochemical demonstration of membrane-located EGFRs in biopsies may
give some indications for these agents’ efficacy in the individual patient. However,
these receptors’ final association with response rates has to be determined in larger
series of patients with known growth factor receptor status.