Download Systemic effects of surgery: quantitative analysis of circulating basic

 Springer 2005
Breast Cancer Research and Treatment (2005) 93: 35–40
DOI 10.1007/s10549-005-3381-1
Report
Systemic effects of surgery: quantitative analysis of circulating basic fibroblast
growth factor (bFGF), vascular endothelial growth factor (VEGF) and transforming
growth factor beta (TGF-b) in patients with breast cancer who underwent limited
or extended surgery
G. Curigliano1,2, J.Y. Petit3, F. Bertolini4, M. Colleoni1, G. Peruzzotti2, F. de Braud2,
S. Gandini5, A. Giraldo3, S. Martella3, L. Orlando1, E. Munzone1, E. Pietri1, A. Luini6,
and A. Goldhirsch1
1
Department of Medicine, Division of Medical Oncology; 2Clinical Pharmacology and New Drugs Development Unit;
Division of Plastic Surgery; 4Laboratory of Haemato-Oncology; 5Division of Epidemiology; 6Division of Senology,
European Institute of Oncology, Milan, Italy
3
Key words: angiogenesis, breast cancer, bFGF, surgery, TGF-b, VEGF
Summary
Background. To assess if feature, extent and duration of surgery could influence levels of systemic proangiogenic
cytokines vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming
growth factor beta (TGF-b).
Patients and methods. We collected blood samples from 82 consecutive breast cancer patients who underwent
various types of surgery, classified according to the magnitude of tissue injury in: minimal (quadrantectomy),
moderate (mastectomy without reconstruction), and heavy [mastectomy followed by reconstruction with transversus recto-abdominal muscle cutaneous flap (TRAM)]. Samples were collected one day before surgery (D)1), at
the end of surgical tumor removal (D0), and on 1st (D+1), 2nd (D+2) and 5th (D+5) day after surgery. Serum
VEGF, bFGF and TGF-b levels were measured by the enzyme immunoassay method.
Results. On average a continuous decrease was observed for all growth factors from the day before operation to
the 5th day after operation. On day (D+5) an increase was observed for patients who underwent extended respect to
moderate surgery. These differences were found statistically significant for bFGF and VEGF (p = 0.05 and
p = 0.025 respectively). A statistically different trend for type of operation was observed also for TGF-b at
24–48 h: a minor reduction, compared to time of operation, was observed for minimal surgery, an intermediate
reduction for moderate surgery and a higher decrease for extended surgery.
Conclusions. Angiogenic cytokines perioperative levels could be increased on 5th day (D+5) by extent of surgery
and should induce perioperative stimulation of residual cancer cells. A better understanding of the time interval
during which the sequelae of events in wound healing occur may be the basis for defining new therapeutic strategies
that can interfere with tumor outgrowth sparing wound healing processes.
Introduction
Surgery is still the main curative therapeutic modality
for breast cancer. Angiogenesis plays a key role in both
wound healing and the ability of a cancer to survive and
grow. Investigations into the angiogenic response may
help in guiding surgical approaches and in timing antiangiogenic therapy [1]. Normal wound repair generates
an angiogenic response to deliver nutrients and inflammatory cells to injured tissue. The angiogenic response
enables the removal of debris and is central to the
development of a granulation tissue framework for
wound closure [2]. The mediators of wound angiogenesis
include soluble factors such as VEGF, tumor necrosis
factor, TGF-b, bFGF, and platelet-derived growth
factor (PDGF) identified in several wound models [3].
Angiogenic agonists (e.g. VEGF) and antagonists (e.g.
thrombospondin-1) have been described at various times
during repair [4–6], suggesting that the net angiogenic
stimulus may be a balance of factors changing to favor
either vessel growth or regression [7]. Previous studies
have shown that surgical wound fluid collected within a
few hours of operation is potently angiogenic. bFGF
levels have been shown to peak immediately after surgery and then fall by the second post-operative day [8].
This immediate release has been suggested to function to
36
G Curigliano et al.
initiate wound angiogenesis. In later wounds, VEGF is
the predominant angiogenic mediator [4]. An up-regulation of VEGF production in wound repair has been
demonstrated in keratinocytes in skin wounds in rat,
guinea pig, and mouse models [9]. TGF family is involved in several steps of wound healing: monocyte
chemoattraction, formation of granulation tissue and
fibroblast stimulation, neovascularization, wound contraction and extracellular-matrix reorganization. The
response of the body to a cancer is not a unique mechanism but has many parallels with inflammation and
wound healing. It has been suggested that the inflammatory cells and cytokines found in tumors are more
likely to contribute to tumor growth, progression, and
immunosuppression than they are to mount an effective
host antitumor response [10]. If genetic damage is the
‘match that lights the fire’ of cancer, some types of
inflammation may provide the ‘fuel that feeds the
flames’. Moreover cancer susceptibility and severity may
be associated with functional polymorphisms of
inflammatory cytokine genes, and deletion or inhibition
of inflammatory cytokines inhibits development of
experimental cancer. Work on the production of proangiogenic cytokines in early human wound fluid has
been done using drain fluid from patients undergoing
cancer surgery [4, 8]. These studies are based on the
premise that wound fluid would be generally representative of the growth environment of the wound. However, none have studied if feature, extent and duration of
surgery could affect systemic perioperative levels of
angiogenic cytokines in patients with breast cancer. A
better understanding of the time interval during which
the sequelae of events in wound healing occur may be
the basis for defining new therapeutic strategies that can
interfere with tumor outgrowth sparing wound healing
processes. After surgical resection of a tumor, the
microenvironment of the wound site differs from that of
normal tissue in several ways. Hypoxia, fibroblast activation, and various growth factors released after
wounding make the wounded site different from nonwounded tissue. Patients undergoing major oncological
resections might develop cytokine production disregulation and subsequent post-surgical immunosuppression, especially when the operation is of long duration.
The aim of this study was to determine the effect of
wounding on tumor growth. To better understand the
mechanism of wound angiogenesis and its significance in
tumor biology and surgical intervention, we specifically
evaluated the temporal profile of serum VEGF, bFGF
and TGF-b in breast cancer patients who underwent
minimal, moderate or heavy surgery.
European Institute of Oncology, Milan, between March
2001 and September 2001. All patients signed a written
informed consent form approved by the Institutional
Review Board of our Institute. Only those patients
without any pre-operative treatment were included in the
current study. Patients underwent minimal (quadrantectomy: Quad), moderate (mastectomy without reconstruction: Mast), and heavy surgery [mastectomy
followed by reconstruction with transversus rectoabdominal muscle cutaneous flap: (TRAM)]. Pathological assessment included evaluation of the primary tumor
size, histological type, lymph node status, including
sentinel node biopsy procedure. Tumor grading and
perivascular invasion have been assessed. Estrogen (ER)
and progesterone (PgR) receptor status, Ki67 labeling
index, determined with the MIB1 monoclonal antibody
and HER2/neu overexpression was evaluated by Dako
test (Dako, Glostrup, Denmark). Samples were obtained
from peripheral vein one day before surgical resection
(D)1), at the end of surgical tumor removal (D0), and on
1st (D+1), 2nd (D+2) and 5th (D+5) day after surgery.
VEGF, bFGF and TGF-b levels were measured by the
enzyme immunoassay method in serum samples.
Approximately 10 ml of peripheral venous blood was
drawn into a serum separator tube and centrifuged at
3000 revolutions per minute. Serum samples were aliquoted and preserved at )80 C until further use. Serum
VEGF, bFGF, and TGF-b levels were measured using a
commercially available competitive Enzyme Immunoassay (EIA) kit (Accucyte; Cytimmune Sciences, Inc.,
College Park, MD). Briefly, appropriate dilutions of
standards (provided with the kit) and a 4-fold dilution of
samples were added to the designated wells of a 96-well
plates that were precoated with goat antirabbit antibody.
Subsequently, competitive ligands and antibodies
against the respective angiogenic factors were added
serially and incubated for 3 h at room temperature. After
five washings, the streptavidin–alkaline phosphatase
complex was added into each well and incubated for
30 min at room temperature. Washing steps were repeated and color reagents (alcohol dehydrogenase and
diaphorase) were added. Absorbance of the 0 dose was
monitored at 492 nm (Molecular Devices Corporation,
Sunnyvale, CA) and when the optical density reached
between 1.5 and 2.0, the final reading was taken. Each
sample was analyzed in duplicate and the average was
considered as the final value. Data plotting and curve
fitting (logit-log plot) were accomplished using a computer-assisted program (SOFTmax PRO, version 2.4,
Molecular Corporation Devices, Sunnyvale, CA) on a
MacIntosh computer. All samples and standards were
assayed in duplicate.
Patients and methods
Statistical analysis
Blood samples were collected prospectively from 84
consecutive pre- and post-menopausal patients who had
presented with primary or recurrenced operable (T1–T4)
node negative/positive (N0–N2), breast cancer to the
Twenty-five observations presented measures for the
growth factors below the minimum possible value
therefore they have been replaced by the minimum
37
Surgery and angiogenesis in breast cancer
values found in the datasets. All variables were graphically checked for normal distribution. Linear models are
applied to verify statistical relevance of clinicopathological feaures on growth factors before the operation.
Such analysis was restricted to patients with invasive
lesions. Percentage changes from operation were evaluated at three time points: before operation, at 24–48 h
after operation and at 5 days. Observations at 24 and
48 h were considered taking the latest observation because at 48 h after operation many missing values were
found. For the same reason, at five days after operation
growth factors, evaluated for quadrantectomy, were
taken out from the analysis becase too many missing
values were found and a problem of selection bias might
arise. Comparisons between types of surgery were performed using linear models and adjusting for values at
intervention (ANCOVA). Time effect was evaluated
analyzing significance of changes from operation
with t-test. Two sided p values £ 0.05 were considered significant. All calculations were performed using
SAS software (SAS for Windows version 8.02. SAS
Institute Inc. Cary NC USA, 2000).
Table 2. Clinicopathologic features and median value of pre-operative
serum angiogenic growth factors
bFGF
VEGF
TGF-b
(pg/ml)
(pg/ml)
(pg/ml)
Median
p
Median
0.336
84.50
82.60
p
Median
p
Age (years)
<65 (n = 49)
‡65 (n = 7)
10.00
6.25
18.56
0.690 25.03
0.820
Tumor size (cm)
<5 (n = 40)
7.38
81.34
18.31
‡5 (n = 2)
9.91
0.775 185.50
0.361 20.99
0.735
Lymph node status
No (n = 18)
4.50
Yes (n = 24)
9.55
Vascular invasion
No (n = 27)
Yes (n = 21)
0.617
85.60
22.54
68.00
0.390 15.84
8.14
82.60
20.95
11.51
0.305 104.10
0.898 18.11
0.828
0.256
ER & PgR status
Negative (n = 15)
8.26
Positive (n = 38)
9.80
76.10
18.08
0.591
83.30
0.311 22.52
87.47
19.75
0.254
67.71
0.474 19.03
0.079
c-erb-B2
Results
Forty-three (52%) patients underwent minimal (quadrantectomy), 18 (22%) moderate (mastectomy without
reconstruction), and 21 (26%) heavy surgery [mastectomy followed by reconstruction with transversus rectoabdominal muscle cutaneous flap (TRAM)]. Median age
of the patients was 51 years. The pre-operative median
values (n = 82) of serum VEGF, bFGF and TGF-b
levels were 84.50 pg/ml (range 14.97–573.66 pg/ml),
10.21 pg/ml (range 0.44–74.70 pg/ml), and 21.45 pg/ml
(range 6.34–135.94 pg/ml), respectively for each type of
surgery. Mean values at baseline (D)1), surgical removal
Table 1. Mean value of angiogenic cytokines respect to timing of
operation
Pre-
Post-
24 h
48 h
120 h
Absent (n = 33)
9.87
Present (n = 19)
8.14
0.359
(D0), and on (D+1), (D+2) and (D+5) after surgery are
reported in Table 1.
Clinicopathologic features and median value of preoperative serum angiogenic growth factors are reported
in Table 2. No relationship has been found between preoperative levels of angiogenic factors and features of
disease.
Plots of median values for VEGF, bFGF and TGF-b
at each time point, related to different types of surgery,
are presented in Figures 1, 2 and 3. Confidence intervals
are not presented in the plots because they overlap.
In Table 3 are reported median values of percentage
changes of angiogenic factors in relation to extent of
surgery for all time points from operation. Significance
of extent of surgery and relevance of changes from
operation are evaluated including p-values in the table.
operative operative (pg/ml) (pg/ml) (pg/ml)
(pg/ml)
(pg/ml)
TRAM
136.2
152.8
130.7
Mastectomy
139.9
190
208
VEGF
VEGF
Quadrantectomy 111.5
bFGF
102.1
94.3
81.6
111
78.1
158.0
85
250
TRAM
Quadr.
Mastect.
200
87.7
150
TRAM
15.7
14.9
14.1
8.0
15.8
Mastectomy
21.9
17.9
21.6
4.2
11.4
Quadrantectomy 111.5
102.1
94.3
78.1
87.7
50
0
100
TGF-b
TRAM
24.3
18.7
14.8
9.4
13.3
Mastectomy
34.3
21.4
21.8
14.4
13.3
Quadrantectomy
25.4
17.2
18.6
15.1
6.9
Baseline
Surgery
24 h
48 h
5 days
Figure 1. Temporal trends of serum VEGF in breast cancer patients
according to type of surgery.
38
G Curigliano et al.
bFGF
30
TRAM
Quadr.
25
Mastect.
20
15
10
5
0
Baseline
Surgery
24 h
48 h
5 days
Figure 2. Temporal trends of serum bFGF in breast cancer patients
according to type of surgery.
TGFbeta
40
35
TRAM
Quadr.
30
Mastect.
25
20
15
10
5
0
Baseline
Surgery
24 h
48 h
5 days
Figure 3. Temporal trends of serum TGF-b in breast cancer patients
according to type of surgery..
When comparing D)1 with D0, no significant differences among various types of surgery were observed for
any angiogenic marker (data not reported). For TGF-b,
a significant effect of time was detected; levels at D)1
were on average 48% higher than levels at D0 (Figure 3).
Table 3. Median of percentage changes from operation for bFGF,
VEGF and TGF-b according to extent and timing from surgery
n
bFGF
VEGF
TGF-b
D)1
n
24–48 h
n
5 days
Quad
43
4
39
19
4
NA
Mast
18
54
18
)29
13
)40
TRAM
21
6
19
)58
12
80
Overall
82
8
76
)12
29
)6
p for time
0.338
0.526
p for surgery
Quad
43
0.170
9
0.329
)6
Mast
18
)20
18
)24
13
TRAM
20
4
19
)42
12
117
Overall
81
2
75
)23
30
)33
38
p for time
0.428
0.028
p for surgery
0.87
0.468
0.892
4
0.050
NA
)47
0.167
0.025
Quad
42
27
41
)18
5
NA
Mast
TRAM
18
21
71
57
18
20
)38
)44
12
12
)38
20
Overall
81
48
79
)29
29
)27
p for time
p for surgery
<0.0001
0.001
0.062
0.321
0.005
0.174
At 24–48 h after surgery (D+1–D+2), a percentage
decrease from D0 was observed for all growth factors.
The percentage change was statistically significant for
TGF-b and VEGF (on average a 29 and 23% reduction
with p = 0.001 and p = 0.028, respectively). Looking
at trends by type of surgery, it can be observed that the
percentage reduction for any angiogenic factor was
higher for mastectomy and TRAM and lower for
quadrantectomy.
In patients who underwent quadrantectomy bFGF
showed, on average, even an increase (19%) at D+1–
D+2, a but this change was not statistically different
respect to the reduction found for mastectomy and
TRAM. Any type of surgery was associated with significant (p = 0.005) reduction of TGF-b at D+1–D+2.
Reduction of perioperative levels of TGF-b is higher for
TRAM and mastectomy than for quadrantectomy
(p = 0.005).
On D+5 after surgery, we observed an increase of all
angiogenic factors in patient who underwent TRAM,
and a reduction for patients who underwent mastectomy. The increase in patients who underwent TRAM
was lower for TGF-b; in fact the difference by type of
operation was statistically significant only for bFGF
and VEGF (at 5 and 2.5%, respectively).
Discussion
Tumor growth is angiogenesis dependent. Perioperative
levels of endogenous stimulators (bFGF, VEGF,
cathepsin, copper, interleukin 1, 6 and 8), inhibitors
(plasminogen activator inhibitor-1, tissue inhibitor of
metalloprotease, zinc, interleukin 10 and 12) and modulators of angiogenesis (TGF-b, tumor necrosis factor
alpha) may indicate the switch to the angiogenic phenotype of neoplasia that depends on a net balance
between positive and negative angiogenic factors released by the tumor. In particular, there is an alteration
in the circulating levels of acute phase reactants that are
believed to play an important role in the perioperative
period, at the time of enhanced release of malignant cells
into the circulation, with risk of metastasis induction.
Endothelial growth factors have a cell mitogen effect
and act as regulator of vascular permeability. Several
retrospective studies reported that VEGF is significantly
associated with relapse-free survival, overall survival, or
both. Patients with early stage breast cancer who have
tumors with elevated levels of VEGF, TGF-b or bFGF
have a higher likelihood of recurrence than patients with
low-angiogenic tumors, even if treated with conventional adjuvant therapy [1]. Preoperative levels of
VEGF, bFGF and TGF-b described in our study are
similar to previously reported [11–13]. Other studies
reported a correlation between clinical pathological
features of disease and preoperative levels of angiogenic
factors [14]. In our study, no relationship has been
observed between age, stage, biological features and
levels of preoperative angiogenic factors. Median values
Surgery and angiogenesis in breast cancer
of VEGF, bFGF and TGF-b usually have a drop out at
24–48 h after surgery. Reduction of TGF-b levels form
pre-operative to post-operative time was statistically
significant. Kong et al. [13] showed that plasma TGF-b
levels were elevated pre-operatively in 81% of the
patients. The mean plasma TGF-b level in breast cancer
patients normalized after surgery (19.3 ± 3.2 versus
5.5 ± 1.0 ng/ml, p < 0.001) in the majority of subjects;
levels were persistent in case of presence of lymph node
metastases or overt residual tumor. No data are reported on bFGF in correlation to timing or extent of
surgery in patients with breast cancer. An overall percentage change with 23% reduction after surgery has
been described for VEGF. In a previous report [15], a
significant change in serum VEGF levels compared with
pre-operative values has been described with time with
an initial drop over the first 3 days; thereafter levels
recovered. In this study has been also performed analysis of the local wound response, showing that VEGF
levels in the wound environment are much higher than
the serum equivalent from as early as the first postoperative day. They then peak at day 2 and remain at a
higher level thereafter for several days. This observation
fits well with wound vascular mechanisms in animal
models. Acute wound response could act as a ‘molecular
trap’ for angiogenic factors and reduction of serum
levels of VEGF, TGF-b and bFGF in our patients could
be a result of this ‘trapping’. Another explanation for
reduction of angiogenic factors, especially for VEGF,
should be considered platelet count drop after surgical
injury. Since platelets are the main source of serum
VEGF, it can be reasonable to suppose that their trapping in wound healing could explain VEGF levels drop
after surgery. The surgical wound itself is a unique
extravascular compartment with increased vascular
permeability and a high surface area:volume ratio. If
reabsorption occurs freely from the surgical wound site
changes in local VEGF concentrations should be
reflected in the circulation, i.e. serum levels. Subsequent
increase of VEGF (specifically in patients who underwent TRAM surgery) should be related to massive local
wound VEGF production. TRAM surgery creates a
wound with a larger surface area than wide local excision. This effect may mark an interaction between
residual tumor-derived local inhibitors resulting in an
initially depressed normal stromal angiogenic response
that recovers over time. This would be in keeping with
the evidence that tumor cells secrete factors that provide
negative ‘feedback’ regulation and serve to suppress
vascular growth, restraining the growth of secondary
tumors or metastases [25–27]. Surgical clearance of
cancer involves regional extirpation, and residual tissues
may still be under the influence of tumor-derived
inhibitors delaying the normal angiogenic wound response. The mechanism underlying these observations
requires additional investigation and may relate to the
half-life of angiogenic stimulators compared with
inhibitors, to a local effect on the stroma when the
angiogenic drive from the tumor is removed or because
39
of impaired influx of blood and platelet release at the
time of injury. This muted response in cancer patients
may represent an opportunity to complete surgical
treatment while minimizing stimulation of metastatic
disease, a biological argument in favor of immediate
reconstruction after, for example, breast cancer surgery.
Experimental evidence suggests that a growth factorrich environment enables the survival of cancer cells left
in an area of cancer extirpation or in the circulation
[25–27]. However, as wounds age the surgical site
becomes less favorable to tumor implantation, and when
healing is complete injected tumor cells do not localize
to the surgical site [26]. Thus, local recurrence found in
conjunction with widespread metastatic disease is likely
to have been established by perioperative seeding rather
than as a late phenomenon. Furthermore, a growth
factor-stimulated microenvironment affects growth of
established residual tumor foci in vivo and cell lines
in vitro [27]. Antiangiogenic therapy is currently undergoing clinical trials, and in the future perioperative
systemic therapy or local therapy may include use of
such therapy. However, our findings indicate that very
high local concentrations may need to be antagonized.
Quantification of this in vivo biological response should
facilitate the design of wound healing experiments to
more closely represent the ‘human’ response to surgical
stress. Drainage systems may offer an opportunity to
manipulate the early wound environment and reduce
local cancer recurrence rates in the future. A better
understanding of the time interval during which the sequelae of events in wound healing occur may be the
basis for defining new therapeutic strategies that can
interfere with tumor outgrowth sparing wound healing
processes.
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Address for offprints and correspondence: G. Curigliano, Division of
Medical Oncology, Clinical Pharmacology and New Drugs Development Unit, European Institute of Oncology, Via Ripamonti 435, 20141
Milano, Italy; Tel.: 39.02.57489482; Fax: 39.02.57489.581; E-mail:
[email protected]