Blood vessels have comparable functions during organogenesis and tumour growth. These
include the maintenance of blood flow necessary for delivery of oxygen and nutrients,
as well as bidirectional paracrine interactions between endothelium and epithelium
that influence proliferation, migration and differentiation of both cell populations
(Cleaver and Melton, 2003). In most malignant human tumours, the vasculature results
from angiogenesis, as evidenced by the formation of a desmoplastic stroma containing
small immature blood vessels clustered in vascular ‘hot spots’, and by a relatively
high fraction of proliferating endothelial cells. Vascularisation in human solid tumours
is measured in these hot spots, which arise through angiogenesis and ongoing vessel
remodelling, and this measure has a widely confirmed prognostic value in breast cancer
(Vermeulen et al, 2002). Blood vessel growth is an invasive process that destroys
the surrounding tissue architecture (Masson et al, 1998). Angiogenesis has traditionally
been regarded as the sole mechanism for a malignant tumour to obtain a functional
vasculature. The dormancy concept consolidated this view: when the number of capillaries
in the tumour tissue in an animal model decreased, tumour cell proliferation was not
affected but the apoptotic fraction increased, leading to ‘dormant’ tumours (Holmgren
et al, 1995).
In 1997, however, simple morphological observations in human primary non-small-cell
lung carcinomas led to the introduction of a new concept of tumour vascularisation:
‘co-option’ of blood vessels of the surrounding normal parenchyma appeared to be an
efficient alternative for angiogenesis (Pezzella et al, 1997). Subsequently, experiments
in a rat glioma model suggested that even angiogenic tumours initially co-opt normal
blood vessels after which a host defence response, governed by angiopoietin-2 expression
on the co-opted endothelial cells, causes blood vessel regression with concomitant
hypoxia and vascular endothelial growth factor (VEGF)-mediated angiogenesis (Holash
et al, 1999).
The liver has a comparably dense vasculature as the lungs and both organs frequently
host metastases of carcinomas. The hypothesis of our first study therefore was that
metastases that would be capable of preserving the stromal structure of the liver
might not become hypoxic and thus would not be dependent upon angiogenesis for survival
(Vermeulen et al, 2001). One of three growth patterns of colorectal adenocarcinomas
(CRC) metastases in the liver, the replacement pattern, in which hepatocytes were
merely replaced by tumour cells, forming muralia that were in continuity with the
liver cell, was indeed characterised by co-option of sinusoidal blood vessels at the
tumour–liver interface, by a lack of inflammation and desmoplastic stroma, and by
a low fraction of proliferating endothelial cells. Sinusoidal blood vessels do not
express CD34 and the co-opted vessel endothelium only started to express this endothelial
cell marker when they were engulfed by a few rows of CRC cells. Tumour cell plasticity
is also exemplified by recently described alternative mechanisms of intravasation
(tumour cells express an endothelial-like phenotype during vasculogenic mimicry (Hendrix
et al, 2003) and are part of the vessel wall) and of extravasation (intravascular
growth of tumour cell nests results in metastasis without the need for extravasation;
Alpaugh et al, 2002).
We have shown that cutaneous breast adenocarcinoma (BC) deposits are a heterogeneous
group with different degrees of hypoxia-driven angiogenesis reflected in an infiltrative
and an expansive growth pattern (Colpaert et al, 2003a). Using the marker carbonic
anhydrase 9 (CA9) (Wykoff et al, 2000), we found evidence of hypoxia in only 17% of
the cutaneous deposits with an infiltrative growth compared with 71% of the tumours
with an expansive, nodule-forming growth (P=0.02). The infiltrative growth pattern
was further characterised by a relatively low endothelial cell proliferation index
(5 vs 18%; P=0.004), and fibrin deposition in a smaller fraction of the metastases
(44 vs 100%; P=0.07). The cutaneous deposits with an infiltrative growth respected
the dermal architecture and co-opted pre-existing collagen bundles, blood vessels
and skin adnexa.
The aim of this study was to analyse the architecture of liver metastases of BC and
CRC with emphasis on the presence or absence of angiogenesis and hypoxia. Radiological
imaging studies have revealed subgroups of liver metastases with different contrast
enhancement and delineation, supporting the hypothesis of morphological heterogeneity
(Yamaguchi et al, 2002).
MATERIALS AND METHODS
Tissue specimens of formalin-fixed, paraffin-embedded human liver metastases from
49 patients with BC and from 28 patients with CRC were retrieved from the files of
the pathology departments of the General Hospital Sint-Augustinus and of the University
Hospital of Antwerp. Twenty-eight BC metastases were necropsy-derived. The other metastases
were obtained after elective surgery or needle biopsy. One tissue block, containing
a representative fraction of the tumour–liver parenchyma interface, was used per patient.
Sections 5 μm in size were cut. A standard haematoxylin and eosin stain was carried
out to evaluate the growth pattern based on the morphology of the tumour–liver parenchyma
interface, as described before (Vermeulen et al, 2001).
In the ‘desmoplastic’ growth pattern, the metastases were separated from the surrounding
liver parenchyma by a rim of desmoplastic stroma in which a dense mononuclear infiltrate
and numerous capillaries were present. Often tumour cell nests were infiltrating the
stroma. The tissue architecture of the liver was not conserved within the metastases.
In the ‘pushing’ growth pattern, liver plates were pushed aside and ran in parallel
with the circumference of the metastases at the tumour–liver parenchyma interface.
There was no desmoplastic stroma formation, and the tumour cells were separated from
the hepatocytes by a thin layer of connective tissue fibres. A mild inflammatory infiltrate
was nearly always present at the interface. The tissue architecture of the liver was
not conserved within the metastases.
In the ‘replacement’ growth pattern, tumour cells were replacing hepatocytes in the
liver plates, at the interface or throughout the metastasis, conserving the tissue
architecture of the liver, without inflammation or fibrosis. Tumour cells and hepatocytes
had intimate cell–cell contact. Intra- and interobserver variability of this classification
was limited, as has been shown before (Vermeulen et al, 2001).
Glandular differentiation was graded according to the system of Elston and Ellis for
BC (Elston and Ellis, 1991). For CRC metastases, the same grading system was used.
Immunohistochemical staining for CA9 was performed at the Weatherall Institute of
Molecular Medicine (John Radcliffe Hospital, Headington, Oxford, UK) with the murine
monoclonal antibody M75 at a dilution of 1 : 50, as has been described before (Chia
et al, 2001). Bile ducts were used as an internal positive control, given the constitutive
hypoxia-independent expression of CA9 by bile duct epithelium. The immunostaining
was quantified by semiquantitative scoring: a score of 0–3 for the intensity of staining
in the majority of the tumour cells was given (0, no staining; 1, weak staining; 2,
moderate staining; 3, strong staining). The fraction (%) of immunostained tumour cells
was estimated. The product of intensity score and percentage yielded a final global
CA9 score of 0–300. The interface (about 20 tumour cell rows adjacent to the liver
parenchyma) and the centre of the metastases were also evaluated separately for CA9
expression: any percentage of immunostaining in the respective regions was scored
as positive CA9 expression.
Macrophages were immunostained with an anti-CD68 monoclonal antibody (clone KP-1,
dilution of 1 : 80, DakoCytomation, Glostrup, Denmark) on the Ventana NexES automated
immunostainer. Quantification of the relative immunostained area, as a measure of
the number of macrophages, was performed with the Chalkley method, a point counting
method using a microscope eyepiece graticule (Chalkley et al, 1943). The × 400 field
at the tumour–liver parenchyma interface giving the impression of the highest number
of CD68-positive cells on low magnification was selected and the Chalkley count was
performed. From this field, an imaginary cross through the centre of the metastasis
and with 90° angles was constructed, and the three other fields at the intersection
of this cross and the interface were analysed. If a field contained a portal tract,
the adjacent × 400 field was taken, given the high number of inflammatory cells present
in portal tracts independent of the degree of inflammation at the interface elsewhere.
The mean of the four counts was used for further analyses.
The presence of fibrin was detected immunohistochemically with the NYB.T2G1 monoclonal
antibody (Accurate Chemical and Scientific Corp., Westbury, NY, USA; dilution 1 : 100),
which reacts with the amino-terminal part of the Bß chain only after removal of fibrinopeptide
B by thrombin, and hence binds to fibrin but not to fibrinogen. The staining was performed
on the DAKO Autostainer (DakoCytomation) after pretreatment at 98°C for 30 min in
Target Retrieval Solution (DakoCytomation). Fibrin deposition due to tissue damage
during excision/biopsy of the metastases was usually present at the cut surface and
was used as internal positive control. Negative staining in the absence of internal
positive control staining resulted in exclusion of the result for further analysis.
In all other cases, fibrin staining was graded from 0 to 3 (0: no staining; 1: minimal
staining; 2: moderate staining; 3: extensive staining). This evaluation was performed
both for the interface and the centre of the metastases.
Immunohistochemical staining for LYVE-1 was carried out as described previously (Beasley
et al, 2002; Williams et al, 2003). In brief, antigen retrieval at 98°C for 30 min
in Target Retrieval Solution (DakoCytomation) was followed by incubation with the
affinity-purified LYVE-1 Ig (0.5 μg ml−1) for 30 min. The staining was performed using
the DAKO Autostainer (DakoCytomation peroxidase Envision kit).
Statistical analyses were performed using JMP-5 software (SAS Institute, North Carolina,
USA) on an Apple PowerBook G4 (Apple Computer, California, USA).
RESULTS
In four BC metastases, the interface, and thus the growth pattern, could not be evaluated
properly, and these cases were excluded from further analysis. The majority of the
BC liver metastases showed a replacement growth pattern (96%) (Figure 1
Figure 1
Breast cancer liver metastasis, replacement growth pattern (haematoxylin and eosin
stain): the tissue architecture of the liver is preserved within the tumour tissue.
There is close contact between tumour cells and hepatocytes at the interface and no
inflammation.
; Table 1
Table 1
Distribution of the growth patterns of the 73 liver metastases according to the site
of the primary tumour (χ
2 analysis; Pearson test)
Desmoplastic
Pushing
Replacement
Breast
(n=45)
1 (2%)
1 (2%)
43 (96%)
Colorectal
(n=28)
14 (50%)
5 (18%)
9 (32 %)
Total
(n=73)
15 (21%)
6 (8%)
52 (71%)
P<0.0001
). In contrast, only one-third of the CRC liver metastases had these growth characteristics
(32%), while 50% clearly induced a desmoplastic tissue reaction at the liver parenchyma–tumour
interface (χ
2 test P<0.0001) (Figure 2
Figure 2
Colorectal cancer liver metastasis, desmoplastic growth pattern (haematoxylin and
eosin stain): a rim of desmoplastic stroma separates the liver parenchyma from the
tumour tissue. A dense inflammatory cell infiltrate is present in the stroma nearby
the liver parenchyma.
). The characteristics of the replacement growth pattern were often present from interface
up to the centre in the BC cases, while they were limited to the interface in all
CRC liver metastases. In 15 metastases (eight of BC origin and seven of CRC origin),
a mixed growth pattern was found. Only the dominant pattern was considered for further
analysis.
When comparing BC liver metastases with CRC liver metastases (Table 2
Table 2
Comparison of glandular differentiation, fibrin deposition, CA9 expression and the
macrophage content of breast cancer and colorectal cancer liver metastases
Breast
Colorectal
Glandular differentiation
(n=45)
(n=28)
1
1 (3%)
21 (75%)
2
8 (17%)
5 (18%)
3
36 (80%)
2 (7%)
P<0.0001
Fibrin, central
(n=37)
(n=24)
0
16 (43%)
6 (25%)
1
10 (27%)
4 (17%)
2
2 (6%)
4 (17%)
3
9 (24%)
10 (41%)
P=0.15
Fibrin, interface
(n=38)
(n=25)
0
30 (79%)
11 (44%)
1
6 (15%)
10 (40%)
2
1 (3%)
1 (4%)
3
1 (3%)
3 (12%)
P=0.037
CA9, central
(n=44)
(n=24)
−
32 (73%)
1 (4%)
+
12 (27%)
23 (96%)
P<0.0001
CA9, interface
(n=45)
(n=24)
−
38 (84%)
11 (46%)
+
7 (16%)
13 (54%)
P=0.002
Global CA9 score
14.4±8.6 (0)
74.5±14.9 (52.5)
P<0.0001
Macrophage count
4.57±0.28 (4.25)
8.25±0.60 (7.50)
P<0.0001
Differences in categorical variables are validated by two-tailed Fisher's exact test.
Continuous variables are expressed as mean±s.e. (median). Differences are validated
by Wilcoxon test.
), the former showed less glandular differentiation (80% grade 3 vs 7% grade 3; P<0.0001),
less frequently had fibrin deposition at the tumour–liver parenchyma interface (21
vs 56%; P=0.037), expressed CA9 only in a minority of cases at the interface (16 vs
54%; P=0.002) or in the central portion of the metastases (28 vs 96%; P<0.0001) (Figure
3
Figure 3
Carbonic anhydrase 9 immunostaining of a colorectal cancer liver metastasis: strong
staining (brown; score 3+) of the tumour cells. Constitutive expression by bile duct
epithelium (internal positive control).
), and had a significantly lower macrophage Chalkley count (4.3 vs 7.5 (median); P<0.0001).
After excluding the 28 necropsy-derived BC liver metastases, results were comparable
(data not shown), with P-values of <0.0001 (glandular differentiation), 0.02 (central
fibrin), 0.08 (fibrin at the interface), <0.0001 (central CA9), 0.009 (CA9 at the
interface), 0.0004 (global CA9 score) and 0.001 (macrophage count).
Taking all liver metastases, there was a positive correlation between the global CA9
score and the Chalkley count of the macrophages (r=0.43; P=0.002).
Analysis of the influence of growth patterns on the different parameters was performed
in the CRC liver metastases group and the BC liver metastases group separately. Forty
per cent of the CRC liver metastases with a pushing growth pattern vs only 5% of the
other liver metastases had extensive (grade 3) fibrin deposition at the interface
(P=0.09) (Figure 4
Figure 4
Immunostaining of fibrin (brown) in a desmoplastic colorectal cancer liver metastasis:
fibrin deposits mainly in the liver parenchyma surrounding the metastasis.
). Desmoplastic CRC liver metastases had a mean macrophage Chalkley count of 9.3 (s.e.:
0.94; median: 9.5) vs 7.3 (s.e.: 0.69; median: 7.0) in the other CRC liver metastases
(P=0.09) (Figure 5
Figure 5
Anti-CD68 immunostaining demonstrating numerous macrophages (brown) in the desmoplastic
rim surrounding a colorectal cancer liver metastasis. The Kupffer cells in the liver
parenchyma are also immunoreactive.
). Although the majority of the non-replacement CRC liver metastases had CA9 expression
at the tumour–liver parenchyma interface and only a minority of the replacement CRC
liver metastases, this difference was not significant. Of the BC liver metastases
with a replacement growth, only 24% had central CA9 expression, in contrast to all
BC liver metastases with a non-replacement growth (P=0.06). Carbonic anhydrase 9 expression
at the tumour–liver parenchyma interface was present in only 12% of the BC liver metastases
with a replacement growth, in contrast to all BC liver metastases with a non-replacement
growth (P=0.02). Other associations were not found.
The lymphatic endothelial/sinusoidal marker LYVE-1 was expressed in the midzonal sinusoidal
blood vessels of normal liver parenchyma, as was reported previously (Banerji et al,
1999). This constitutive expression was used as internal positive control of the immunohistochemical
staining. In the desmoplastic CRC liver metastases, there were few if any LYVE-1-positive
vessels in the connective tissue capsule or in the metastases. In most of the metastases
with this growth pattern, LYVE-1 expression was absent or attenuated in the midzonal
sinusoids of the surrounding liver parenchyma. In contrast, in the BC and CRC liver
metastases with a replacement growth pattern, LYVE-1 expression in the sinusoids of
the liver parenchyma that made contact with the tumour cells was not attenuated. Moreover,
sinusoidal blood vessels engulfed by up to about 20 tumour cell rows still expressed
LYVE-1 at the tumour–liver interface (Figure 6
Figure 6
Expression of LYVE-1 (brown, immunostaining) by sinusoidal endothelial cells within
the liver parenchyma at the interface with a replacement-type breast cancer liver
metastasis (arrow heads). Sinusoids engulfed by tumour cells express LYVE-1 at the
tumour–liver interface (arrows) and lose this expression towards the centre of the
metastasis (towards the right on the microphotograph).
), whereas those towards the centre of the metastases expressed less of the receptor.
DISCUSSION
The liver is a target organ for metastasis of BC and CRC. The parenchyma is supported
by a dense vasculature composed of branches from both venous and arterial blood vessels.
Liver cell plates, usually composed of only two rows of hepatocytes, are intimately
associated with sinusoidal blood vessels, in this way minimising the development of
acute or chronic hypoxia in a tissue with a high cell density and a high metabolic
activity. As hypoxia is the most important stimulus of angiogenesis, it was the hypothesis
of this and a former study (Vermeulen et al, 2001) that liver metastases in which
tumour cells would be able to preserve the architecture of the liver stroma could
grow without hypoxia and subsequent angiogenesis. We have indeed shown that a minority
of CRC liver metastases display a replacement pattern, characterised by low endothelial
cell proliferation, a high tumour cell proliferation to endothelial cell proliferation
ratio and only weak expression of CD34 in the constitutively CD34-negative endothelial
cells of the co-opted sinusoidal blood vessels.
In the present study, we have shown that BC liver metastases have different growth
characteristics to CRC metastases: nearly all BC metastases had a replacement pattern
that, in contrast to CRC metastases, was often also present in the centre of the metastases.
In addition, this was characterised by minimal fibrin deposition, lack of CA9 expression
in most of the BC metastases and a lower macrophage content compared to CRC metastases
(Figures 7
Figure 7
Carbonic anhydrase 9 immunostaining of a breast cancer liver metastasis: replacement
growth and no immunostaining (constitutive expression by bile duct epithelium (internal
positive control) was present in the section (not shown)).
, 8
Figure 8
Immunostaining of fibrin (brown) in a replacement breast cancer liver metastasis:
no staining at the tumour–liver interface (internal positive control: fibrin in a
sinusoidal blood vessel (arrow)).
and 9
Figure 9
Anti-CD68 immunostaining demonstrating the Kupffer cells in the liver parenchyma.
No macrophages at the tumour–liver interface of this breast cancer metastasis with
replacement growth.
).
The induction of angiogenesis by breast cancer has been shown to be highly variable
when comparing patients. In a study of cutaneous breast cancer deposits, an infiltrative
growth pattern, in which cancer cells respect the dermal architecture and co-opt pre-existing
blood vessels, was characterised by CA9 expression in only 17% of the tumours (Colpaert
et al, 2003a). Fibrin was present in the dermal stroma in 33% of the deposits with
an infiltrative growth pattern, and the endothelial cell proliferation was 4.2% (median
value). For the expansive growth pattern, the respective values were 71, 89 and 16.4%
(respective P-values: 0.02; 0.02; 0.004). Fifty-one per cent of the cutaneous deposits
expressed the infiltrative, less or non-angiogenic growth pattern vs 18% with an expansive
and angiogenic growth. The remainder of the metastases had a mixed growth pattern
with intermediate characteristics. Also, primary BC has a variable angiogenic profile:
inflammatory BC, a highly aggressive subtype with extensive dermal lymphovascular
permeation, has been shown to have an endothelial cell proliferation fraction two-fold
higher than non-inflammatory BC (19 vs 11%, respectively; P=0.01) (Colpaert et al,
2003b). Liver metastases of BC apparently are at the other end of the angiogenic spectrum:
the majority of the BC liver metastases co-opt the pre-existing sinusoidal blood vessels
in a replacement growth pattern that has been shown to have a low proliferative activity
of the endothelial cells (Vermeulen et al, 2001).
LYVE-1 is a receptor for hyaluronan mainly expressed on lymphatic endothelial cells
(Banerji et al, 1999). Together with the liver sinusoids, the lymphatic system is
responsible for the degradation of hyaluronan via LYVE-1. Expression of LYVE-1 has
indeed been demonstrated on the endothelial cells of liver and spleen sinusoidal blood
vessels (Banerji et al, 1999). In the liver metastases of BC and CRC with a replacement
growth pattern, the blood vessels close to the interface, but well surrounded by tumour
cells, continued to express LYVE-1. This observation, together with the conserved
stromal architecture within the replacement-type metastases, strengthens the blood
vessel co-option hypothesis. Interestingly, the co-opted sinusoidal blood vessels
in the replacement growth pattern started to express CD34, which is not expressed
constitutively on sinusoidal endothelial cells, at a distance of a few cell layers
from the interface (Vermeulen et al, 2001). Both the apparent loss of LYVE-1 expression
and the gain of CD34 expression indicate paracrine interactions between tumour cells
and co-opted endothelial cells, yet without eliciting angiogenesis or desmoplasia.
Recently, Williams et al (2003) have also reported the apparent loss of LYVE-1 from
lymph vessels that were engulfed by invasive breast cancer cells. The possible mechanisms
of this loss and its physiological consequences are currently under investigation
(DG Jackson, unpublished). The desmoplastic-type metastases contained very few, if
any, LYVE-1-expressing vessels, supporting the other data of angiogenesis as means
of vascularisation in this growth pattern (Figures 10
Figure 10
Expression of LYVE-1 (brown, immunostaining) by sinusoidal endothelial cells in the
liver parenchyma surrounding a desmoplastic colorectal liver metastasis (arrows).
No staining within the tumour tissue.
).
A possible explanation for the higher fraction of angiogenic, desmoplastic liver metastases
of CRC compared to BC might be that primary CRC are more angiogenic than primary BC.
Indeed, using the same method for both tumour histiotypes to quantify angiogenesis
by counting proliferating endothelial cells, the median proliferation fraction was
more than six times higher in primary CRC than in BC (8.9 vs 1.4%, respectively; P<0.0001)
(Vermeulen et al, 1997). This supports the rationale of counting microvessels in vascular
‘hot spots’ as a prognostic marker in solid human tumours: stroma containing more
newly formed blood vessels is more likely to give rise to angiogenic metastases. Treatment
of patients with metastatic colorectal cancer with bevacizumab, an anti-VEGF monoclonal
antibody, has shown a clear synergy with chemotherapy, supporting the high angiogenic
activity measured in our studies of primary colorectal cancer and its metastases in
the liver (Kabbinavar et al, 2003).
The angiogenic proteome of the metastases seems to be similar to that of the primary
tumours, since even in the less-vascularised skin, about half of the BC metastases
grow with minimal induction of vessel sprouting, reflected by low endothelial cell
proliferation. Taken together, the ‘seed’ might have more influence on the growth
pattern than the ‘soil’. Nevertheless, the more angiogenic CRC can induce liver metastases
with a blood vessel co-opting replacement growth pattern, as shown by us (this study;
Vermeulen et al, 2001) and by Prall et al (2003). Three types of invasion of the liver
parenchyma have been described by the latter authors, based on the number of apoptotic
hepatocytes at the tumour–liver interface and on the degree of compression of the
reticular connective tissue of the liver parenchyma surrounding the metastases. A
pattern that resembles the replacement pattern of our study was characterised by prominent
apoptosis of hepatocytes at the interface, with conservation of the reticular connective
tissue architecture of the liver, with debris, but without fibrosis. The other two
invasion patterns showed decreasing destruction of hepatocytes and increasing compression
of the stroma surrounding the metastases with the formation of a pseudocapsule. These
invasion patterns correspond to our pushing and desmoplastic growth patterns.
Other interesting observations concerning growth patterns of liver metastases originate
from animal tumour models. Solaun et al (2002) have described two subtypes of colon
carcinoma liver metastases that differ regarding their position in the liver tissue
and regarding their connection to the local microvasculature: the portal type and
the sinusoidal type. In the sinusoidal type of metastases, the blood vessels were
recruited, without disturbing the liver architecture. The portal type induced a desmoplastic
stromal reaction that surrounded the metastases and disturbed the liver architecture.
Necrotic areas frequently developed in these metastases. The histological microphotographs
in this publication show similar growth patterns as the replacement-type and desmoplastic-type
metastases of our study. The main difference, however, is that the two types of metastases
develop in the same animal after injection of a single tumour cell line, while patients
in our study have liver metastases with a single growth pattern, independent of their
position in relation to a portal tract. Whether the source of the blood vessels of
human liver metastases is associated with the growth pattern or with the route of
entrance of tumour emboli, arterial or venous, is not clear.
In a second animal tumour model (Griffini et al, 1997), 27% of the colon carcinoma
liver metastases were small and totally encapsulated by stroma, which was always connected
with adjacent portal tracts. All these metastases grew distant from the liver surface
and consisted of well-differentiated acini. The majority of the metastases, however,
were larger in diameter, and were not surrounded by a capsule. The metastases consisted
mainly of undifferentiated cancer cells in direct contact with hepatocytes and without
much desmoplastic stromal reaction. Most of these metastases were in contact with
the liver capsule. In our study, human BC liver metastases were poorly differentiated
and in most of these metastases, there was no desmoplastic reaction. The low degree
of differentiation in the replacement growth patterns might be the consequence of
less reciprocal interactions between cells from the desmoplastic stroma and epithelial
tumour cells. On the other hand, less-differentiated tumour cells might more easily
adopt a hepatocyte-like phenotype, induced by the sinusoidal blood vessels, a process
analogous to liver development during foetal life (Zaret, 2002).
If the replacement growth pattern is indeed composed of less-differentiated tumour
tissue, a worse prognosis would be expected. We have investigated an analogous phenomenon
in primary lung cancer (Sardari Nia et al, 2003), and found that the alveolar growth
pattern according to Pezzella et al (1997) in lung tumours indeed predicts shorter
disease-free and overall survival. These tumours co-opt the blood vessels of the alveolar
septa and are comparable to the liver metastases with a replacement growth pattern.
Computed tomographic (CT) imaging of liver metastases has been shown to be predictive
for recurrence after hepatic resection (Yamaguchi et al, 2002). CT contrast-enhanced
images of liver metastases were subtyped according to the shape of the metastases
and the irregularity of the outline of the nodules. Liver metastases with the most
irregular shape and contour were predictive of reduced 5-year disease-free survival.
Although this implies that growth and vascularisation patterns might influence prognosis,
it is not clear how the images relate to the histological findings. We are currently
performing studies to elucidate this important question.
Liver metastases of CRC had a significantly higher number of macrophages at the interface
with the liver than BC metastases. Inflammation and cancer progression are intimately
linked (Coussens and Werb, 2002) and, for instance, in primary BC, the degree of vascularity
was positively associated with the number of hot spots of macrophages expressing HIF-2alpha
(Leek et al, 2002). Since the HIF-2alpha expression in macrophages is induced by hypoxia
(Burke et al, 2002), and since macrophages in tumours migrate to hypoxic areas, their
presence in CRC liver metastases indicates a lower oxygen tension, which accords with
the elevated CA9 expression compared to BC liver metastases.
Both CA9 and VEGF-A are regulated by transcriptional HIF complexes (Wykoff et al,
2000). VEGF induces blood vessel hyperpermeability resulting in extravasation of fibrinogen.
Fibrin is then formed upon contact with the subendothelial matrix, supporting angiogenesis
and inducing a wound-healing response with tumour stroma generation (Dvorak, 1986).
The angiogenic CRC liver metastasis indeed frequently contained fibrin in contrast
to the non-angiogenic BC metastases.
Finally, since 28 of 49 BC metastases were necropsy-derived compared to none of the
CRC liver metastases, which were usually resected together with the primary colorectal
tumour, it might be that advanced stage and multiple chemotherapy courses have influenced
the growth pattern of BC metastases. Exclusion of the necropsy-derived BC liver metastases
and statistical re-analysis did however not change the results.
In conclusion, this study shows that liver metastases of BC mainly co-opt sinusoidal
blood vessel during their growth, in contrast to most of the CRC metastases, that
expand with concomitant hypoxia-driven angiogenesis. The vessel-co-opting replacement
growth pattern and the angiogenic desmoplastic growth pattern have also been observed
in animal tumour models. This histopathological study opens perspectives for the study
of the mechanisms responsible for the differences in tumour vascularisation of liver
metastases. Whether metastases with a replacement growth pattern can sustain a more
intense hypoxic stress before inducing angiogenesis, or whether hypoxia is not an
issue due to the co-option of highly functional sinusoidal blood vessels, is not clear.
Another possible mechanism is that angiogenesis is suppressed by endogenous angiogenesis
inhibitors, which overrule local VEGF production. Additionally, some angiogenesis
inhibitors seem to be able to inhibit HIF-1-induced transcriptional activation of
VEGF expression (Mabjeesh et al, 2003).
The clinical relevance of our study is corroborated by the observation in one of the
animal models that endostatin, an endogenous angiogenesis inhibitor, inhibits the
growth of liver metastases with an efficacy that varies according to the growth pattern
(Solaun et al, 2002).