Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-alpha-based isolated hepatic perfusion.

Abstract

Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-alpha) is currently performed in clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-alpha in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-alpha contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-alpha. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-alpha resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-alpha, ruling out a direct interaction of TNF-alpha with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-alpha was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-alpha mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-alpha in this setting.

Full text

Page 1

Degree of tumour vascularity correlates with drug accumulation
and tumour response upon TNF-a-based isolated hepatic
perfusion
B van Etten1, MR de Vries1, MGA van IJken1, TE Lans1, G Guetens2, G Ambagtsheer1, ST van Tiel1, G de Boeck3,
EA de Bruijn3, AMM Eggermont1and TLM ten Hagen*,1
1Department of Surgical Oncology, University Hospital Rotterdam-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands;2Laboratory for Organic
Analytical Chemistry, University of Antwerp, Belgium;3Laboratory for Experimental Oncology, University of Leuven, Belgium
Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-a) is currently performed in
clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-a in isolated limb
perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature.
However, whether TNF-a contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model
we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit
different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-a. IHP with melphalan
alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-a resulted in a
strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity
studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-a, ruling out a direct interaction of
TNF-a with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-a was coadministrated with melphalan was
strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was
observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD
for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and
TNF-a mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for
the utility of TNF-a in this setting.
British Journal of Cancer (2003) 88, 314–319. doi:10.1038/sj.bjc.6600707
& 2003 Cancer Research UK
www.bjcancer.com
Keywords: liver metastases; isolated hepatic perfusion; TNF-a; melphalan; microvessel density
??????????????????????????????????????????????????????
Tumour necrosis factor alpha (TNF-a) is a cytokine with an
interesting potential in the treatment of cancer (ten Hagen et al,
2001). When administered systemically it is accompanied with
severe toxicity; however, especially when TNF-a in combination
with chemotherapy is used locoregionally without systemic
exposure, it has very potent antitumour effects. Clinical trials of
isolated limb perfusion (ILP) with recombinant human TNF-a and
melphalan resulted in high complete response rates of 75–90% in
patients with in-transit melanoma and unresectable sarcoma of the
extremities (Lienard et al, 1992; Eggermont et al, 1996a,b). This
is in contrast to ILP with melphalan alone, which is relatively
effective against small in-transit melanoma metastases (Lejeune
et al, 1989), but achieves very poor results against large tumours
such as soft-tissue sarcomas (McBride, 1974; Hoekstra et al, 1987;
Thompson and Gianoutsos, 1992).
In order to elucidate the mechanism of TNF-a, several studies
have been performed. In our preclinical ILP model, we observed
drastic alterations in tumour microvasculature integrity (Nooijen
et al, 1996). Ru ¨egg et al (1998) demonstrated elegantly that TNF-a
in combination with IFN-g induced functional downregulation of
avb3, resulting in detachment of the endothelial cells of the tumour
vasculature. Moreover, angiographic studies performed in patients
pre- and post-TNF-a perfusion showed selective destruction of
tumour-associated vasculature and histologic studies demon-
strated haemorrhagic necrosis of the tumour (Olieman et al,
1997). Recently, we demonstrated, what we consider a key
explanation for the potent synergy between TNF-a and chemo-
therapy, an up to six-fold increased intratumoural melphalan or
doxorubicin concentration in rat sarcomas after ILP when high-
dose TNF-a was coadministrated (de Wilt et al, 2000; van der Veen
et al, 2000). These findings led to the hypothesis that TNF-a causes
specific destruction of tumour endothelial cells and thereby
induces an increased permeability of tumour vasculature.
As a result of the favourable experience with the ILP system,
other isolated perfusion settings have been developed (van der
Veen et al, 1999; van Ijken et al, 2000). Especially, the liver offers
superb opportunities for isolated perfusion. Irresectable liver
metastases are a significant clinical problem. Isolated hepatic
Received 24 May 2002; revised 7 October 2002; accepted 18 October
2002
*Correspondence: Dr TLM ten Hagen, Department of Surgical
Oncology, Laboratory Experimental Surgical Oncology, Erasmus Uni-
versity Rotterdam, Room Ee 102, PO Box 1738, 3000 DR Rotterdam,
The Netherlands; E-mail: tenhagen@heel.fgg.eur.nl
British Journal of Cancer (2003) 88, 314 – 319
& 2003 Cancer Research UKAll rights reserved 0007– 0920/03$25.00
www.bjcancer.com
Experimental Therapeutics

Page 2

perfusion (IHP) with melphalan with or without TNF-a is
technically feasible and is currently performed in clinical trials
in patients with hepatic metastases (Alexander et al, 1998a, b;
Vahrmeijer et al, 2000). Whether TNF-a contributes to the
therapeutic efficacy in IHP still remains unclear.
Based on our findings in the ILP studies, it is indicated to study
whether TNF-a can improve tumour response in different tumours
after IHP and, if so, to investigate the capability of TNF-a to
augment drug accumulation in this perfusion setting. By addres-
sing this issue, the usefulness of TNF-a in IHP might become clear.
Since the tumour-associated vasculature is the target of TNF-a, we
expect that tumour microvessel density (MVD) is a predictor of the
potentiating effect of TNF-a during isolated perfusions. Here we
present data that indicate that the antitumour effect of TNF-a is
correlated with the tumour microvessel density.
MATERIALS AND METHODS
Rat liver metastases model
We used male inbred WAG/RIJ or Brown-Norway (BN) strain rats,
weighing 250–300g, obtained from Harlan-CPB (Austerlitz, The
Netherlands). The rats were fed a standard laboratory diet. All
animals were housed under standard conditions of light and
accommodation. The protocol was approved by the committee for
animal research of the Erasmus University, Rotterdam, The
Netherlands. The experimental protocols adhered to the rules
outlined in the Dutch Animal Experimentation Act of 1977 and the
published Guidelines of the UKCCCR for the Welfare of Animals in
Experimental Neoplasia (UKCCCR, 1998).
Three different tumours were used in this study. The weakly
immunogenic colon carcinoma CC531 is a 1,2-dimethylhydrazine-
induced, moderately differentiated adenocarcinoma transplantable
in syngeneic WAG/RIJ rats. The estimated doubling in vivo is
about 6–8 days. The spontaneously originated nonimmunogenic
osteosarcoma ROS-1 is also transplantable in the WAG-RIJ rat and
in the liver metastases model it has a mean doubling time of about
4–5 days. The spontaneously originated nonimmunogenic soft-
tissue sarcoma BN-175 is the fastest growing tumour of the
tumours tested, with an estimated doubling time in vivo of about
2–3 days and is transplantable in syngeneic BN rats. Following a
standardised protocol, small viable tumour fragments of CC531,
ROS-1 or BN-175 tumour fragments of 1?2mm2were implanted
under the liver capsule, one on the left and one on the right side of
the left liver lobe, using a 19 G Luerlock needle. Experiments
started at a fixed tumour diameter between 5 and 6mm. When
tumours reached a size of 20mm in diameter or animals showed
obvious signs of discomfort the animals were killed.
Drugs
Recombinant human TNF-a (4.9–5.8?107Umg?1) was provided
as a kind gift by Boehringer Ingelheim GmbH, Ingelheim/Rhein,
Germany. Melphalan (L-pam, Alkeran, Wellcome Ltd, London,
UK) was obtained as a sterile powder (100mg) that was dissolved
aseptically using solvent and diluent provided by Burroughs
Wellcome (London, UK).
Isolated hepatic perfusion
This rat isolated liver perfusion model has been described in detail
earlier by van IJken et al (2000). A schematic representation is
shown in Figure 1. Anaesthesia was induced and maintained with
ether (Merck, Darmstadt, Germany). During the surgical proce-
dure, with an average duration of 60–75min, rats were kept at a
constant temperature using a warmed mattress. A mid-line
laparotomy was performed and the hepatic ligament exposed.
The gastroduodenal side branch of the common hepatic artery was
cannulated, positioning the tips of the cannula (0.025 outer
diameter (OD), 0.012in inner diameter (ID) (Dow Corning, MI,
USA)) in the proper hepatic artery. Through a small inguinal
incision the femoral vein was exposed. To collect hepatic venous
outflow a silicon cannula (0.047 OD, 0.025in ID) (Dow Corning,
MI, USA) was introduced in the femoral vein and moved up into
the caval vein positioning the tip of the cannula at the level of the
hepatic veins.
Isolation of the hepatic vascular bed was obtained by
temporarily ligating the common hepatic artery and the portal
vein. The venous outflow limb was isolated by temporarily
clamping the supra-hepatic caval vein and by applying a
temporary ligature around the infra-hepatic caval vein containing
the cannule, cranial to the right adrenal vein. The mesenteric
artery was temporarily clamped in order to reduce splanchnic
blood pressure. The circuit was primed with 10ml Haemaccel
(Behring Pharma, Amsterdam, The Netherlands). Arterial flow of
5mlmin?1was maintained with a low-flow roller pump (Watson
Marlow type 505 U, Falmouth, UK). Rats were perfused for ten min
with oxygenated Haemaccel in which melphalan and/or TNF-a was
dissolved. This short perfusion time was used as we observed rapid
clearance of melphalan from the perfusate in this time frame.
Secondly, perfusion of the liver beyond 10min may increase the
risk for tissue damage to the liver, but also to the gut as blood flow
to the gut is impaired during the perfusion. Afterwards a washout
was performed by perfusing with 10ml of oxygenated Haemaccel.
Heparin (50 IU) (Heparine Leo, The Netherlands) was added to the
perfusate. The perfusate was oxygenated in a reservoir with a
mixture of O2/CO2(95%:5%) and was kept at 38–391C by means
of a heat exchanger and a warm water bath. A temperature probe
was positioned in the lumen of the arterial catheter, 5cm from the
catheter tip.
Following the washout procedure, the clamps on caval vein,
portal vein, hepatic artery and mesenteric artery were released. The
gastroduodenal artery and femoral vein were ligated and the
gastroduodenal and femoral cannulas were removed.
Figure 1Schematic representation of an IHP.
IHP with TNFa increases intratumoural melphalan concentration
B van Etten et al
315
British Journal of Cancer (2003) 88(2), 314–319
& 2003 Cancer Research UK
Experimental Therapeutics

Page 3

In vivo antitumour efficacy study
Treatment started at a fixed tumour size of 5–6mm in diameter.
Rats were perfused in random order. In a pilot dose finding study
performed for each tumour type the melphalan dose inflicting a
partial tumour response was chosen for this study. So in the case of
additive or synergistic effect of TNF-a on melphalan this could still
be demonstrated in the growth curves of the tumours. All animals
underwent IHP only once. CC531-bearing rats were treated with
50mg melphalan (n¼6), 20mg TNF-a (n¼6) or a combination of
50mg melphalan and 20mg TNF-a (n¼6). ROS-1-bearing rats were
perfused with 50mg melphalan (n¼6), 20mg TNF-a (n¼8), or a
combination of 50mg melphalan and 20mg TNF-a (n¼6). In the
BN-175-bearing rats perfusions were carried out with 200mg
melphalan (n¼6), 20mg TNF-a (n¼6), or a combination of 200mg
melphalan and 20mg TNF-a (n¼6). After IHP tumour size was
measured via a small midline laparotomy every fourth day.
Tumour volume was calculated by using the following formula:
tumour volume¼A2?B?0.4, where B is the largest diameter and
A the diameter perpendicular to B, measured with a standardised
calliper. In every treatment group, sham perfused rats (n¼6) and
untreated control rats (n¼5) were included.
In vitro cytotoxicity assay
CC531 and BN-175 cells were grown in RPMI 1640 and ROS-1 cells
in modified Eagle’s medium (Gibco BRL, Paisley, UK) supple-
mented with 10% foetal calf serum (Harlan/Sera-Lab, UK), 1%
penicillin (5000IUml?1), 1% streptomycin (5000IUml?1) and 1%
L-glutamine (200mM) (all Gibco BRL) in a humidified incubator at
371C and 5% CO2. Before usage, the cells were trypsinised (1min,
371C), centrifuged (5min, 700g), resuspended and the viability
measured by trypan blue exclusion. For in vitro testing of
proliferation inhibition, 1.0?104viable cells were seeded in flat-
bottomed 96-well microtiter plates (Costar, USA). After 24h the
cells were incubated with different concentrations of TNF-a for
72h ranging from 0 to 10mgml?1. Afterwards, cells were washed
with PBS and fixed for 1h with 10% trichloroacetic acid at 41C.
Growth of tumour cells was measured using the sulpharhodamine-
B assay according to the method of Skehan et al (1990). Tumour
cell proliferation was measured using the formula: tumour
growth¼(test well/control)?100%. Five independent tests were
performed for each point on the line.
Measurement of melphalan in tissue
After 5min of the restoration of the circulation, the perfused tumour
and part of the liver were excised. The tissues were immediately
frozen in liquid nitrogen to stop metabolism of melphalan and
stored at –801C. Tumour and liver tissues were homogenised in 2ml
acetonitrile (Pro 200 homogenizer, Pro Scientific, CT, USA) and
centrifuged at 2500g. Melphalan was measured in the supernatant
by gas chromatography–mass spectrometry (GC–MS). p-[Bis(2-
chloroethyl)amino]-phenylacetic acid methyl ester was used as an
internal standard. Samples were extracted over trifunctional C18
silica columns. After elution with methanol and evaporation, the
compounds were derivatised with trifluoroacetic anhydride and
diazomethane in ether. The stable derivates were separated on a
methyl phenyl siloxane GC capillary column and measured
selectively by single-ion monitoring GC–MS in the positive EI
mode described earlier by Tjaden and Bruijn (1990).
Assessment of tumour microvessel density by
immunohistochemistry
Cryosections of tumours were fixed for 15min with 4%
formaldehyde. After rinsing with PBS, sections were incubated
for 1h with 1:10 PBS diluted, mouse-anti-rat-endothelial cell
antibody (RECA-1, Instruchemie, Hilversum, The Netherlands).
For the negative control an aspecific mouse IgG was used (Santa
Cruz Biotechnology, Santa Cruz, CA, USA). Thereafter, sections
were rinsed with PBS and incubated for 1h with 1:100 diluted, in
5% normal rat serum in PBS, goat-anti-mouse peroxidase-labelled
antibody (DAKO, Carpinteria, CA, USA). After rinsing with PBS,
positive cells were revealed by immunoperoxidase reaction with
DAB solution (DAB-kit, DAKO) and counterstained with haema-
toxylin. For microvessel quantification two independent persons
performed a blinded analysis. Positive cells were counted in three
different high-power fields (magnification ?160) in each slide
according to the method of Bosari et al (1992). In total, three slides
per tumour and three tumours per tumour type were evaluated.
Statistical analysis
In vitro bioassays and in vivo tumour response results were evalu-
ated for statistical significance with the Mann–Whitney U-tests
with SPSS8.0 for Windows 98. Mann–Whitney U-test was used to
compare melphalan concentrations in different groups and
Kruskal–Wallis test to compare number of positive cells in different
tumours. A significance level of Po0.05 was used in all analyses.
RESULTS
Tumour response after isolated hepatic perfusion
The antitumour efficacy of IHP with melphalan with or without
TNF-a was evaluated for the CC531, ROS-1 and BN-175 tumour
starting at an equal size of 5–6mm in diameter. In all groups,
sham IHPs with only perfusion medium were performed. The
graphs in Figure 2 show the growth curves of CC531 tumour (a),
ROS-1 (b) and BN-175 (c) after IHP with melphalan, TNF-a, both
or after sham perfused rats. Perfusion with melphalan alone
significantly reduced tumour growth rates compared with sham
perfused animals in all tumour types. When IHP was performed in
BN-175-bearing rats with the combination of melphalan and TNF-
a, a dramatically enhanced tumour response was observed in all
animals. This is a significant reduction of mean tumour volume
compared with rats perfused with either TNF-a only or melphalan
alone (Po0.005 and o0.01, respectively). In the CC531 or ROS-1
tumours, synergy between TNF-a and melphalan was not observed.
In vitro cytotoxicity assay
The effect of TNF-a on the growth of tumour cells in vitro was
determined to evaluate whether the synergistic effect of TNF-a could
be related to direct tumour cell toxicity. The calculated concentra-
tion of TNF-a in the perfusate during IHP in vivo is about
1.5mgml?1. So in vitro tumour cells were exposed to a range of
TNF-a concentrations varying from 0 to 10mgml?1. The growth
curves are shown in Figure 3. It is demonstrated that the BN-175
and the CC531 tumour cell line did not show significant sensitivity
to TNF-a. Only the ROS-1 tumour cells were moderately sensitive to
TNF-a, a growth inhibition of up to 30% at 10mgml?1was observed.
Melphalan concentration in tumour and liver tissue
In this perfusion setting, in which the dose of TNF-a is 20% of the
dose used in ILP, an enhanced drug accumulation in tumour tissue
might take place as well, as observed after TNF-a based ILP. In
order to investigate this mechanism, melphalan concentrations
were measured in tumour and liver tissues after IHP with
melphalan with and without TNF-a. In the CC531 and ROS-1,
tumours, melphalan concentration did not increase significantly
after IHP with melphalan and TNF-a (Figure 4A,B). After IHP with
melphalan alone in the BN175 tumour-bearing rats the melphalan
concentration in tumour and liver tissue was equal (Figure 4C).
IHP with TNFa increases intratumoural melphalan concentration
B van Etten et al
316
British Journal of Cancer (2003) 88(2), 314–319
& 2003 Cancer Research UK
Experimental Therapeutics

Page 4

Figure 3
CC531 (K), ROS-1 (E), BN-175 (’). Six independent assays were
performed in duplicate for each point on the line. Mean values (7s.e.m.)
are shown.
In vitro growth curves of tumour cells upon exposure to TNF-a;
Figure 2
contained at least six animals. Mean values (7s.e.m.) are shown; (A)
CC531, (B) ROS-1, (C) BN-175.
Growth curves of in vivo tumours after IHP. Each group
Figure 4
with melphalan with or without TNF-a. Six IHPs were performed per
tumour type. Mean values (7s.d.) are shown. (*¼Po0.05 vs tumour
melphalan concentration after IHP with melphalan alone); (A) CC531, (B)
ROS-1, (C) BN-175.
Melphalan concentrations in liver and tumour tissue after IHP
IHP with TNFa increases intratumoural melphalan concentration
B van Etten et al
317
British Journal of Cancer (2003) 88(2), 314–319
& 2003 Cancer Research UK
Experimental Therapeutics

Page 5

After IHP with TNF-a however a more than 5-fold increase of
melphalan in tumour tissue is measured compared to tumour
tissue after IHP without TNF-a; (Po0.05). So an augmented drug
accumulation can also be achieved in the IHP setting when TNF-a
is coadministered.
Assessment of tumour microvessel density
We already hypothesised that TNF-a by increasing leakage of
tumour vessels enhances intratumoural concentrations of che-
motherapeutics. The increased uptake of melphalan might there-
fore be correlated with the microvessel density (MVD) of the
tumour. Quantification of the MVD was performed by immuno-
histochemical staining of endothelial cells. The microvessel count
of the colon carcinoma CC531 and the osteosarcoma ROS-1 was
equal (Figure 5). The soft-tissue sarcoma BN-175 however showed
a significantly higher MVD than CC531 and ROS-1. These results
indicate a relations between vascularity of the tumour and TNF-a-
mediated effects.
DISCUSSION
In the present study, we demonstrated that addition of TNF-a to
IHP with melphalan results in strongly improved response rates in
a tumour with high vascular density. In vitro, no or only minor
sensitivity of tumour cells to TNF-a was found. Even in ROS-1
tumours, which are moderately sensitive to TNF-a in vitro, IHP
with TNF-a alone showed no tumour response. These data indicate
strongly that in vivo indirect mechanisms mediated by TNF-a in
combination with melphalan determine antitumour effects in IHP.
Our data support the notion that this indirect mechanism is the
selective destructive effect of TNF-a on the tumour-associated
vessels, thereby increasing vascular permeability (Nooijen et al,
1996; Ruegg et al, 1998). To investigate this hypothesis, the
melphalan uptake in liver and tumour tissue was measured after
IHP with or without TNF-a. Tumour melphalan concentrations
were increased in all tumours but varied significantly in a tumour-
type-dependent way. Moreover, enhanced uptake of melphalan
by healthy liver was not observed. With TNF-a alone, at the
most some tumour growth was observed. Only the combination of
TNF-a and melphalan resulted in a complete tumour response in
the BN175 tumour. To elucidate this tumour-type-dependent
response, the MVD of the tumours was determined. We expected a
higher tumour vascularity in this tumour. Indeed a significantly
higher MVD compared to the CC531 and ROS-1 tumours could be
demonstrated. This indicates that TNF-a has specific tumour
vascular mediating capacity in this perfusion model, which results
in enhanced tumour responses in highly vascularised tumours. As
a result of our findings in ILP and now also in IHP, we know that
TNF-a is able to augment the accumulation of melphalan. The
presence or lack of TNF-a-mediated synergy appeared to be
independent of tumour size as also in smaller (diameter 3–4mm)
or bigger (7–8mm) tumours comparable tumour responses were
observed (data not shown). We are of the opinion that this
observation is essential in understanding and explaining the
impressive responses demonstrated.
Changes in vascular permeability in patients who underwent
IHP with TNF-a was studied by Alexander et al (1998).
Vascular permeability was measured by diffusion of radiolabelled
131I albumin in liver and tumour tissue. A significant increase of
the131I albumin postperfusion could be demonstrated compared
to levels131I albumin measured before perfusion. However, this
rise was equal in tumours perfused with or without TNF-a. A TNF-
a independent mechanism of the increased endothelial perme-
ability was suggested by the authors. However, in the present
study, we demonstrated that TNF-a is effective in increasing
vascular permeability for melphalan selectively in tumour tissue. A
more important finding, however, is that this effect could only be
found in the highly vascularised BN-175 tumour. The results of
Alexander et al reported on intratumoural131I albumin concen-
trations were mainly based on colorectal carcinoma liver meta-
stases. In hypovascular rat colon carcinoma, we also could not find
an increase of melphalan intratumourly. We therefore hypothesize
that the usual hypovascularity of colorectal metastases in patients
explains the lack of TNF-benefit in the experience as described by
Alexander in patients, which correlates closely to our observations
in our hypovascular colon cancer liver metastases model in rats.
IHP with melphalan and TNF-a performed in patients with
metastases of ocular melanoma or leiomyosarcoma showed overall
response rates of 50–52% (Lindner et al, 1999; Alexander et al,
2000). Both tumour types are highly vascularised. A prolonged
duration of response was found in melanoma patients: 14 months
after IHP with TNF-a vs 6 months after IHP without TNF-a
(Alexander et al, 2000). After IHP with melphalan with or without
TNF-a in patients with colorectal liver metastases the mean
duration of response was in both groups 8–10 months (Alexander
et al, 1998a,b; Bartlett et al, 2001). The data we now present and
the first reports of IHP in melanoma and sarcoma liver metastases
strongly indicate that in these patients TNF-a has therapeutic
potential in IHP. In patients with colorectal liver metastases
however, IHP with melphalan alone may well be just as effective as
combined with TNF-a. Assessment of the degree of tumour
vasculature of liver metastases would be a way of establishing an
indication for the utility of TNF-a in this setting.
ACKNOWLEDGEMENTS
We thank Boehringer Ingelheim GmbH for the generous supply of
rhTNF. This study was supported by a grant of the Dutch Cancer
Society/Queen Wilhelmina Foundation.
REFERENCES
Alexander HR, Brown CK, Bartlett DL, Libutti SK, Figg WD, Raje S, Turner
E (1998a) Augmented capillary leak during isolated hepatic perfusion
(IHP) occurs via tumour necrosis factor-independent mechanisms. Clin
Cancer Res 4: 2357–2362.
Alexander HR, Libutti SK, Bartlett DL, Puhlmann M, Fraker DL,
Bachenheimer LC (2000) A phase I-II study of isolated hepatic perfusion
using melphalan with or without tumour necrosis factor for patients with
ocular melanoma metastatic to liver. Clin Cancer Res 6: 3062–3070.
Figure 5
Mean values (7s.e.m.) are shown (*¼Po0.001 vs CC531 and vs ROS-1).
Microvessel count of CC531, ROS-1 and BN-175 tumours.
IHP with TNFa increases intratumoural melphalan concentration
B van Etten et al
318
British Journal of Cancer (2003) 88(2), 314–319
& 2003 Cancer Research UK
Experimental Therapeutics

Page 6

Alexander HRJ, Bartlett DL, Libutti SK, Fraker DL, Moser T, Rosenberg SA
(1998b) Isolated hepatic perfusion with tumour necrosis factor and
melphalan for unresectable cancers confined to the liver. J Clin Oncol 16:
1479–1489.
Bartlett DL, Libutti SK, Figg WD, Fraker DL, Alexander HR (2001) Isolated
hepatic perfusion for unresectable hepatic metastases from colorectal
cancer. Surgery 129: 176–187.
Bosari S, Lee AK, DeLellis RA, Wiley BD, Heatley GJ, Silverman ML (1992)
Microvessel quantitation and prognosis in invasive breast carcinoma.
Hum Pathol 23: 755–761.
de Wilt JH, ten Hagen TL, de Boeck G, van Tiel ST, de Bruijn EA,
Eggermont AM (2000) Tumour necrosis factor alpha increases melphalan
concentration in tumour tissue after isolated limb perfusion. Br J Cancer
82: 1000–1003.
Eggermont AM, Schraffordt KH, Klausner JM, Kroon BB, Schlag PM,
Lienard D, van Geel AN, Hoekstra HJ, Meller I, Nieweg OE, Kettelhack C,
Ben-Ari G, Pector JC, Lejeune FJ (1996a) Isolated limb perfusion with
tumour necrosis factor and melphalan for limb salvage in 186 patients
with locally advanced soft tissue extremity sarcomas. The cumulative
multicenter European experience. Ann Surg 224: 756–764.
Eggermont AM, Schraffordt KH, Lienard D, Kroon BB, van Geel AN,
Hoekstra HJ, Lejeune FJ (1996b) Isolated limb perfusion with high-dose
tumour necrosis factor-alpha in combination with interferon-gamma
and melphalan for nonresectable extremity soft tissue sarcomas: a
multicenter trial. J Clin Oncol 14: 2653–2665.
Hoekstra HJ, Schraffordt KH, Molenaar WM, Oldhoff J (1987) Results of
isolated regional perfusion in the treatment of malignant soft tissue
tumours of the extremities. Cancer 60: 1703–1707.
Lejeune FJ, Lienard D, el Douaihy M, Seyedi JV, Ewalenko P (1989) Results
of 206 isolated limb perfusions for malignant melanoma. Eur J Surg
Oncol 15: 510–519.
Lienard D, Ewalenko P, Delmotte JJ, Renard N, Lejeune FJ (1992) High-dose
recombinant tumour necrosis factor alpha in combination with
interferon gamma and melphalan in isolation perfusion of the limbs
for melanoma and sarcoma. J Clin Oncol 10: 52–60.
Lindner P, Fjalling M, Hafstrom L, Kierulff-Nielsen H, Mattsson J,
Schersten T, Rizell M, Naredi P (1999) Isolated hepatic perfusion with
extracorporeal oxygenation using hyperthermia, tumour necrosis factor
alpha and melphalan. Eur J Surg Oncol 25: 179–185.
McBride CM (1974) Sarcomas of the limbs. Results of adjuvant
chemotherapy using isolation perfusion. Arch Surg 109: 304–308.
Nooijen PT, Manusama ER, Eggermont AM, Schalkwijk L, Stavast J,
Marquet RL, de Waal RM, Ruiter DJ (1996) Synergistic effects of TNF-
alpha and melphalan in an isolated limb perfusion model of rat sarcoma:
a histopathological, immunohistochemical and electron microscopical
study. Br J Cancer 74: 1908–1915.
Olieman AF, van Ginkel RJ, Hoekstra HJ, Mooyaart EL, Molenaar WM,
Koops HS (1997) Angiographic response of locally advanced soft-tissue
sarcoma following hyperthermic isolated limb perfusion with tumour
necrosis factor. Ann Surg Oncol 4: 64–69.
Ruegg C, Yilmaz A, Bieler G, Bamat J, Chaubert P, Lejeune FJ (1998)
Evidence for the involvement of endothelial cell integrin alphaVbeta3 in
the disruption of the tumour vasculature induced by TNF-a and IFN-
gamma. Nat Med 4: 408–414.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren
JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity
assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107–1112.
ten Hagen TL, Eggermont AM, Lejeune FJ (2001) TNF-a is here to stay–
revisited. Trends Immunol 22: 127–129.
Thompson JF, Gianoutsos MP (1992) Isolated limb perfusion for
melanoma: effectiveness and toxicity of cisplatin compared with that of
melphalan and other drugs. World J Surg 16: 227–233.
Tjaden UR, de Bruijn EA (1990) Chromatographic analysis of anticancer
drugs. J Chromatogr 531: 235–294.
United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR)
(1998) Guidelines for the Welfare of Animals in Experimental Neoplasia
(Second Edition). Br J Cancer, 77: 1–10.
Vahrmeijer AL, van Dierendonck JH, Keizer HJ, Beijnen JH, Tollenaar RA,
Pijl ME, Marinelli A, Kuppen PJ, van Bockel JH, Mulder GJ, van de velde CJ
(2000) Increased local cytostatic drug exposure by isolated hepatic
perfusion: a phase I clinical and pharmacologic evaluation of treatment
with high dose melphalan in patients with colorectal cancer confined to
the liver. Br J Cancer 82: 1539–1546.
van IJken MG, van Etten B, de Wilt JH, van Tiel ST, ten Hagen TL,
Eggermont AM (2000) Tumour necrosis factor-alpha augments tumour
effects in isolated hepatic perfusion with melphalan in a rat sarcoma
model. J Immunother 23: 449–455.
van der Veen AH, de Wilt JH, Eggermont AM, van Tiel ST, Seynhaeve AL,
ten Hagen TL (2000) TNF-alpha augments intratumoural concentrations
of doxorubicin in TNF-alpha-based isolated limb perfusion in rat
sarcoma models and enhances anti-tumour effects. Br J Cancer 82: 973–
980.
van der Veen AH, Seynhaeve AL, Breurs J, Nooijen PT, Marquet RL,
Eggermont AM (1999) In vivo isolated kidney perfusion with tumour
necrosis factor alpha (TNF-alpha) in tumour-bearing rats. Br J Cancer 79:
433–439.
IHP with TNFa increases intratumoural melphalan concentration
B van Etten et al
319
British Journal of Cancer (2003) 88(2), 314–319
& 2003 Cancer Research UK
Experimental Therapeutics