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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 987
_____________________________ _____________________________
Hepatic and Peritoneal Colorectal Metastases
Aspects of Prognosis and Treatment
BY
HAILE MAHTEME
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001
Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Surgery presented atUppsala University in 2001
ABSTRACT
Mahteme, H. 2001. Hepatic and peritoneal colorectal metastases. Aspects of prognosis and treatment.Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Facultyof Medicine 987. 70pp. Uppsala. ISBN 91-554-4912-3.
Although two-thirds of colorectal cancer patients are cured by surgery, approximately 50% of the
patients with this disease develop locally recurrent or distant metastases during the course of their
illness. The aim of this study was to identify metastatic sites associated with poor prognosis in rectal
cancer and then to investigate methods that can prevent the development and growth of metastases and
optimise uptake of drugs at these sites in animal models.
In a defined population, 151 patients with irresectable metastatic or local rectal cancer were identified.
Bilateral liver involvement, abnormal liver function tests, peritoneal growth or abdominal lymph node
metastases implied a poor prognosis.
In a study on Wistar rats with liver metastases from colorectal cancer, blocking of hyaluronan uptake
and elimination by the liver enhanced the hyaluronan uptake in liver metastases. Hyaluronan may thus
be used to promote uptake of drugs in specific hyaluronan receptor-positive tumour sites.
Adjuvant intravenous radioimmunotherapy delivered as a specific or unspecific monoclonal antibody
prevented human colonic cancer cells inoculated into the portal vein of nude rats from developing into
liver metastases. Furthermore, intraperitoneally administered radioimmunotherapy inhibited the
growth of peritoneal metastases.
Blocking of 5-FU absorption with a vasoconstrictive agent enhanced the uptake of 5-FU in peritoneal
metastases. In addition, the uptake of 5-FU in peritoneal metastases could be improved when these
tumours were mechanically disintegrated by surgical tumour reduction and the drug was given
intraperitoneally.
Key words: Liver metastases, peritoneal metastases, hyaluronan, radioimmunotherapy, cytoreductivesurgery, intraperitoneal administration, 5-FU.
Haile Mahteme, Department of Surgical Sciences, Uppsala University Hospital, SE-751 85 Uppsala,Sweden
Haile Mahteme 2001
ISSN 0282-7476ISBN 91-554-4912-3
Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001
To Charlotte
Josef
Christopher and
Salomon
And in memory of my mother
This thesis is based on the following papers, which will be referred to by their Romans
numerals:
I Mahteme H, Påhlman L, Glimelius B, Graf W. Prognosis after surgery in
patients with incurable rectal cancer: a population- based study. Br J Surg. 1996,
83, 1116-1120.
II Mahteme H, Graf W, Larsson BS, Gustafson S. Uptake of hyaluronan in hepatic
metastases after blocking of liver endothelial cell receptors. Glycoconjugate
Journal 1998, 15, 935-939.
III Mahteme H, Lövqvist A, Graf W, Lundqvist H, Carlsson J, Sundin A. Adjuvant131I-anti-CEA-antibody radioimmunotherapy inhibits the development of
experimental colonic carcinoma liver metastases. Anticancer Res 1998, 18, 843-
848.
IV Mahteme H, Sundin A, Larsson BS, Khamis H, Arow K, Graf W. 5-FU Uptake
in peritoneal metastases after pretreatment with radioimmunotherapy or
norbormide: An autoradiographic study in the rat (Manuscript)
V Mahteme H, Larsson BS, Sundin A, Khamis H, Graf W. Uptake of 5-FU in
peritoneal metastases in relation to mode of administration and surgical tumour
reduction: An autoradiographic study in the rat (Manuscript)
Reprints were made with the permission of the publishers.
5
CONTENTS
ABBREVIATIONS ……………………………………………………... 7
1. INTRODUCTION ……………………………………………………. 8
1.1 Prognostic features in colorectal cancer …………………………... 8
2. BACKGROUND ……………………………………………………… 10
2.1 Mechanisms of metastatic dissemination …………………………. 10
2.1.1 Direct spread ………………………………………………… 10
2.1.2 Lymphatic spread ……………………………………………. 10
2.1.3 Blood borne spread ………………………………………….. 10
2.1.4 Transcoelomic spread ……………………………………….. 11
2.2 Treatment of metastases …………………………………………... 11
2.2.1 Surgery……………………………………………….………. 11
2.2.2 Chemotherapy ……………………………………………….. 12
2.2.2.1 Intravenous chemotherapy ……………………………… 14
2.2.2.2 Hepatic arterial infusion ………………………………... 14
2.2.2.3 Intraportal chemotherapy ………………………………. 15
2.2.2.4 Intraperitoneal chemotherapy ………………………….. 16
2.2.2.4.1 Hyperthermic intraperitoneal chemotherapy………. 17
2.2.3 Radio-labelled chemotherapy………………………………… 17
2.2.4 Radioimmunotherapy ………………………………………… 18
2.2.5 Radiofrequency ………………………………………………. 18
2.2.6 External beam and intra-operative irradiation therapy ……... 19
3. AIMS OF THE INVESTIGATION …………………………………. 20
4. MATERIALS AND METHODS …………………………………….. 21
4.1 Animals and tumour cells …………………………………………. 21
4.2 Hyaluronan and chondroitin sulphate …………………………….. 22
4.3 Monoclonal antibodies and radiolabelling ………………………... 22
4.4 14C-labelled 5-fluorouracil ………………………………………… 23
6
4.5 Autoradiography and Phosphoimaging .…………………………... 23
4.6 Paper I - V…….…………………………………………………… 24
5. STATISTICAL METHODS ………………………………………… 29
6. RESULTS …………………………………………………………….. 31
6.1 Paper I - V…..……………………………………………………… 31
7. DISCUSSION ………………………………………………………… 39
8. SUMMARY AND CONCLUSIONS ……………………………….. 40
9. ACKNOWLEDGEMENTS …………………………………………. 44
10. REFERENCES ……………………………………………………… 46
7
ABBREVIATIONS
CEA Carcinoembryonic antigen
CS Chondroitin sulphate
EBRT External beam irradiation therapy
5-FU 5-fluorouracil
HA Hyaluronan
HAI Hepatic artery infusion
HIPEC Hyperthermic intraperitoneal chemotherapy
IFP Interstitial fluid pressure
IORT Intra-operative irradiation therapy
i.p. Intraperitoneal
IPO Intraportal
i.v. Intravenous
kDa Kilo Dalton
MAb Monoclonal antibody
Mw Molecular weight
MVP Microvascular pressure
RF Radiofrequency
RIT Radioimmunotherapy
TR Tumour reduction
8
1. INTRODUCTION
1.1 Prognostic features in colorectal cancer
It is estimated that adenocarcinoma of the colon and rectum is currently
diagnosed in about 678 000 new cases annually (Parkin el al 1993) and that 394 000
patients die of the disease each year (Pisani el al 1993) worldwide. It is one of the major
health problems in the western world, and with about 5000 new cases of the disease per
year in Sweden, it is the third commonest form of cancer in this country (Swedish
Cancer Registry 1999). Although in the great majority - in about two-thirds - of these
patients the diagnosis is made at a stage when all apparently diseased tissue can be
surgically removed (Gordon et al 1993, Singh et al 1995, Klein et al 1996), metastases
are present in about 25% of the patients at the time of diagnosis (McArdle 1990) and the
disease will recur in about 40% at some point in time (Goldberg et al 1998, Laurie et al
1989, Moertel et al 1990). In a substantial proportion of the patients, recurrent disease is
likely to be due to residual cancer already present at surgery, existing in an occult and
microscopic stage. The overall 5-year survival rate has been reported to vary between
50 and 60% (McArdle 1990, Semmens et al 2000, Ries el al 2000). Postoperative
adjuvant chemotherapy has been associated with improved prognosis in colonic cancer
(Moertel et al 1995, O´Connel et al 1998), and preoperative radiotherapy in combination
with total mesorectal excision (TME) has been shown to improve survival in rectal
cancer (Dahlberg et al 1998). However, the prognosis in advanced colorectal cancer is
poor, especially when metastasis to the liver or peritoneal layer has occurred (Graf et al
1991, Newland et al 1993, Yamamura et al 1997, Assersohn et al 1999, Sadeghi et al
2000), and treatment remains a challenging problem (Nordic gastrointestinal adjuvant
therapy group 1992, Scheithauer et al 1993, Van Halteren et al 1999). Intravenous 5-
9
fluorouracil (5-FU) either alone or modulated with leucovorin, methotrexate or
oxaliplatin is used with the aim of achieving regression of the tumour and thereby
improved survival (Nordic gastrointestinal adjuvant therapy group 1992, Glimelius et al
1998, de Gramont et al 2000). However, the response rate has been limited (Glimelius
et al 1992, Graf et al 1994, Szelényi et al 2000, de Gramont et al 2000). This poor
outlook underlines the importance of increasing the efficacy of the available treatments.
10
2. BACKGROUND
2.1 Mechanisms of metastatic dissemination
2.1.1 Direct spread
Direct spread of colorectal cancer may occur longitudinally, transversely or radially, but
as adequate proximal and distal clearance by surgery is technically feasible, it is the
radial spread that is of most importance. An intraperitoneal tumour may involve
adjacent organs such as the small intestine, stomach, gallbladder, urinary bladder and
anterior or lateral abdominal wall. In a retroperitoneal colonic cancer, radial spread may
involve the ureter, duodenum, pancreas, large vessels and muscles of the posterior
abdominal wall, and rectal cancer may involve seminal vesicles, the prostate, vagina or
bladder, or pelvic bones.
2.1.2 Lymphatic spread
In general, the lymphatic spread of colonic cancer progresses from the paracolic nodes
along the main colonic vessels and eventually reaches the para-aortic glands. This
orderly process does not always occur, however, and in about 20% of the cases
perineural or perivascular tumour deposits are found, which can easily be mistaken for
lymph nodes (Goldstein et al 2000). In contrast to rectal disease, it is rare for colonic
cancer that has not breached the muscle wall to give rise to lymph node metastases
(Newland et al 1987).
2.1.3 Blood borne spread
The most common site for blood borne spread of colorectal cancer is the liver (Weiss et
al 1986), presumably arriving by the portal venous system (Fisher et al 1955). About
11
25-30% of the patients may have detectable liver metastases at the time of operation
(Bergmark et al 1969, Finlay 1982), and around 50% of patients may be expected to
develop overt disease (Patanaphan et al 1993, Kjeldsen et al 1997). The lung is the next
most common site of haematogenic spread, with around 10% of patients developing
lung metastases at some stage (Patanaphan et al 1993, Kjeldsen et al 1997). Other
reported sites include ovary-uterus, brain, bone, adrenals and skin (Patanaphan et al
1993, Kjeldsen et al 1997, Goldberg et al 1998).
2.1.4 Transcoelomic spread
Colonic cancer may spread throughout the peritoneum, either via subperitoneal
lymphatics or by virtue of viable cells being shed from the serosal surface of a tumour,
giving rise to malignant tumour deposition (Moore et al 1961, Hase et al 1998).
Colorectal cancer is the most common origin of peritoneal metastases (about 45%), next
to gynaecological malignancy (Chu et al 1989).
2.2 Treatment of metastases
2.2.1 Surgery
Surgery is the fundamental treatment of primary colorectal cancer and, when possible,
also of metastases. In a study by Scheele et al (1990) of patients with resectable solitary
hepatic metastases who did not undergo resection, there were no 5-year survivors.
However, a small proportion (about 25%) of patients with localised hepatic (Scheele et
al 1995) and lung metastases can possibly be cured by surgery. In selected patients
without extra-hepatic disease in whom liver resection has been performed, 5-year
survival of about 30% has been achieved (Harms et al 1999, Seifert et al 2000,
12
Minagawa et al 2000). Furthermore, resection of pulmonary metastases has proved to be
curative in about 30-60% (Goldberg et al 1998).
The main mode of presentation in advanced locoregional disease is intestinal
obstruction (Glass et al 1973, Annest et al 1979). Moreover, in the past this disease has
been regarded as a terminal condition, and most patients have been left to a palliative
procedure or no surgery at all. However, in locoregional metastases of ovarian cancer,
cytoreductive surgery has been applied since the 1970s (Griffiths 1975) and is still
being used (Scarabelli 2000) with claimed survival benefit. Perhaps by reason of these
results and the fact that in rectal cancer combined preoperative radiotherapy and surgery
has been found to improve survival and reduce local recurrence rates (Swedish Rectal
Cancer Trial 1997), there has been renewed interest in treating locoregional colorectal
cancer by an aggressive surgical approach (Esquivel et al 1993, Sugarbaker 1995, Elias
et al 1997, Cavaliere et al 1999, Loggie et al 2000) in combination with other therapy
modalities with the aim of improving survival.
2.2.2 Chemotherapy
The effect of adjuvant chemotherapy after curative resection of colorectal carcinoma
has been investigated in a large number of trials (Wolmark et al 1988, Moertel et al
1995). At a consensus conference in April 1990 the national cancer institute
recommended 5-FU plus levamisole as the standard treatment after resection of stage III
colonic cancer. However, colorectal cancer is usually resistant to chemotherapy
(Houghton et al 1981). In order to exert its effect on the target organ, an anti-cancer
drug must gain entrance into the tumour. Insufficiency of drug levels at target organs
may be due to pharmacokinetic factors, such as poor vascular supply (Jain 1988),
elevated interstitial fluid pressure (IFP), (Less 1992), a microvascular pressure (MVP),
13
which is low in relation to the raised IFP (Boucher et al 1992), a higher oncotic pressure
(Stohrer et al 2000), altered extracellular matrix, e.g. anomalous assembly of the
collagen network (Netti et al 2000), and high drug clearance (Los et al 1991). Further
reasons for resistance of the tumours to currently available anti-cancer drugs, which
may be at least partly responsible for the generally dismal outcome of therapy may be
related to cellular mechanisms. Examples of these are over-expression of transport
molecules such as P-glycoprotein, a so-called “primary active” drug pump and
multidrug resistance-associated protein (MRP), a glutathione (GSH)-dependent increase
in the activity of cellular detoxification systems, altered function of nuclear target
enzymes such as topoisomerase II, and a change in tubulin binding or function
(Germann 2000, Keppler et al 2000, Wright et al 1998, Cole et al 1992, Jedlitschky et al
1996).
As a possible means of obtaining a maximum drug concentration in the target organ,
administration routes other than intravenous (i.v) have been suggested (hepatic artery
infusion [HAI], intra-portal [IPO] injection, intraperitoneal [i.p]). Different therapy
modalities based on radiation, e.g. radio-labelled chemotherapy, external beam
irradiation therapy (EBRT) and intra-operative irradiation therapy (IORT) have been
used in colorectal cancer. This is discussed separately later.
Another possible way to promote drug penetration into tumours would be to use a
carrier substance that specifically targets the tumour. Hyaluronan (HA) is an
endogenous macromolecule that is cleared from the circulation via receptor-mediated
uptake in liver endothelial cells. The HA receptors in these cells are not down-regulated
after ligand binding and also recognise chondroitin sulphate (Gustafson et al 1997). In
addition to binding to these “normal” receptors, HA has the ability to accumulate in
tumours (Gustafson et al 1995) and an in vitro study of rat colorectal adenocarcinoma
14
has shown that the cells carry saturable HA binding sites that do not bind chondroitin
sulphate (Samuelsson et al 1998).
2.2.2.1 Intravenous chemotherapy
Intravenously administered 5-FU has a central role in the treatment of advanced
colorectal cancer (Glimelius et al 1992, Graf et al 1994, Van Halteren el tal 1999). To
exert its anti-neoplastic activity, 5-FU has to be anabolised into its active forms. 5-FU
may be converted along the deoxyribonucleotide pathway to the active metabolite, 5-
fluoro-deoxyuridine monophosphate (Pinedo et al, 1988), which is a potent inhibitor of
the target enzyme thymidylate synthase, leading to inhibition of DNA synthesis. 5-
fluoro-uridine diphosphate and 5-fluoro-uridine triphosphate are also produced along
the ribonucleotide pathway and these compounds are incorporated into RNA, leading to
fraudulent RNA and causing cell death (El Hag et al 1990). However, when 5-FU has
been used as a single agent, the response rate is usually low (Carter et al 1976,). The
limited activity of 5-FU alone in advanced colorectal cancer has prompted efforts to
improve its effect either by altering the rate of administration (by using bolus or pulse
therapy) (Conti et al 1995, Leichman et al, 1995, Glimelius et al 1998) or by
combination with other cytostatics or addition of agents that will increase its
effectiveness, i.e. biochemical modulation (Nobile et al, 1992, Glimelius et al 1993,
Leichman et al, 1995, Blijham et al 1996).
2.2.2.2 Hepatic arterial infusion
In non-resectable liver metastases, regional chemotherapy has been advocated.
Chemotherapy administered by HAI is based principally on the following rationale: 1)
liver metastases larger than a few millimeters derive most of their blood supply from the
15
hepatic arterial circulation, while the normal liver is supplied mainly from the portal
circulation (Ridge et al 1987); and 2) clearance of chemotherapeutic agents by first-pass
through the liver can increase the drug level in the liver and reduce systemic toxicity
(Collins 1984). Lorenz et al (2000) found that HAI therapy was associated with
prolonged time to tumour progression and longer survival than i.v. chemotherapy.
Besides the marginal effect of HAI on survival, the number of patients who switched to
i.v. administration and the problems related to the HAI catheter in the study by Lorenz
et al, the problem of hepatotoxicity causing sclerosing cholangitis (Hohn et al 1988)
warrants further investigation of HAI before it can be recommended as a routine
therapeutic measure.
2.2.2.3 Intraportal chemotherapy
The blood supply to liver metastases up to a size of a few mm predominantly comes
from the portal vein (Ackermann 1974) and initial promising clinical results of adjuvant
IPO treatment have been reported (Taylor et al 1985), but other studies have shown
conflicting results (Liver infusion meta-analysis group 1997, Rougier et al 1998).
Among patients with non-resectable liver metastases, Taylor (1978) found a mean
survival of 4 months in those who received only IPO 5-FU, whereas patients who had
both hepatic artery ligation and IPO infusion of 5-FU survived for a mean of 10 months.
However, because of the small number of patients in that study (n=24), no firm
conclusions can be drawn about the value of IPO infusion. In a randomised study by
Hafstrom et al (1994) in 60 patients with hepatic metastases, a survival benefit was
found in those who underwent hepatic artery occlusion and IPO infusion of 5-FU
compared with patients given systemic treatment. However, the high mortality rate in
16
the study by Gerard et al (1991), who compared hepatic artery occlusion with and
without IPO needs to be considered.
2.2.2.4 Intraperitoneal (i.p.) chemotherapy
The rationale of i.p. chemotherapy is the lower drug clearance from the peritoneal
cavity compared with that from the plasma, higher peritoneal drug concentrations, and
to some extent avoidance of the vascular barrier associated with i.v. drug administration
(Dedrick et al 1978). Furthermore, studies by Speyer et al (1981) of 5-FU showed a
uniform distribution within the peritoneal cavity and a high drug concentration in the
portal blood and hepatic parenchyma when 5-FU was delivered in a large volume of
fluid. An experimental study (Mahteme et al 1998) on the uptake of 5-FU in rat liver
metastases after i.v., IPO and i.p. administration and different times of sacrifice showed
that early (both 20 min and 2 h) tumour 5-FU uptake was inferior after i.p. and IPO
administration as compared with the i.v. route. However, after 24 hours, i.p.
administration gave a better result than i.v. injection.
Early initiation of i.p. treatment has been considered essential for avoiding adhesive
processes, which can limit the drug distribution in the peritoneal cavity (Schellinx et al
1996). However, one of the concerns of i.p. chemotherapy is anastomotic dehiscence. In
an experimental study, Graf et al (1992) compared the influence of early i.p. 5-FU and
i.v. folinic acid on colonic anastomotic healing. They found impaired healing after i.p.
5-FU, but when folinic acid was added, no further deterioration occurred. However, this
problem and other chemotherapy-related toxicities have been investigated in several
clinical studies and this form of administration has not been associated with an
increased complication rate (Graf et al 1994, Kelsen et al 1994, Scheithauer et al 1998,
Vaillant et al 2000). Furthermore, in selected patients with peritoneal metastases, i.p.
17
treatment following complete cytoreduction has shown promising results (Portilla et al
1999, Horsell et al 1999). In our own non-randomised study (unpublished data) in 28
patients with peritoneal carcinomatosis, who underwent a cytoreduction procedure and
received i.p. 5-FU and i.v. leucovorin, 18 survived 1.5 to 9 years.
2.2.2.4.1.Hyperthermic intraperitoneal chemotherapy (HIPEC)
This method was first applied clinically in 1980 by Spratt et al (1980), in a patient with
pseudomyxoma peritonei. The anti-tumoural effect of chemotherapy is believed to be
enhanced by hyperthermia (41-420 C), possibly through an increase in cell membrane
permeability, alteration of active drug transport, a change in cell metabolism and a
decrease in IFP (Hahn 1979, Hahn et al 1983, Leunig et al 1992, Kong et al 2000).
Recent clinical studies have shown promising results (Stephens et al 1999, Cavaliere et
al 2000, Beaujard et al 2000). However, the lack of consensus about the optimal target
temperature, a report on an increased anastomotic dehiscence rate when HIPEC is
combined with preoperative radiotherapy in a rat experimental study (Biert et al 1996),
and a finding of increased morbidity and mortality when a cytoreduction procedure has
been followed by HIPEC (Jacquet et al 1996), all warrant further studies.
2.2.3 Radio-labelled chemotherapy
By combining a short-range beta-emitting radionuclide with chemotherapy (111 In
bleomycin) in an in vitro study by Hou et al (1989), apoptosis was achieved in human
lung cancer cells. In colorectal cancer patients with liver metastases, F-18 labelled 5-FU
has been used in a combination with positron emission tomography (PET), for
pharmacokinetic and diagnostic studies (Kissel et al 1999).
18
2.2.4 Radioimmunotherapy
Theoretically radioimmunotherapy (RIT) appears promising, as the irradiation can be
directed against antigen-expressing tumour cells, whereas the normal host tissue is less
exposed (Welt et al 1994). Furthermore, the inverse relationship between tumour size
and uptake of RIT is of importance (Sigel et al 1990, Blumenthal et al 1994). An
experimental study in mice (comparing conventional chemotherapy with RIT) and a
Phase I study in patients with small-volume liver metastases (<2.5 cm) receiving RIT
were performed by Behr et al (1999). In mice, RIT led to an 80% permanent cure rate
when this therapy was initiated 10 days after tumour inoculation, and in the phase I
study 2 out of 11 patients had partial remissions, while 5 patients showed a minor/mixed
response. This outlines the potential importance of RIT as an adjuvant tool, since the
anti-tumour effects of RIT are most impressive in minimal residual disease. One of the
major limitations of the clinical usefulness of RIT is the small percentage of injected
MAb that is selectively delivered to the tumour (Buchegger et al 1995), since bone
marrow depletion is a major factor for dose-limiting toxicity (Ychou et al 1998).
2.2.5 Radiofrequency (RF)
This technique involves percutaneous or intraoperative insertion of an RF electrode into
the centre of a metastasis under ultrasonic or CT guidance. RF energy is emitted from
the electrode and absorbed by the surrounding tissue. This process generates extreme
heat, leading to coagulative necrosis of treated tissue (Goldberg et al 1995). A study of
RF treatment in patients with hepatic metastases was carried out recently by Goldberg et
al (2000) who found that this mehod induced irreversible cellular damage. However, at
19
present this treatment is in an experimental phase, and further studies are needed in
order to define its role in the therapeutical arsenal.
2.2.6 External beam irradiation therapy (EBRT) and intra-operative
irradiation therapy (IORT)
At the less favourable end of the clinical spectrum of rectal cancer, i.e. locally advanced
tumours that are adherent to adjoining structures such as the sacrum, lateral pelvic
walls, prostate or bladder, pre-operative EBRT has been used to promote tumour
regression (Ahmad et al 1997). Furthermore, an attempt to increase the dose within a
restricted area without introducing significant toxicity to the small bowel and ureter,
IORT has been suggested (Wiig et al 2000).
20
3. AIMS OF THE INVESTIGATION
The general aims of this investigation were to identify clinical factors with influence on
survival in advanced rectal cancer and then use experimental models to study methods
that might optimise treatment. The specific aims were to answer the following
questions:
* What is the prognosis in advanced rectal cancer and what clinical factors
influence survival? What is the outcome after surgical and non-surgical
treatment of non-curable rectal tumours in a defined population? (Paper I).
* Is the uptake of hyaluronan in hepatic metastases influenced by blocking of liver
endothelial cell receptors? (Paper II)
* What is the effect of adjuvant radioimmunotherapy on the development of liver
metastases? (Paper III)
* Is 5-FU uptake in peritoneal metastases influenced by pre-treatment with
radioimmunotherapy or a locally vasoconstrictive agent? (Paper IV)
* Can cytoreductive surgery optimise 5-FU uptake in peritoneal metastases?
(Paper V)
* Is intraperitoneal 5-FU administration superior to intravenous infusion with
respect to 5-FU uptake in peritoneal metastases? (Paper V)
21
4. MATERIALS AND METHODS
4.1 Animals and tumour cells
Rats were used for the experimental studies (II-V). All animals were treated in
compliance with the “Principles of Laboratory Animal Care” and the “Guide for the
Care and Use of Laboratory Animals” (National Institute of Health Publication No. 80-
23, revised 1985). In all experiments, a 10-day acclimatisation period preceded the first
surgical procedure. All animals had free access to standard laboratory diet and water
throughout the experiments. Approval of the study was obtained from the local Ethics
Committee for animal research.
The rats used in study II were anesthetised with fentanyl:midazolam:sterile water
(1:1:2), 3.3 ml kg-1 i.p., and in the rats in studies III-V the anaesthetic agent was chloral
hydrate 36 mg/ml in a mixture of sodium pentobarbital 0.97 mg/ml and manganese
oxide 2.1 mg/ml in distilled water, given i.p. In studies II and V, Wistar FU rats were
inoculated with a chemically induced rat colon carcinoma (NGW cell line, Steele et al
1974), and in studies III and IV nude rats were inoculated with a CEA-producing human
colonic adenocarcinoma, LS 174T (Tom et al 1976). Both tumour cell lines were grown
as a monolayer culture in a nutrient mixture composed of Ham´s F10 medium (Flow
Laboratories Swedish AB, Stockholm, Sweden) supplemented with 10 % fetal calf
serum, 2 mM L-glutamine, penicillin (20 U/ml) and streptomycin (20 µg/ml). Tumour
cell aggregates were achieved mechanically with a scraper (Cell Lifter, Costar
Corporation, Cambridge, MA, USA). Previously it has been shown that this preparation
comprises 40% single cells and 60% tumour cluster, up to 200 µm in diameter, each
containing an average of 200 cells (Graf et al 1992).
22
4.2 Hyaluronan and chondroitin sulphate (CS)
The HA used for labelling and turnover studies was of bacterial origin (Hyal
Pharmaceutical Corporation, Toronto, Canada). The mean molecular weight (Mw) was
400 kDa (range 100 – 2000 kDa), as determined by size exclusion chromatography
(Lebel et al 1989). The HA was labelled with 125I-tyrosine, which does not change the
Mw of HA or its binding to cell surface receptors (Gustafson et al 1994). CS extracted
from a bovine trachea (Sigma Chemical Company, St Louis, MO, USA) was used. The
Mw was approximately 30 kDa as determined by gel filtration chromatography on
Sephacryl S-1000 and S-300 calibrated with hyaluronan standards under conditions
previously described (Gustafson 1997).
4.3 Monoclonal antibodies and radiolabelling
In studies III and IV, the previously investigated (Ahlström et al 1987, Sundin et al
1991, 1993) anti-CEA MAb 38S1 was used. In addition, the anti-TSH MAb 79C was
used in study III. These mouse-derived monoclonal antibodies of the IgG1 kappa
isotype were a gift from Pharmacia AB (Uppsala, Sweden). The 131I-38S1 direct
labelling of the MAbs was achieved by the chloramine-T method (Primus et al 1973).
Briefly, 3.5 mg MAb 38S1 and anti-TSH MAb 79C were separately mixed with 1.5
GBq of a highly concentrated 131I preparation (Amersham International, Little Chalfont,
Buckinghamshire, England), and 150 mg chloramine-T in 750 µl phosphate buffer, pH
7.4, was added. After 10 min of incubation at 00C, the reaction was terminated by
adding 300 mg sodium metabisulphite in 50 µl of 50 mM phosphate buffer, pH 7.4. Gel
filtration was used to separate labelled MAb from unreacted 131I.
23
4.4 14C-labelled 5-fluorouracil
In studies IV and V tracer amounts of labelled 5-FU were used. Gerard et al (1990)
reported that the drug uptake did not differ after injection of tracer amounts of 5-FU
compared with therapeutic doses of labelled 5-FU. The major catabolites of 5-FU are
fluoro-ureidoproprionic acid and fluoro-β-alanine. Further, Chadwick et al (1972)
showed that the radioactivity represents 5-FU as well as drug anabolites and catabolites.
In study IV, for each animal a dose of 17 µCi 5-fluoro[2-14C]uracil, (Amersham,
Buckinghamshire, England), and in study V, a dose of 23 µCi 5-fluoro[2-14C]uracil,
(ICN, Pharmaceuticals, Irvine, Calif, USA) was dissolved in 2 ml 0.9% NaCl (370C).
The injected volume, 2000 µl (i.p.) or 300 µl (i.v.), was administered in 30 seconds.
4.5 Autoradiography and phosphoimaging
In studies IV and V, a whole body autoradiography procedure was carried out to
determine the radioactivity concentration in tumours and various organs. In brief;
immediately after the rats were sacrificed, they were frozen in hexane (paper II) or in
ethanol (papers IV and V) that was cooled with dry ice to –780C in 10 min. The frozen
rats were then mounted in an aqueous gel of carboxymethyl cellulose, which was
rapidly frozen around the animals. Sagittal whole-body sections 10 or 20 µm thick were
attached onto a tape (No. 810, Minnesota Mining & Manufacturing Co., USA). The
sectioning was performed at –200C with a cryomicrotome (PMV Co, Stockholm,
Sweden) as previously detailed by Ullberg et al (1982) and Dencker et al (1991). The
sections were freeze-dried and apposed to Bio-Rad phosphoimager screen and exposed
for 10 days (paper II), or exposed to X-ray film (Agfa Structurix D7, Agfa-Geavert,
Belgium) for 8 weeks (paper IV) or 12 weeks (paper V). In study II, the developed
24
image was analysed on a Bio-Rad GS-525 Molecular Imager and the average
radioactivity was expressed in phosphoimaging density units. In studies IV and V, for
subsequent quantification of autoradiograms, carbon-14 standard staircases
(Autoradiographic 14C-Micro-Scales, Amersham, UK) were co-exposed with the
sections, and to evaluate the sections objectively, computer-based image analysis was
performed as previously described (d´Argy et al 1990).
Paper I
Between 1973 and 1992, 927 patients in the province of Uppsala were identified in the
Swedish Cancer Registry as having been given a diagnosis of rectal cancer. A total of
290 patients were excluded for various reasons (histopathological diagnosis of polyp or
carcinoma in situ (144), other tumours than a rectal adenocarcinoma (72), not living in
the province of Uppsala at the time of diagnosis (5), diagnosis made at autopsy (37), or
their records could not be found (32)), leaving 637 patients for the analysis. Of these,
151 patients (24%) had surgically non-curable disease (unresectable metastatic or
locally advanced disease (Fig 1, Table 1).
Figure 1. Patients with rectal cancer and surgical treatment. APR = abdominoperineal
resection. AR = anterior resection
637 study patients
Incurable rectalcancer, resection ofprimary tumour(n=81) (Group I)
Incurable rectalcancer, noprimary tumourresection (n=70)(Group II)
Curativelyresected (n=444)(Group III)
Resectable primarytumour, nometastases, noprimary tumourresection (n=42)(Group IV)
APR= 36AR = 37Hartmann´s = 8
Stoma= 31Laparotomy =6No surgery = 33
25
Table 1. Characteristics of patients with surgically incurable rectal
adenocarcinoma.
Group 1 Group 2
(n=81) (n=70)
Sex ratio (M : F) 42 : 39 36 :34Mean age (range), years 68 (42-91) 73 (50-94)Tumour site
Distant* 48 33Local† 11 10Abdominal‡ 4 5Distant and local 10 9Distant and abdominal 8 10Abdominal and local 0 1Distant, abdominal and local 0 2
____________________________________________________________*Liver, lung, extra-abdominal lymph nodes, skeleton and brain†Lateral pelvic wall, sacrum, urinary bladder, vagina, uterus and prostate‡Peritoneal surfaces or para-aortic lymph nodes
Paper II
In 36 inbred female Wistar FU rats (mean weight 193 g, range 170-210 g, Möllegaards
Ltd., Denmark), a peripheral branch of the superior mesenteric vein was cannulated
with a plastic catheter and a tumour suspension (5 x 106 viable tumour NGW cells) was
injected as previously described (Graf et al 1992). After 3 weeks, 17 rats with hepatic
metastases (19 had no hepatic metastases and were excluded from further analysis) were
randomly allocated to three groups and given the following treatment i.v. in the caudal
vein or i.p. In group I, the animals received 25 µg 125I-labelled HA by i.v. injection. The
animals in group II received 2.5 mg CS i.v. 3 min prior to i.v. labelled HA. The animals
of group III received treatment similar to that in group II, with the addition of 2.5 mg
CS i.p., 30 min after i.v. injection of labelled HA. Seven animals were sacrificed after
30 min, and nine after 5 hours. One animal was killed 1-2 min after injection of a high
dose of unlabelled HA followed by a tracer dose of labelled HA to observe the
26
distribution of circulating HA. The animals were sacrificed in a CO2 chamber and were
frozen immediately for subsequent evaluation with phosphoimaging.
Paper III
Thirty-three male nude rats (mean weight 249 g, range 180 – 318, Rowett nu/nu,
Wallenberg Laboratory, Lund, Sweden) were inoculated with a LS 174T tumour cell
suspensions (5 x 106), using the same procedure as mentioned above (paper II). The
right femoral vein was catheterised (for i.v. administration purposes) and animals were
randomly allocated to five different groups. Within half an hour, 20 MBq (n=2), 75
MBq (n=5) or 150 MBq (n=10) of the 131I-labelled anti-CEA MAb (38S1) was injected
i.v., and control groups received either i.v. saline injections (n=12) or 150 MBq of the
irrelevant 131I-labelled Mab 79C (n=4). Body weight was recorded every 5 days and
immediately before sacrifice. Whole-body and blood clearance (blood samples taken
from a tail vein) of 131I activity was monitored every 2-4 days. Whole-body
radioactivity was measured by means of a solid-state Ge-detector and blood clearance in
a well-type NaI-detector. After a period of 5-7 weeks, all rats were sacrificed in a CO2
chamber, and blood was obtained by cardiac puncture for determination of
haemoglobin, haematocrit, leucocytes and platelets. The abdomen was inspected for the
presence and extent of hepatic metastases and for extrahepatic tumour growth.
27
Paper IV
A total of 32 nude rats (17 females and 15 males, mean weight 205 g, range 151-341 g,
Rowett nu/nu, Wallenberg Laboratory, Lund, Sweden) underwent abrasion of the right
lateral abdominal peritoneum (1 cm2), using a scalpel, and were inoculated with 1.0 x
107 LS 174T. Two weeks later the animals were randomly allocated to five groups.
Group I (n=5) and group IV (n=4) received 3 ml NaCl/rat i.p. and served as control
groups, and the animals in groups II (n=6) and V (n=8) received 131I-Mab 38S1 i.p. in
the same volume. Six days (groups I, II and III) or 2 days (groups IV and V) later, a
second laparatomy was performed and verified that all animals had a macroscopic
tumour in the peritoneal cavity. One rat in group I died immediately after the second
laparatomy and was excluded. Two rats, one from group IV and one from group V,
underwent tumour dry-weight analysis. In the remaining 29 animals, a plastic catheter
(Polyethylene Tubing, PP 380, Swevet AB, Stockholm, Sweden) was inserted i.p. and
14C-labelled 5-FU was injected. The animals in group III (n=6) received 2.5 mg /kg U/V
Norbormide (rat specific, vasoconstrictive agent) i.p. 10 minutes prior to injection of
labelled 5-FU. In all groups, the animals were killed in a CO2 chamber 2 hours after 5-
FU injection, for subsequent evaluation with autoradiography.
Paper V
Forty-six inbred female Wistar rats (mean weight 201 g, range 150 – 240 g,
Möllegaards Ltd., Denmark) were used in study V. A suspension of an adenocarcinoma
cell line (NGW), containing 1.0 x 107 viable tumour cells, was applied by injection
either into non-abraded (n=6) or into the abraded (1 cm2) (n=6) right lateral abdominal
peritoneum. Since abrasion was associated with more reproducible tumour growth, this
method was used in the remaining animals. Three weeks after tumour inoculation, a
28
second laparatomy was performed. Totally 34 animals had macroscopic tumours
scattered in the abdomen. They were randomly allocated into eight groups and injected
with 14C-labelled 5-FU either by the i.v. or the i.p. route, with or without an
immediately preceding tumour debulking procedure, and sacrificed after 2 or 8 hours. In
the i.v. group the right femoral vein was cannulated, and in the i.p. group a catheter was
introduced as outlined above (papers II and IV respectively). The debulking procedure
encompassed tumours that were located in the right half of the abdomen. These tumours
were divided in the midline and half of the tumour was excised. To minimise blood loss,
tumours in the left half of the abdomen were left in situ. The animals were killed in a
CO2 chamber and subjected to autoradiography.
29
5. STATISTICAL METHODS
In study I, differences in proportions were evaluated with the X2 test and numerical
differences with Student’s t-test. The log rank test and the Cox´s proportional hazards
model (Cox 1972) were used to evaluate the influence of categorical and of continuous
clinical variables, on survival in a univariate analyses.
To evaluate differences between groups in study II, non-parametric analyses were
performed using the Mann-Whitney U test.
In study III, the proportions of animals with metastases in the groups were compared by
Fisher´s exact test. A one-way analysis of variance was used to evaluate weight
development and blood data differences between groups.
In studies IV and V, a two-factor repeated measure analyses of variance was performed
(with or without stratification by tumour area) to determine whether the drug uptake in
tumours differed between the groups. In these analyses, group and rat are treated as the
“between” factors and tumour as the “within” factor. In study IV, Dunnett´s test was
used to compare treated groups with the control group, and in study V Tukey´s
studentized range test was used to conduct multiple comparisons among the groups. In
both tests, a Bonferroni correction was used to control the overall type I error rate of
0.05 (Fleiss 1986). In all studies, statistical significance was accepted at P < 0.05.
30
6. RESULTS
Paper I
The median survival was 7.5 months in group I, and 3.5 and 1.9 months in surgically
and non-surgically treated patients, respectively, in group II. The incidence of
postoperative ileus was higher in group I than in the patients treated with a curative
resection (P=0.02). Analysis of patients with surgically non-curable disease (groups I
and II combined) showed that bilateral hepatic, lymph node and peritoneal metastases as
well as abnormal liver function tests correlated with poor survival (Table 2 and 3).
Furthermore, a shorter survival was found in patients with abdominal metastases than in
those with distant metastases (P=0.01, Table 2).
Table 2 Association between clinical variables and survival at univariateanalysis in patients with surgically non-curable rectal cancer (n=151)._____________________________ Groups I and II
n survival. P*_____________________________
Bilat hepaticYes 75 4.3 0.04No 52 5.7
Lymph nodesYes 15 2.4 0.01No 136 5.7
PeritonealYes 18 3.6 0.02No 133 5.5
Abdominal†Yes 70 4.1 0.01No 81 5.7
___________________________________Survival (median survival in months)†Metastatic growth confined to peritonealsurfaces, local sites or para-aortic lymph nodes.* Log rank test.
31
Table 3 Relationship between liver function tests and survival at univariateanalysis in patients with surgically non-curable rectal cancer (n=151).____________________________________
Group I and IIn Relative P*
hazard________________________________________________Serum bilirubin 114 1.02 0.002Serum alkalinephosphate 115 1.07 0.001Serum ASAT 116 2.32 0.001Serum ALAT 116 3.17 0.001Serum albumin 121 0.95 0.004_____________________________________ASAT: aspartate aminotransferase.ALAT: alanine aminotransferase.* Cox proportional hazards model.
Paper II
Sixteen rats had a total of 50 hepatic metastases (Table 4). At 5 hours, the radioactivity
in hepatic metastases was higher in group II than in group I (P=0.01, Table 5, Fig 2).
The liver/tumour ratio was higher in group I than in groups II and III (P=0.01).
Table 4 Number of rats (and hepatic metastases) in the three groups.__________________________________________________________________
Group I II III__________________________________________________________________
4(9) 8(34) 4(7)__________________________________________________________________Group I= Not inhibited by chondroitin sulphateGroup II= Inhibited by i.v. chondroitin sulphateGroup III= Inhibited by i.v. and i.p. chondroitin sulphate
32
Table 5 Concentration of radioactivity in hepatic metastases and skeletalmuscle after 5 hours. Figures are mean (SD) in the experimentalgroups and individual animals.
________________________________________________________________Tumour Muscle
________________________________________________________________Group I 1.8 (0.6) 0.1 (0.1)
Animal 1 1.4 (-) 0.3 (-)Animal 2 1.9 (0.7) 0.1 (-)
Group II 4.8 (1.8) 0.5 (0.2Animal 1 5.7 (2.7) 0.7 (-)Animal 2 4.3 (1.8) 0.5 (-)Animal 3 4.5 (-) 0.2 (-)
Group III 3.6 (1.1) 0.7 (0.4)Animal 1 2.8 (0.4) 0.4 (-)Animal 2 3.9 (0.8) 0.5 (-)Animal 3 4.6 (1.3) 1.2 (-)Animal 4 2.4 (-) 0.4 (-)
________________________________________________________________For definition of groups, see Table 4
Figure 2. Autoradiographs of whole body sections of two rats. The left autoradiographis from a rat which received chondroitin sulphate prior to labelled HA (group II), andthe right one is from a rat which was given labelled HA alone (group I). The arrowspoint to liver metastases.
33
Paper III
In the 20 MBq, 75 MBq and 150 MBq 131I-38S1 groups, 2/2, 3/5 and 0/10 rats,
respectively, developed liver metastases, compared with 6/12 animals in the group
injected with saline only and 0/4 animals in the 150 MBq 131I-79C group (Table 6). The
difference in tumour induction between the saline group and the groups injected with
the 150 MBq dose of MAb (either specific or non-specific) was significant (P=0.03).
Furthermore, a mean weight loss was found in both 150 MBq groups, whereas a mean
weight gain occurred in the other groups (P=0.01,Table 6). The highest doses of 131I-
38S1 and 131I-79C were associated with depletion of B-haemoglobin, B-erythrocyte
volume fraction and B-white blood cell count (P=0.001).
Table 6 Proportion of rats with metastases in the different groups, and haematologicalparameters and weight change immediately before sacrifice. Figures are mean ±SE.__________________________________________________________________Group Rats with Hb B-EVF B-TRC B-WBC Weight
metastases (g/L) (%) (x109/L) (x109/L) Change (g)
__________________________________________________________________
NaCl 6/12 160±16 47±4 653±342 6.1±3.3 80±113
20 MBq 2/2 151±4 -- 575±6 5.0±0.4 139±30
75 MBq 3/5 148±14 44±1 570±81 4.5±1.7 61±5
150 MBq 0/10 139±19 42±7 560±297 3.8±2.0 -7±1-0
150 MBq‡ 0/4 126±15 38±4 623±272 3.2±2.0 -35±25__________________________________________________________________20 MBq, 75 MBq and 150 MBq refer to labelling of 131I-38S1 MAb‡150 MBq 131I-79C (unspecific) MAbEVF= erythrocyte volume fractionTRC= thrombocyte countWBC= white blood cell count
34
Paper IV
Totally 243 peritoneal metastases were observed (Table 7). A high uptake of 131I in
peritoneal tumours and skin was found in group II after 1 week of exposure (Fig. 3).
After 8 weeks of exposure, the tumour size was reduced in group II compared with
group I (P=0.01), whereas the size did not differ between groups IV and V. The tumour
uptake of 5-FU was higher in group III than in group I (Table 8, P=0.04). When the
tumours were divided into small tumours (<627 pixels) and large tumours > 627), where
627 was the median tumour area, large tumours in group III were found to have a higher
radioactivity concentration than group I (Table 8, P=0.002), whereas the difference was
less pronounced in small tumours. There was no difference in the tumour fluid content
between group IV (79%) and group V (77%).
Table 7 Number of rats (and peritoneal metastases) in the experimental groups. The
numbers of tumours larger or smaller than 627 pixels (median tumour area) are shown
separately.
__________________________________________________________________
Group I II III IV V
__________________________________________________________________
n 5(58) 6(70) 6(50) 4(19) 8(46)
Small tumours 28 54 15 8 15
Large tumours 30 16 35 11 31__________________________________________________________________Group I = Treated with NaCl, 6 days prior to 14C-5-FU injection.
Group II = Treated with 131I-MAb 38S1, 6 days prior to 14C-5-FU injection.
Group III = Treated with Norbormide, 10 min prior to 14C-5-FU injection.
Group IV = Treated with NaCl, 2 days prior to 14C-5-FU injection.
Group V = Treated with 131I-MAb 38S1, 2 days prior to 14C-5-FU injection.
35
Table 8 Concentration of 14C-originated radioactivity in peritoneal metastases in all
tumours, in small and large tumours separately, and in the liver and intestine (kBq/g
tissue), values are mean (SD).
__________________________________________________________________
All tumours Small Large Liver Intestine
n n
__________________________________________________________________
Gr I 11.8 (11) 30 11.9 (13) 28 11.8 (7) 21.3 (27) 9.1 (5)
Gr II 9.8 (11) 54 11.2 (12) 16 4.8 (2) 7.1 (4) 9.7 (10)
Gr III 21.4 (17) 15 17.1 (11) 35 23.3 (19) 17.3 (15) 34.4 (33)
Gr IV 12.1 (3) 8 12.1 (4) 11 12.1 (3) 15.6 (9) 11.4 (8)
Gr V 10.1 (10) 15 10 (4) 31 10.2 (4) 16.2 (11) 11.6 (5)__________________________________________________________________For definition of groups, see Table 7
Figure 3. Autoradiographs of whole body section of a rat after one week of exposureshowing radioactivity originating from 131I in peritoneal metastases (arrow).
36
Paper V
A total of 360 peritoneal metastases were eligible for this study (V) of the uptake of 5-
FU in relation to the mode of administration and surgical tumour reduction (Table 9).
After 8 hours, tumours treated by i.p. injection after a debulking procedure (i.p.TR) had a
higher uptake than those treated by i.p. injection without such a procedure or tumours
treated by i.v. injection with such a procedure (i.v.TR), (P=0.002, Table 10, Fig. 4). The
95% confidence intervals for the mean differences i.p.TR-i.p. and i.p.TR-i.v.TR were [10.6
– 50.2] and [21.0 – 68.3], respectively. After 8 hours, small tumours (< 571 pixels,
where 571 was the median tumour area) subjected to i.p.TR had a higher uptake than i.p.
or i.v.TR tumours (P=0.004 Table 11). Among larger tumours (> 571 pixels), after 2
hours i.p.TR tumours had higher radioactivity than i.p. or i.v.TR tumours (P=0.03, Table
11), and after 8 hours i.p.TR tumours showed higher uptake than i.p. or i.v.TR tumours
(P=0.001, Table 11).
Table 9 Number of rats (and peritoneal metastases) studied 2 and 8 hours after
injection of labelled 14C-5-FU, by route of administration and tumour debulking.
______________________________________________________________________
2 hours 8 hours
______________________________________________________________________
i.v. 4 (43) 4 (68)
i.p. 6 (98) 4 (62)
i.v.TR 4 (26) 4 (22)
i.p.TR 4 (22) 4 (19)
______________________________________________________________________
i.v. = Intravenous injection
i.p. = Intraperitoneal injection
i.v.TR =Intravenous injection after debulking procedure
i.p.TR =Intraperitoneal injection after debulking procedure
37
Table 10 Radioactivity concentration in peritoneal metastases (kBq/g tissue) 2 and 8
hours after injection of labelled 14C-5-FU in relation to route of administration and
tumour debulking. Values are mean and (SD).
______________________________________________________________________
2 hours 8 hours
______________________________________________________________________
i.v. 73.2 (94) 22.1 (13)
i.p. 82.2 (113) 32.8 (14)
i.v.TR 26.1 (20) 18.5 (18)
i.p.TR 157.8 (81) 63.2 (28)
______________________________________________________________________
For definition of groups, see Table 9
Figure 4. Autoradiographs of whole body sections of rats, showing uptake of 14C-
labelled 5-FU in peritoneal metastases (arrows). A rat treated with i.p. 5-FU and
debulking (i.p.TR) is seen to the left and a rat treated with i.v. 5-FU and debulking
(i.v.TR) to the right.
38
Table 11 Radioactivity (kBq/g tissue) in peritoneal metastases, stratified according to
tumour area (small and large tumours; median tumour area = 571 pixels), 2 and 8 hours
after injection of labelled 14C-5-FU, by route of administration and tumour debulking.
Values are mean (and SD).
______________________________________________________________________
Small tumours Large tumours
2 hours 8 hours 2 hours 8 hours
n n n n
______________________________________________________________________
i.v. 30 86 (107) 34 25 (17) 13 40 (38) 34 19 (7)
i.p. 49 116 (141) 29 35 (17) 49 48 (59) 33 31 (10)
i.v.TR 15 31 (24) 8 23 (17) 11 20 (13) 14 16 (18)
i.p.TR 6 176 (121) 9 77 (26) 16 151 (63) 10 50 (24)
______________________________________________________________________
For definition of groups, see Table 9
39
7. DISCUSSION
Many factors influence survival after the development of metastases from colorectal
cancer. In this investigation (study I), the clinical factors most strongly associated with a
poor prognosis were found to be massive involvement of the liver and abdominal
tumour growth, supporting previous findings (Graf et al.1991, Assersohn et al 1999,
Sadeghi 2000). This result indicates that at the time of diagnosis there is a need to select
a “subgroup” of patients, who may not benefit from surgical treatment. The median
survival in patients who underwent resection was reduced by one-third when abdominal
growth was present compared with those without abdominal involvement. On the other
hand, patients with peritoneal metastases may benefit from therapeutic strategies other
than conventional surgery or chemotherapy. To control local symptoms (bleeding,
intestinal obstruction), palliative resection has been recommended (Tacke et al 1993,
Fitzgerald et al 1993). However, in our study, about 15% of the patients who did not
undergo either resection or a non-resectional procedure developed symptoms from the
primary tumour that necessitated surgery. Moreover, the need for late surgery was not
lower in patients in whom the primary tumour was resected than in those in whom it
was left in situ, and it is therefore doubtful whether the risk of intestinal obstruction is a
good argument for a palliative resection.
In the light of the above finding, there is a need to find a way to optimise the uptake of
currently available drugs in liver metastases. A previous in vitro study by Samuelsson et
al (1998) had shown that NGW cell lines had the ability to bind HA with high affinity.
Furthermore, in our in vivo study we found that HA targeted liver metastases and that
40
pretreatment with CS enhanced the tumour uptake of the labelled HA. The differences
in specificity between HA binding sites can be utilised in attempts to block uptake of
exogenous HA in healthy liver without reducing targeting to metastases. It is possible
that targeting of HA and HA/drug complexes to HA binding sites can result in more
effective drug treatment, with probably enhanced uptake and reduced side-effects. Klein
et al (1993) compared the effects of 5-FU alone with those of 5-FU + HA on liver
metastases from rat mammary carcinoma. They found an increased tumour drug uptake
in rats treated with 5-FU + HA. However, the affinity between HA and 5-FU is believed
to be low and the possibility of drug-targeting using HA needs to be investigated
further. Another study indicating the potentiality of HA as a drug carrier was reported
by Coradini et al (1999) who found that when sodium butyrate was linked to HA, the
availability of the sodium butyrate increased in a breast cancer cell line.
The superior penetration of MAb into smaller lesions compared to larger ones has been
demonstrated previously (Pervez et al 1988, Sato et al 1999). By contrast, Sundin et al
(1993) reported that there was no relationship between MAb uptake by liver metastases
and their size. However, the ability of MAb to prevent further progression of small
metastases has been observed in both clinical and experimental studies (Juweid et al
1996, Behr et al 1999). In the study by Behr et al (1999), in which mice with liver
metastases were treated with MAb 10 or 20 days after tumour inoculation, a definite
cure was observed in the 10-day group but not in the 20-day. In our study III, in which
RIT was given as adjuvant treatment, in experimental colonic cancer the development
of liver metastases could be totally prevented by administration of 150 MBq of 131I-
38S1 or 131I-79C, while in the control group (NaCl) and the groups that received 20
MBq or 75 MBq, liver metastases developed. The unexpected tumour-preventing effect
41
of the unspecific MAb (150 MBq) in our study might have been due to non-specific
radiation, contributing to the inactivation of tumour cells. In a previous experimental
study in mice, unspecific MAb labelled with a lower of 131I (14.8 MBq) exerted a slight
inhibitory effect on the growth of inoculated human ductal mammary carcinoma
(Senekowitsch et al 1989). These differences may be due to the total radiation dose.
The relation between peritoneal metastases from rectal cancer and a poor prognosis
(paper I) raises the question as to how we can optimise the uptake of cytotoxic drugs in
such metastases. It was found in study IV that RIT treatment given 6 days prior to
sacrifice induced a decrease in tumour size but did not affect or perhaps negatively
affected the uptake of 5-FU. The effect on tumour size is most likely due to the β-
radiation, which reduces the capacity of the tumour cells to proliferate, as reported from
previous experimental models (Blumenthal et al 1994). Theoretically, oedema can be
initiated by the radiation, leading to an increase in intratumoral pressure. However,
when we compared the water content in tumours from groups IV and V (see Table 7 for
definition of groups), no difference was found. Clinically, especially in ovarian cancer,
intraperitoneal RIT has been used in combination with debulking surgery or
chemotherapy, but the results from these studies are not yet convincing (Meredith et al
1996, Mahe et al 1999).
The uptake of 5-FU in the peritoneal metastases, especially in “large tumours”, was
improved after pretreatment with a vasoconstrictive agent (Norbormide). This drug,
which is rodent-specific, produces an irreversible local vasoconstriction through an α-
adrenergic effect (Roszkowski 1965). The higher uptake in peritoneal metastases
observed after administration of Norbormide may probably be due to reduced peritoneal
absorption of 5-FU, leading to prolonged exposure of the tumours to 5-FU and thereby
42
promoting local diffusion of 5-FU. One possible reason for failure of intraperitoneally
administered drugs to cure larger tumour masses is the poor penetration into tumour
tissue. For example, Collins et al (1982) found that the concentration of 5-FU measured
at a depth of 0.6 mm from the peritoneal surface was only 5% of that in the peritoneal
fluid. Our results are consistent with those of Duvillard et al (1999), who reported that
the anti-tumoral effect of i.p. chemotherapy was enhanced by a combination with i.p.
epinephrine.
In paper V, the debulked, i.p. treated tumours overall showed a higher uptake at 8 hours
than the native tumours treated i.p., and the uptake was also higher than in debulked
tumours treated by the i.v. route. It is reasonable to conclude that 5-FU uptake is
improved by breaking biological barriers and thereby possibly by decreasing the
interstitial pressure to promote regional drug delivery. A previous study on the uptake
of Cisplatin with a molecular weight (Mw) of 300.6 kDa and of Carboplatin with a Mw
of 317.3 kDa, in rat peritoneal tumours, showed a favourable uptake of Cisplatin (Los et
al 1991). 5-FU, which has a lower Mw (130.1 kDa) could be ideal to use
intraperitoneally. However, the influence of the Mw of drugs on their tissue diffusion
has been found to be limited (Flessner et al 1985). The enhanced uptake of 5-FU in the
peritoneal tumours in the present study may have been due to mechanically
disintegrating membranes, with different solubility for drugs (Kerr et al 1988), which
the drug has to cross. An increased drug accumulation due to local diffusion was also
indicated by the lower radioactivity concentration in the centre of the tumour compared
to its periphery (data not shown), which is consistent with the results of Collins et al
(1982).
43
The present study has thus confirmed that patients with bilateral liver metastases,
peritoneal growth or abdominal lymph node metastases have a short survival. The
experimental finding of adjuvant RIT in preventing liver metastases is promising, and
this may be clinically applicable in the future. The observation of enhanced uptake of
HA in liver metastases may be applied clinically, with use of HA as a drug-delivery
molecule. In selected patients with peritoneal carcinomatosis, cytoreductive surgery and
i.p. chemotherapy may be associated with a prolonged survival (Gough et al 1994,
Schellinx et al 1996, Horsell et al 1999). Our finding of a higher drug uptake in
debulked, i.p. treated tumours is encouraging. Moreover, the enhanced uptake of 5-FU
observed in peritoneal metastases after pretreatment with a local vasoconstrictive agent
is promising. Clinically, epinephrine injected intratumorally together with Cisplatin in
patients with solid tumours located in subcutaneous tissue has yielded favourable results
(Burris et al 1998). However, the risk of systemic (mainly cardiovascular) and local
(intestinal infarction) effects of epinephrine limits intraperitoneal application of this
drug in patients with peritoneal carcinomatosis, and the concept of peritoneal
vasoconstriction merits further investigation.
44
8. SUMMARY AND CONCLUSIONS
The aims of this work were to identify metastatic sites associated with poor prognosis in
rectal cancer patients and to investigate methods that might prevent the development of
metastases and optimise the uptake of drugs at these sites in animal models. The main
results were:
♦ Bilateral hepatic involvement, peritoneal growth or abdominal lymph node
metastases implies a poor prognosis.
♦ Blocking of uptake and elimination of hyaluronic acid by the liver enhances the
hyaluronan uptake in liver metastases. Hyaluronan may thus be of future value as a
delivery-molecule for chemotherapy, targeting specific hyaluronan receptor-
positive tumour sites.
♦ Radioimmunotherapy may prevent the development of liver metastases from
colorectal carcinoma in an “adjuvant” setting, although the unspecifically delivered
radiation dose is most likely the major contributing factor. Furthermore,
radioimmunotherapy can inhibit the growth of peritoneal metastases.
♦ The uptake of intraperitoneally administered 5-FU in peritoneal metastases can be
potentiated by blocking the 5-FU absorption with a vasoconstrictive agent.
Moreover, uptake of 5-FU in peritoneal metastases can be improved if the tumours
are surgically reduced and the drug is given intraperitoneally.
45
9. ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to everyone involved in this study, for alltheir encouragement and assistance. My special thanks are due to:
Wilhelm Graf, my tutor, for initiating this study, for introducing me to the field ofresearch, for his never-ending enthusiasm and for his unbeatable skill in “targeting” theright questions and comments on all my manuscripts.
The late Bengt Larsson, my co-tutor, for sharing his profound knowledge andexperience in an unprestigious manner, and for his patience and friendship.
Anders Sundin, my co-tutor, for giving me scientific education in a constructive andfriendly way, with patience and optimistic encouragement.
Lars Påhlman, my co-author, for allowing me the opportunity to work in the colorectalteam and being supportive in my surgical training, and listening to all my “complaints”.
Bengt Glimelius, my co-author, for sharing his knowledge and for his interested
support.
Ulf Haglund, head of the Department of Surgery, for providing conditions and facilitiesto make these studies possible.
David Bergqvist and Lars Wiklund, former and present Heads of the Department ofSurgical Sciences, for providing the necessary conditions for carrying out this doctoralwork.
All my co-authors, for valuable help: Harry Khamis, Stefan Gustafson, JörgenCarlsson, Anna Lövqvist, Hans Lundqvist, and Kiril Arow.
Lennart Dennker, for placing facilities at my disposal to allow completion of the lasttwo experiments, in the absence of Bengt Larsson.
Raili Engdahl, Veronica Asplund, Lena Norgren, and Tomas Björkman, for excellenttechnical assistance.
Yngve Raab, former senior colleague, for his outstanding guidance, patience (evenwhen surgery went at Ångstoms speed), encouragment and great support in my surgicaltraining. It was a pleasure to work with you.
Asbjörn Österberg, former colleague, for valuable friendship, interesting philosophicaldiscussions and some good laughs.
Former and current colleagues in the colorectal team, Sten Ejerblad, MichaelDahlberg, Ulf Kressner, Urban Karlbom, Magnus Thörn, Ulla-Maria Gustafsson,Erik Lundin, George Dafnis, Pia Jestin, and Ulf Gunnarsson, for their support andfriendship.
46
Ola Hessman, Richard Henriksson, and Agnetha Westling, colleagues and room-mates, for god friendship, especially Ola for being my PC-consultant, without gettingtired of my computer “problems”.
Friends and colleagues at the Department of Surgery, Uppsala.
My former colleagues at the Department of Surgery, Sala, for their friendship, and Iespecially thank, Jan Vannfält, former head, for giving me opportunities, support andencouragement, and Bengt Andersson, for teaching me general surgical principles andintroducing me to colorectal surgery.
Maud Marsden, for skilful linguistic revision.
My father, Mahtemework, and my late mother, Almaze for giving me a good start inlife and for their incredible love and concern.
My sisters and brothers, Aster, Admase, Daniel, Azeb, Michael, Simon, Yohannes,Abebech, and Berhane, for their love.
And most of all
To my beloved Charlotte, no words are enough to tell you how grateful I am to you, foryour love and support, and for taking care of the most important things in life, our threewonderful boys, Josef, Christopher and Salomon, from whom I have my greatestsupport and love and are sources of energy and joy.
This study was supported by a grant from the Swedish Cancer Society, project no.
3764-B99-04XBB.
47
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