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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