university of groningen different aspects of hyperthermic

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Different aspects of hyperthermic isolated limb perfusionGinkel, Robert Johannes van

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DIFFERENT ASPECTS OF HYPERTHERMIC

ISOLATED LIMB PERFUSION

This research was financially supported by the Dutch Cancer Society (Nederlandse

Kankerbestrijding KWF) grant GUKC 90-06.

ISBN: 90-367-1716-7

Page lay out: P. van der Sijde, Groningen, The Netherlands

Printed by: Ponsen en Looijen BV, Wageningen, The Netherlands

RIJKSUNIVERSITEIT GRONINGEN

DIFFERENT ASPECTS OF HYPERTHERMIC ISOLATED LIMB

PERFUSION

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus, dr. F. Zwarts,

in het openbaar te verdedigen op

woensdag 20 november 2002

om 16.00 uur

door

Robert Johannes van Ginkel

geboren op 12 mei 1964

te Amsterdam

.

Promotores Prof. dr. H.J. Hoekstra

Prof. dr. H. Schraffordt Koops

Prof. dr. W. Vaalburg

Beoordelingscommissie Prof. dr. B.B.R. Kroon

Prof. dr. M.F. von Meyenfeldt

Prof. dr. W.M. Molenaar

Voor opa Hans

Paranimfen Drs. D.J. Klees

Drs. R.P. Winkel

Contents

Chapter 1 General introduction and aim of the thesis 9

Chapter 2 Hyperthermic isolated limb perfusion with cisplatin in the localtreatment of spontaneous canine osteosarcoma:Assessment of short term effectsJournal of Surgical Oncology 1995; 59: 169-176. 29

Chapter 3 Hyperthermic isolated limb perfusion with TNF and cisplatinin the treatment of osteosarcoma of the extremities:A feasibility study in healthy dogsSarcoma 1999; 3: 89-94. 45

Chapter 4 Hyperthermic isolated limb perfusion with cisplatin in fourpatients with sarcomas of soft tissue and boneEuropean Journal of Surgical Oncology 1996; 22: 528-531 57

Chapter 5 Isolated limb perfusion of an irradiated foot with TNF,interferon and melphalanArchives of Surgery 1996; 131: 672-674. 67

Chapter 6 FDG-PET to evaluate response to hyperthermic isolated limbperfusion for locally advanced soft-tissue sarcomaJournal of Nuclear Medicine 1996; 37: 984-990. 77

Chapter 7 [1-11C]-Tyrosine PET to evaluate response to hyperthermicisolated limb perfusion for locally advanced soft-tissuesarcoma & skin cancerJournal of Nuclear Medicine 1999; 40: 262-267. 93

Chapter 8 Value of Continuous Leakage Monitoring with RadioactiveIodine-131 Labeled Human Serum Albumin DuringHyperthermic Isolated Limb Perfusion with TNFand MelphalanAnnals of Surgical Oncology 2002; 9: 355-363. 107

Chapter 9 Summary and conclusionsSamenvatting en conclusies 125

Dankwoord 140

Curriculum vitae 142

Publications 143

9

General introduction and aim of the thesis

10

Chapter 1

Before Tumor Necrosis Factor

The first report of the beneficial effect of intravenously administered nitrogen-mustard

on tumor growth appeared just after the second world war.1 Soon afterwards reports

were published on the advantageous effect of intra-arterially administered nitrogen-

mustard on malignant tumors.2-4 Using technology to support extracorporeal

circulation developed for cardiac surgery in the 1950s, the surgical oncologists Creech,

Krementz, Ryan and Winblad of the Tulane University in New Orleans developed

the technique of isolated limb perfusion (ILP).5 In this procedure the blood circulation

of a tumor bearing limb is isolated from the circulation of the rest of the body by

clamping the major artery and vein and tightening a tourniquet around the root of the

limb. The major artery and vein are subsequently connected to a heart-lung machine

and the cytotoxic drug is administered through this isolated circuit. Key point in ILP

is that the dose of chemotherapeutics used, can be 15-20 fold the maximum systemic

tolerated dose, since vital organs are isolated from the perfusion circuit.6-8

The original patient population treated with ILP was a subgroup of melanoma patients

who had extensive local recurrence in the arm or leg. The initial drug used for ILP to

treat extremity melanoma was melphalan (L-phenylalanine mustard). Melphalan is

an alkylating agent of the bischloroethylamine type comprising nitrogen mustard

and phenylalanine. Phenylanaline is a metabolite of melanin and therefore melphalan

specifically targets melanocytes and melanoma cells. Its cytotoxicity appears to be

related to the extent of its interstrand cross-linking with DNA. Like other bifunctional

alkylating agents, it is effective against both resting and rapidly dividing tumor cells.

In 1959 Creech, Krementz and Ryan described their initial results of patients treated

with regional perfusion. The first patient was a 76 year old male with multiple

melanoma satellites on his upper leg. After regional perfusion with melphalan the

satellites disappeared completely and the patient died at the age of 92 with no local

recurrence. The case history of this patient was frequently illustrated at lectures and

a poster with pictures of this patient decorated the entrance of the surgical ward of

the Tulane University for many years. Cavaliere and co-workers investigated the

addition of hyperthermia in the treatment of cancer and, as this appeared to augment

the anti-tumor effects of melphalan, in doing so they laid the basis for hyperthermic

isolated limp perfusion (HILP).9 At temperatures of 41.5 degrees C and higher a

direct anti-tumor effect was observed however, this was accompanied with

unacceptable local toxicity.10 To avert this increased local toxicity it was established

that mild hyperthermia with temperatures of 39 to 40 degrees C was best used.

Wieberdink introduced the optimal dose calculations of melphalan based on limb

volume instead of patient weight, since the latter may lead to under- or overtreatment

of an individual dependent on body habitus.11 An essential component of HILP is

11

Introduction

monitoring the perfusion leakage to the systemic circulation and being able to make

adjustments during treatment to reduce this leakage. Different methods to measure

leakage are used. Stehlin and associates were the first to describe a method of

continuous external leakage monitoring with radioactive Iodine-131 labeled human

serum albumin (RISA).12 This is still the method most frequently used nowadays. It

places a gamma counter over the precordium with RISA in the perfusion circuit,

which allows continuous readings and estimations of the leak of the perfusion solution

into the systemic circulation.13

From 1969 until recently, ILP with hyperthermia and melphalan was the gold standard

for regional treatment of in-transit melanoma. The response rates to this therapeutic

HILP are considerably higher to any other systemic therapy for this type of tumor.

Objective response rates have been reported as high as 70% to 100%, with complete

response rates between 54% and 65%. The median duration of responses is

approximately 9 months, and some patients experience a long-term disease control

with this regional therapy.14,15

Many publications on HILP for melanoma combine adjuvant perfusions with

therapeutic perfusions, often with different treatment schedules, making the

interpretation of available data very difficult. A publication on the 35-year experience

with HILP of the Tulane Hospital serves as a good example for this problem. Over

1100 cases were reported with a median follow-up longer than 10 years. However,

an evidence based conclusion about the benefit of the procedure could not be made.16

A prospective randomized German study published in the 1980s reported a significant

improvement in survival after adjuvant HILP.17 However, the numbers of patients

treated were small, and the outcome in the control group was much worse than

expected compared to historical controls, which meant that this trial could not be

used in arguing for adjuvant HILP.18 The value of HILP as an adjuvant treatment

modality in patients with high risk stage I disease (more than 1.5 mm Breslow

thickness), was recently evaluated in a prospective randomized trial by the European

Organization for Research on Treatment of Cancer (EORTC).19 This study showed

no overall survival benefit for patients treated with HILP with melphalan followed

by local excision compared to patients that had undergone local excision only.

However, a slight benefit in disease free survival was seen in the perfusion group.

With the publication of this study as a negative trial, no adjuvant HILP should be

performed after resection of primary melanoma. Another patient population that may

benefit from a adjuvant HILP are those who have developed in-transit metastases

that have been excisionally biopsied. These patients are at a much greater risk for

additional recurrences in the limb than patients with high-risk primary cutaneous

melanoma who have not had a regional recurrence. A small prospective study from

12

Chapter 1

Sweden found a significant improvement in tumor free survival in the perfusion

group, however no overall survival benefit was demonstrated.20 In conclusion, adjuvant

HILP with melphalan should not be used for high-risk primary melanoma and should

only be used as an adjuvant in the setting of a clinical trial with patients with in-

transit metastases.

Other chemotherapeutic agents used in HILP for melanoma have shown much lower

subjective response rates often with a higher toxicity. Cisplatin as one of the most

successful alternatives with a 50% to 60% response rate showed a high frequency of

peripheral neuropathy.21-23 The most successful systemic treatment agent for melanoma

is DTIC but used in regional perfusion this agent leads to a complete response rate of

11% and a partial response of only 26%.24

Although HILP was most frequently used in the treatment of extremity melanoma,

the procedure was also applied to soft tissue sarcomas (STS) of the extremity.

Krementz described their initial results in 113 patients. Fifty-four patients treated

with HILP without surgical excision of the tumor showed an early response rate of

83%, however only four patients had a complete regression of the tumor.25 Several

studies were published on the treatment of STS with HILP and melphalan, these

studies also have the problem of being heterogeneous as to the type of STS, disease

stage and therapy performed, making comparison difficult. The local recurrence rates

range from 0% to 25% with a 5-year survival rate of 56% to 69%.26-31 Other perfusion

agents have been investigated in the treatment of STS with HILP. Klaase et al.

described the use of doxorubicin as the sole perfusion agent but this was ineffective.

The complete remissions observed in four patients occurred after perfusion with

doxorubicin combined with melphalan. Local toxicity was high, and tissue necrosis

necessitated amputation in three cases.32 However in a study of Rossi et al, tumor

necrosis was more than 50% in 17 patients (74%) and limb-sparing surgery was

feasible in 20 patients (91%). They concluded that HILP with doxorubicin is an

active and well-tolerated procedure within a multidisciplinary approach of the

treatment of limb sarcomas.33 Pommier and Di Filippo investigated cisplatin as a

perfusion agent in the treatment of STS. 34,35 Seventeen patients whose sarcomas

were measured prior to HILP, none of the patients showed a complete response,

three had a partial response (18%), five had a minimal response (29%), seven had no

change (41%), and two had progression (12%).34 In conclusion, results with HILP

for STS were not impressive and alternative strategies for limb preservation by intra-

venous and intra-arterial adriamycin with preoperative or postoperative radiation

therapy followed by compartmental excisions, were able to provide adequate local

control for most extremity STS.36-39

13

Introduction

Introducing Tumor Necrosis Factor

William Coley, a surgeon who lived and worked in New York City during the second

half of the 19th century, was the first to investigate the phenomenon of tumor necrosis,

occurring in patients suffering from severe infections. By administering preparations

of gram-positive and gram-negative bacteria or their products to patients with

inoperable neoplastic diseases, Coley hoped to bring about an involution of the tumor.

The side effects of Coley’s regimen were unacceptable, however, and his treatment

ultimately fell into disrepute.40,41 Shear and co-workers, seeking to isolate an active

therapeutic fraction from Coley’s toxins, purified what they called the “bacterial

polysaccharide” from Serratia marcescens organisms.42-44 This molecule, now known

as lipopolysaccharide (LPS), was shown to induce hemorrhagic necrosis of

transplantable tumors in mice.45 A major conceptual advance occurred with the work

of O’Malley, et al., who reported that an endogenous factor appeared in the serum of

animals treated with LPS, which could induce hemorrhagic necrosis of tumors grown

in animals that had not been exposed to LPS. This information, though published in

a prominent journal, was largely overlooked for over 20 years.46 The transferability

of tumor-necrotizing activity from one animal to another was then identified by Old

and co-workers, who showed that a factor produced in mice pretreated with Bacillus

Calmette-Guérin (BCG) and subsequently challenged with LPS was capable of

causing hemorrhagic necrosis of the meth A sarcoma, grown in the skin of a recipient

animal.47 The factor was dubbed “tumor necrosis factor” (TNF). A large number of

studies reveal that TNF is produced principally by macrophages.48-51 A long period of

time elapsed between the identification of TNF and its isolation in pure form. TNF

from a human source was first isolated by Aggarwal and colleagues at Genentec.52

The molecular cloning of the TNF DNA was accomplished almost simultaneously

by a number of workers at separate biotechnology firms and the cloning of the human

TNF locus followed soon afterwards.53-56

A lot of articles published both in scientific literature and in popular press claimed,

that this molecule would prove to be a revolutionary tool in the battle against cancer.

However, phase I and II clinical trials of systemic TNF were very disappointing. An

overall response rate of 1-2% was seen in almost 1000 patients treated with systemic

TNF.57-60 The dose-limiting toxicity of TNF was typical hypotension, clearly

delineating the central role of this cytokine as a mediator of the pathophysiology of

septic shock.61-64 This dose-limiting toxicity in patients kept the peak intravascular

level achievable in humans 100-fold lower than the level needed for an anti-tumor

effect in a mouse model.65,66

Because it seemed impossible to achieve effective systemic concentrations of TNF

in patients, and because it appeared to act very rapidly with a short, single treatment

14

Chapter 1

in animal models, TNF was ideally suited for use in HILP. Ferdy Lejeune and Danielle

Lienard, surgical oncologists working in Brussels at the time, were the first to link

high-dose TNF and HILP to treat 19 patients with cutaneous melanoma and 4 patients

with STS in the early 1990s.67 In this setting, the equivalent intravascular levels that

led to responses in mice (1-3 µg/ml) could be achieved in the perfusion circuit.68 In a

pilot study of 3 patients with TNF as the sole perfusion agent, one complete response

of 7 months, one partial response of 21 days, and one minor response lasting for

1 month were observed. Posner described these 3 patients and another 3, treated with

HILP and TNF as the sole perfusion agent. One patient had a complete response,

2 patients had a partial response of less than 1 month’s duration and no response was

seen in 3 patients. HILP with TNF as the sole perfusion agent showed inadequate

activity. Three of these 6 patients had been reperfused with TNF and melphalan

resulting in 2 complete responses and 1 partial response.69 In vitro and vivo studies

had already shown an enhanced cytotoxic activity of TNF when chemotherapeutic

drugs, especially alkylating agents were added.70,71 The treatment regimen conceived

by Lejeune was a combination of preoperative subcutaneous interferon-gamma (IFN)

and perfusion with low-dose IFN, high-dose TNF and melphalan for a 90-minute

treatment period. The IFN was added to the regimen because it synergized with TNF

in pre-clinical studies.72,73 In all 23 cases, an early and spectacular softening of the

tumors was seen within the first 3 days after treatment, consistent with the TNF

effect seen in the murine models. Sixteen of 19 patients with melanoma (84%) and 3

out of 4 patients with a STS (75%) showed a complete response. Three melanoma

(16%) and 1 STS (25%) showed a partial response.67,74

Based on the initial study, two prospective randomized trials were initiated. In Europe,

Lejeune and colleagues started a prospective randomized phase II study of patients

with advanced melanoma of the limbs with in-transit metastasis. They compared 32

patients who received melphalan plus TNF and IFN to 32 patients who received

melphalan plus TNF only. The overall response rate and the complete response rate

were higher for the patients treated with IFN compared to the ones treated with

melphalan TNF only, 100% vs. 91% and 78% vs. 69% respectively, but the differences

were not significant.75 In the United States a trial comparing melphalan alone to the

identical dose of melphalan combined with TNF and IFN was initiated by Fraker in

patients with in-transit melanoma of the extremity with no known disease outside

the extremity. At an interim analysis of this study the complete response rate for

melphalan, TNF and IFN perfusion arm was 80% and 61% for the melphalan alone

perfusion arm. In a subgroup of patients with a high tumor burden of the extremity,

the melphalan, TNF and IFN perfusion arm had a much more dramatic effect (67%

complete responses) than what could be achieved by melphalan alone (17% complete

15

Introduction

responses). Patients with low tumor burden or small tumors showed equivalent results

with both of these two perfusion regimens, 87% complete responses with TNF versus

81% with melphalan only.76 The complete response rate seen with melphalan alone

in this study is somewhat better than that reported by other investigators and in order

to draw conclusions about the value of TNF as an adjunct to HILP in melanoma

patients, more patients need to be included.

When the benefit of TNF with melphalan in HILP for bulky melanoma was observed,

the same regimen was applied to STS.67 The results were much more positive in this

combination compared to melphalan alone, and several series have been published

demonstrating limb preservation in patients deemed to have unresectable tumors

with amputation as the only surgical option.77-79 The overall approach with large

extremity sarcomas that have no local resection options because of their relationship

to neurovascular and bony structures, is to conduct HILP with TNF and melphalan.

This treatment results in significant tumor shrinkage in 6 to 12 weeks. A second

procedure is performed after this period to resect the remaining tumor that is often

reduced in size. Patients with multifocal sarcoma do not undergo the secondary

resection, similar to those patients suffering from in-transit melanoma. The European

trial of 186 patients showed complete responses in 18% and partial responses in 57%

of the cases measuring tumor size.77 HILP with TNF and melphalan was also feasible

in patients with locally advanced extremity STS with disseminated disease as local

control improved the quality of life.80 These studies on bulky extremity sarcomas

demonstrated that TNF acts by attacking the tumor vasculature with rapid elimination

of tumor blood flow within days after treatment.81 Other more unusual tumors of the

extremity such as Merkel cell carcinoma, which often spreads by in-transit metastases

within the limb, as well as eccrine adenocarcinoma and basal and squamous cell skin

carcinoma have been reported to respond to HILP with melphalan plus TNF.82 Again,

because this treatment acts via an apparent antiangiogenic mechanism, it may be

applicable against all solid malignancies, with the tumor endothelium as the target

tissue, which is similar across several histologies.

Toxicity of HILP

Toxicity of HILP can be categorized as a side effect from systemic exposure to the

drugs and as a side effect due to the regional effects of high-dose exposure. The

systemic exposure depends not only on the adequacy of the isolation during HILP,

but is also caused by systemic exposure to the perfused drug during reperfusion.

Although the limb is flushed after perfusion, residual active agents still remain in the

limb either within the intravascular space or in the interstitial fluid, which results in

a systemic peak of drug concentration following the re-establishment of normal

16

Chapter 1

vascular flow to the extremity. Systemic leakage of melphalan has been described

and consisted of nausea and vomiting (22%), bone marrow depression in 4% and

miscellaneous systemic side-effects, including fever and minimal scalp hair loss,

occurring in 19 patients (5%).83 With the introduction of high-dose TNF at levels 10

times the maximum tolerated systemic intravenous bolus, isolation was all the more

important, but it introduced also another path to systemic toxicity namely the induction

of secondary host mediators during HILP that are subsequently released into the

systemic circulation after the perfusion. For standard chemotherapeutics, there is

little or no induction of host mediators.84 The systemic effects of TNF HILP reflect

the reported toxicity present in phase 1 systemic TNF trials. The most serious

complication is hypotension. In the first report by Lienard, 23% (7/31) of the patients

treated experienced hypotension, and 10% (3/31) showed severe hypotension.74 All

patients in this initial trial received dopamine (3 mg/kg/min) at the time of TNF

injection into the perfusate as a prophylaxis against hypotension. The most significant

toxicity of TNF limb perfusions can be summarized as a so called Systemic

Inflammatory Response Syndrome (SIRS). This was observed in all patients and

was accompanied by fever, rise in cardiac output, fall in systemic vascular resistance

and the need for fluid resuscitation and inotropes. Perfusion with melphalan as the

sole perfusion agent did not trigger these effects. Detailed analysis showed positive

correlations between maximum TNF concentrations and systemic vascular resistance

and cardiac index.85 The National Cancer Institute perfusion group demonstrated the

relation between the vascular response and the need for vasopressor support and

systemic TNF levels in patients with TNF leakage as well.86 Lejeune also demonstrated

severe toxicity in patients with leaks of >5%.67,68 Vrouwenraets et al. reported an

absence of severe systemic toxicity of TNF in patients without systemic leakage.87

Stam et al. observed only a mild postoperative toxicity in the event of significant

leakage during perfusion.88 This was easily managed on the ICU with fluid substitution

and, in some cases, with vasopressors. All these systemical side effects of TNF HILP

were minimal, transient, and could easily be managed with appropriate resuscitative

techniques.89,90

The normal tissues in the limb that are perfused such as skin, muscle, peripheral

nerves, blood vessels, bone, cartilage, and synovium comprising the skeletal system,

are also exposed to the same concentrations of anti-neoplastic agents active against

the tumor. Wieberdink developed a grading system to score these regional toxicities.11

The toxicities seen with melphalan are skin erythema, some with areas of blistering

and subcutaneous edema, in virtually all patients.91,92 The skin changes as well as this

edema universally returns to normal after several months. The most important

toxicities are the effects on muscle and peripheral nerves. Myopathy can occur with

17

Introduction

mild muscle discomfort and in the worst case may cause a compartment syndrome

with potential muscle necrosis and subsequent limb loss. This is the main reason

why a prophylactic fasciotomy is performed after HILP at the University Hospital in

Groningen.93 Long term analysis of limb function after fasciotomy showed no impaired

function of the perfused limb compared to the contralateral none perfused limb. 94This

was in contrast with other reports claiming approximately 5% to 10% of the patients

have significant long-term discomfort in their extremity after HILP, a difference that

can be possibly explained by the prophylactic fasciotomy. Initial reports from Lienard

et al. indicate that TNF and IFN add little to the regional toxicity of limb perfusions

compared to melphalan alone. Skin erythema and desquamation, edema, joint stiffness,

and peripheral neuropathy appear to occur in the same number of patients as after

melphalan alone perfusions.

Positron Emission Tomography

Positron Emission Tomography (PET) is a non invasive, diagnostic imaging technique

for measuring the metabolic activity of cells in the human body with the aid of short-

lived positron emitting radiopharmaceuticals. Traditional diagnostic techniques, such

as x-rays, CT scans or MRI, produce images of the body’s anatomy or structure.

The first step in a PET-study is to label a selected compound with a positron emitting

radionuclide. Starting from non-radioactive atoms, a cyclotron is used to produce

radionuclides. In a cyclotron, particles such as protons or deuterons (hydrogen and

deuterium atoms without their orbital electrons) are brought to high energies by

traversing several hundred orbits within the cyclotron. When the protons or deuterons

orbits near the maximum radius of the cyclotron, they are removed through

electrostatic or magnetic deflection and are impinged upon small volume hollow

metallic cylinders filled with a nonradioactive gas or liquid. Nuclear reactions take

place within the cylinder (target) between the high energy particle (proton or deuteron)

and the contents of the target. With different target materials, different radioactive

products can be obtained. These are then separated from the target material and can

be used in the synthesis of more complex radiopharmaceuticals. The most frequently

applied radionuclides in PET are carbon-11 (11C, half-life 20 minutes), nitrogen-13

(13N half-life 10 minutes), oxygen-15 (15O half-life 2 minutes) and fluorine-18 (18F

half-life 110 minutes).

The production of the radiopharmaceutical is performed with the use of automated

synthesis systems. These are located within lead-walled (5-6 cm thick) cabinets so

called “hot cells”. The precise composition of the radiopharmaceutical is assured by

testing the products with e.g. high pressure liquid chromatography before

administrating them to the patient. Sterility and pyrogen testing are performed on

18

Chapter 1

every dose afterwards.

The radionuclides now incorporated within the radiopharmaceutical, have a surplus

of positive nuclear particles. Because this is an unstable situation, these radionuclides

either capture an electron or emit a positron (which is a particle with the same weight

as an electron, but with a positive charge) to achieve stability, depending on the

energy of the nucleus. After a positron is emitted, it is rapidly slowed down by

interactions within the surrounding tissue until all its kinetic energy (velocity) is

lost. At this point, the positron combines momentarily with an electron. The

combination of particles (positron and electron) then totally annihilates or disintegrates

and results in two diametrically (1800 apart) photons of exactly 511 keV energy. The

pairs of photons are emitted equally from the body in all directions. In general, several

million events (photon pairs) are accumulated for each PET image.

The next step in PET is to detect the emitted photons with the PET camera. The PET

camera used for this study at the University of Groningen contains 8192 crystals

oriented into 16 rings arranged in two rings of 64 detector blocks each 512 detectors

per ring. The 16 rings are used to collect 16 planes (slices) of data and an additional

15 cross-planes (slices) are obtained by collecting photon interactions between

adjacent direct planes for a total of 31 planes. The scanner has a 10.4 cm axial field

of view. Patients are positioned comfortably on a table which moves through the

opening of the scanner. Some patients require only one field of view (10 cm) to

visualize a particular area of the body while others are moved through the scanner

using 9-10 bed positions (90-100 cm) to complete whole body imaging. PET cameras

make use of the fact that the two annihilation quanta have opposite directions. Emitted

photons can be absorbed by the detectors in the camera. Each detector has connections

with many opposite detectors. A signal is said to be caused by annihilation if the

capture of a photon by two opposite detectors coincides within 20 nsec. Simultaneous

detection of two of these photons by detectors on opposite sides of an object places

the site of the annihilation or on about a line connecting the centers of the two detectors.

At this point mapping the distribution of annihilations in the field of view by a

computer is possible and an image can be reconstructed. If the annihilation originates

outside the volume between the two detectors, only one of the photons can be detected,

and since the detection of a single photon does not satisfy the coincidence condition,

the event is rejected. The image achieved is generally presented as a gray scale image

of a cross-section of the patient, with the intensity of each picture element proportional

to the isotope concentration at that point in the patient.

Fluorine-18 labeled 2-fluoro-2-deoxy-D-glucose (FDG) is one of the most widely

used radiopharmaceuticals used in PET and has proven to be of value in the

visualization of various types of tumors.95,96 The use of FDG is based on Warburg´s

19

Introduction

observation of increased glycolysis in cancer cells. The citric acid cycle, which is

more efficient in adenosine triphosphate generation, is suppressed.97 As a result, cancer

cells accumulate the glucose analog FDG which is trapped intracellularly as FDG

phosphate. The FDG consumption, and since FDG acts in the same way as glucose,

the glucose consumption can be determined with the use of a three-compartment

model: plasma-FDG, tissue-FDG and tissue-FDG-6-phosphate, as described by

Sokoloff.98 The tissue components can be measured by the PET camera and the plasma

components can be measured by counting the activity in blood samples. With the

compartment model, the glucose consumption can be calculated in µmol per 100

grams of tissue per minute.

The majority of the PET studies with amino acid tracers have been performed with

L-[methyl-11C]-methionine (MET). 99-101 MET reflects amino acid uptake rather than

protein synthesis and because it is involved in other metabolic pathways such as

transmethylation and polyamine synthesis, this may lead to accumulation of a variety

of nonprotein metabolites in tumor tissue.102-104 This complicated metabolism of

methionine has made it impossible to create a precise metabolic model. Carboxyl-

labeled amino acids, such as L-[1-11C]-tyrosine (TYR), L-[1-11C]-methionine and L-

[1-11C]-leucine, appear to be more appropriate compounds to determine protein

synthesis in tumors.103,105 The main metabolite of these amino acids is 11CO2, which

is rapidly cleared from tissue and exhaled and does not contribute to the PET-measured11C radioactivity in tumor tissue. Using a method developed at the PET Center

Groningen, the protein synthesis rate can be determined using 11C labeled L-amino

acids with a four-compartment model: plasma-amino-acid, tissue-nonprotein-amino-

acid, metabolites and protein-incorporated-amino-acid.106

The aim of this thesis

Hyperthermic isolated limb perfusion is a major surgical procedure and over the

years new developments have been initiated and examined. Traditionally the

University Hospital Groningen plays an important role in the history of regional

perfusion and therefore this thesis describes different aspects of regional perfusion

during the last decade.

1. What are the short term effects of HILP with cisplatin in the local

treatment of spontaneous osteosarcoma in dogs?

2. Is HILP with TNF and cisplatin feasible in the canine model?

3. What are the results of HILP with cisplatin in patients with sarcomas of

soft tissue and bone?

20

Chapter 1

4. What is the relation between the tumor vascularization and the vascular

changes after irradiation therapy?

5. How does HILP influence the glucose metabolism and protein metabolism

as studied by PET, and is it possible to predict the outcome of therapy?

6. Is it worthwhile to monitor continuous leakage with RISA during HILP

with TNF and melphalan?

21

Introduction

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26

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29

Robert J. van Ginkel1

Harald J. Hoekstra1

Freek J. Meutstege2

Jan W. Oosterhuis3

Donald R.A. Uges4

Heimen Schraffordt Koops1

Departments of Surgical Oncology1 and Pharmacy4, University Hospital Groningen,

Department of Veterinary Medicine2, State University Utrecht, and

Dr. Daniel den Hoed Cancer Center3, Rotterdam, The Netherlands.

Journal of Surgical Oncology 1995; 59: 169-176.

Hyperthermic isolated limb perfusion with cisplatin

in the local treatment of spontaneous canine

osteosarcoma: Assessment of short-term effects

30

Chapter 2

Abstract

To increase the effect of cisplatin on locoregional osteosarcoma, the short term effect

of hyperthermic isolated limb perfusion (HILP) with cisplatin (30 mg/L extremity

volume) was studied in 28 dogs with spontaneous osteogenic sarcoma using clinical,

radiological, and histological parameters. Thirty days postoperative mortality was

14.3 %. Total platinum levels at the start of perfusion were 28.2 ± 14.3 mg/L. A

significant improvement (p<0.001) in the clinical score was observed in the overall

group at 6 and 12 weeks after perfusion. The radiological parameter showed a

stationary X-ray 2 weeks after perfusion and an improved X-ray 6 weeks after

perfusion. Overall histological scores showed a moderate effect according to the

Huvos classification. No additional therapeutic effect, according to the three

parameters, could be demonstrated by increasing the perfusate temperature by 1°C.

HILP with cisplatin is feasible in the local treatment of spontaneous osteosarcoma

in dogs with acceptable locoregional toxicity. However the histological results were

modest, with none of the dogs showing a complete response 6 weeks after perfusion.

Therefore, the search for the ideal perfusion agent with substantial contribution to

the limb-sparing treatment in human osteosarcoma, continues.

Introduction

Osteosarcoma is the most frequent primary malignant bone tumor in humans. Until

the early 1970s, the most common approach to the management of localized

osteosarcoma was surgical resection, amputation or radiation. In most large series of

patients treated in this manner, long term survival was only 20%.1,2 During the past

few decades, the use and further development of systemic neoadjuvant chemotherapy,

e.g., including high-dose methotrexate (HD-MTX) and cisplatin, appears to have a

definite influence on the disease free and overall survival for patients with

osteosarcoma.3-5 The effect of the systemic neoadjuvant chemotherapy on the primary

bone tumor, the improved surgical resection technique, and the development of

prosthetic replacement techniques also improved the limb salvage rate for

osteosarcomas, especially for the lower extremity. Salvage rates varying from 40 %

to almost 80 % are reported.6

However, the potential local tumor effect of the systemic neoadjuvant chemotherapy

is not always favorable, although a good response of the local tumor to systemic

chemotherapy demonstrated prognostic value.7 Increasing the systemic chemotherapy

dose to achieve a higher local tumor response is limited due to the nephrotoxicity

and ototoxicity of cisplatin. To avoid systemic toxicity but to raise the effect on the

local tumor and thereby facilitate limb preserving procedures, a local treatment of

the primary tumor could be the solution.

31

Cisplatin perfusion for canine osteosarcoma

With hyperthermic isolated limb perfusion (HILP) as a local treatment modality, it is

possible to obtain very high local drug concentrations in a limb with minimal systemic

toxicity.8 The value of cisplatin in HILP has also been demonstrated in humans for

melanoma and various soft tissue sarcomas.9-11 Fletcher and associates showed that

250 mg/m2 was the maximum tolerable dose of cisplatin for lower-extremity

perfusions, with improved local control rates for sarcomas and melanoma of the

extremities without regional nodal metastases.12 Before introducing HILP with

cisplatin in the clinical treatment of osteosarcoma of the limb, the short term effect

of this treatment modality on the primary tumor was investigated by clinical,

radiological, and histological parameters in dogs with spontaneous osteogenic sarcoma

of the limb. Biological behavior of osteosarcoma is similar both in human and in

dogs; a locally aggressive bone tumor predominantly occurring in the long bones

with early hematogenous metastases to the lungs.13,14 The differences between canine

and human osteosarcoma are that in humans a younger age group (adolescence) is

most commonly affected, and the tumor is less common.With the high frequency of

occurrence in dogs, allows canine osteosarcoma is a useful model for evaluation of

new treatment regimens in humans as rapid case accrual and rapid time to reach

measurable end points are possible. The canine osteosarcoma therefore appears to be

a valid model for studying the potential treatment of HILP with cisplatin in the local

treatment of osteosarcomas of the extremity in humans.

Materials and methods

Dogs

Twenty-eight dogs with an average weight of 45 ± 10.0 kg and a mean age of 7 ± 2.5

years with spontaneous, histologically proven, previously untreated, primary

osteosarcoma of the extremity, without radiographic evidence of distant metastases,

underwent HILP with cisplatin. Preoperatively, all dogs were thoroughly clinically

evaluated at the Department of Veterinary Medicine and underwent a complete blood

count (CDC), serum chemistry profile, and X-rays of the primary tumor and thorax.

The perfusion procedure was performed at the Central Animal Facility of the State

University Groningen, while follow-up was performed at the Department of Veterinary

Medicine Utrecht. The study was approved by the Animal Welfare Committee of the

Faculty of Medicine of the State University Groningen.

Anesthetics

All dogs were premedicated with atropine sulfate (0.5 mg, i.m.) and piritramide (15-

17.5 mg, i.m.)(Dipidolor, Janssen Pharmaceutica, Tilburg, The Netherlands). The

dogs were anesthetized with thiopenthal (30 mg/kg BW, i.v.)(Pentothal, Abbott,

32

Chapter 2

Amstelveen, The Netherlands) and after muscle relaxation with pancuroniumbromide

(0.08mg/kg BW, i.v.)(Pavulon, Organon, Oss, The Netherlands), the dogs were

ventilated by means of a Siemens Servo Ventilator 900B, with a mixture of

nitrousoxide and oxygen. The oxygen concentration in the gas mixture, continuously

measured by means of an oxygen analyzer (Taylor Servomex OA 272), and minute

volume (4-6 L/min), were adjusted to maintain an end-expiratory CO2 concentration

of 4-5% (Siemens CO2-analyser 930). The dogs were placed in the supine position

on a heated mattress to maintain their normal body temperature of 38 °C.15 During

the operations, all dogs were given about 500 ml of Isodex through the cephalic vein.

Operation and perfusion techniques

The iliac or axillary vessels of the affected limb were exposed under sterile conditions

and collateral vessels were clipped. Cannulas were inserted into the artery (Bardic,

16F-18F) and vein (Portex, 6-8 mm). Both cannulas were connected to an

extracorporeal circuit consisting of an occlusive roller pump, a cardiotomy reservoir

and a bubble oxygenator with heat-exchanger. A canvas tourniquet was placed around

the base of the extremity to complete isolation of the limb from the systemic

circulation. The perfusate consisted of 350 ml of 5% dextran 40 in glucose 5% (Isodex,

Pharmacia AB, Uppsala, Sweden), 250 ml red blood cells (typed canine blood donors),

250 ml plasma, 30 ml sodium bicarbonate 8.4%, and 0.5 ml 5000 IU/ml heparin

(Thromboliquine, Organon BV, Oss, the Netherlands). A mixture of oxygen, air, and

carbon dioxide through the oxygenator was adjusted to maintain the blood gas values

within the physiological range and, when necessary, bicarbonate was added to adjust

the pH.

All perfusions were performed under hyperthermic conditions. To study the effects

of additional heat to the perfusate, two groups of dogs were randomized. In group I

(14 dogs), HILP of the extremity was performed at 39-40°C limb temperature; and

for group II (14 dogs), HILP was performed at 40-41 °C limb temperature. The arterial

line temperature was kept at 40-41 °C in group I and at 41-42 °C in group II. In

addition a 1000 Watt infrared lamp was placed at a distance of 90 cm to heat the

extremity. Thermistor probes (Electrolaboriet, Copenhagen, Denmark) were inserted

into the subcutaneous tissue and into a muscle of the proximal limb just above the

knee for continuous monitoring of the temperatures during perfusion. The perfusion

time was 1 hour and the perfusion was followed by washout of the extremity with

500 ml of Isodex. Tourniquet, cannulas and clips were then removed and the incisions

in the vessels were repaired. Protamine hydrochloride (Hoffman La Roche, Mijdrecht,

The Netherlands) was administered to neutralize heparin, in a ratio of 1:1 to the

initial dose of heparin. All dogs were observed for one night and allowed to go home

33


with the owner the next day. No anti-inflammatory medications or analgesics were

administered during follow up.

The dosage of cisplatin (Platinol 0.5 mg/ml�, Bristol Myers SAE, Barcelona, Spain)

used in the perfusion had been established in a previous study which showed massive

edema with necrosis and fibrosis with cellular infiltrates in skeletal muscle throughout

the perfused extremity in dogs that received 45 mg cisplatin per liter extremity

volume.16 These local side effects were not seen after a 30% dose reduction to 30

mg/L extremity volume, as used in the present study. The volume of the extremity

was determined by submersion in water to the tourniquet border and measurement of

displaced water volume. Extremity volumes varied from 1.7 ± 0.27 L. Cisplatin was

added to the circulated perfusate in 10 minutes. During perfusion, serum platinum

levels were determined in the regional and systemic circulation at 0, 10, 20, 30, 40,

50, and 60 minutes by flameless atomic absorption spectrophotometry (FAAS).

Local effect parameters

Short-term effects on the tumor were determined using three parameters: clinical,

radiological, and histological. The clinical score was determined by a veterinarian

on the basis of gait analysis: walking on three legs (score I), severe limp (score II),

slight limp (score III), walking normally (score IV). This score was determined 1

week before and at 2, 6 and 12 weeks after perfusion. The radiological score was

determined by a veterinarian radiologist on conventional X-rays in two directions of

the extremity according to methods described earlier.17,18 Preperfusion X-rays were

compared with 2 and 6 week postperfusion X-rays: progression (score I), stationary

(score II), regression (score III), the latter defined as a decrease in tumor volume,

increased ossification of intraosseous tumor osteoid, periosteal new bone and soft

tissue margin more densely ossified and well-defined resulting in a more benign

appearance. Biopsies from the tumor were taken before, and 2 and 6 weeks after

perfusion at random in three directions with a 3.5 mm diameter Coombs bone biopsy

system.19 The histological score was determined by a pathologist on the material

obtained from the biopsies, according to the criteria of Huvos et al.20 no reaction

(score I), moderate effect (score II), good effect (score III), total necrosis (score IV).

The radiological as well as the histological scores describe the response to treatment;

therefore, it was not possible to score these parameters 1 week before treatment, as it

was with the clinical score. All dogs were followed for local and systemic side effects

by the cisplatin perfusion.

34

Chapter 2

Statistical Considerations

Mean clinical, radiological and histological scores of the total group were analyzed

with the Pittman test. Differences between group I and II were analyzed with the

Yates & Cockran test. The survival curve was calculated according to the Kaplan

Meier method.21 P-values <0.05 were considered significant.

Results

Although the dogs underwent a thorough clinical work-up before treatment, the

investigators were confronted with a 30 days postoperative mortality of 14.3%

(4 dogs). The first dog, 10 years of age, died at the end of the perfusion from cardiac

failure. The other two dogs, both 6 years of age, died postoperatively due to pulmonary

and cardiac failure. Postmortem examination of those two dogs was not obtained

from their owners. A fourth dog died 1 week after the perfusion from a large myocardial

infarction. Postmortem examination of this animal showed a completely necrotic

tumor.

No systemic or normal tissue side effects of the perfusion were encountered. The

local reaction of the limb to the perfusion consisted of an initial slight edema that

reached a maximum on the third postoperative day and disappeared completely within

the first week. Total platinum levels in the perfusate ranged from 28.2 ± 14.3 mg/L at

the start of perfusion to 12.1 ± 5.3 mg/L at the end of a 1-hour perfusion in the total

group. There was no significant difference in platinum levels during perfusion between

group I and II. Systemic platinum levels never rose above 0.7 mg/L in both groups

(Fig. 1).

Fig. 1 Concentration of total platinum

(tPt) measured in the perfusate during

cisplatin perfusion. Values are the

mean of all dogs; error bars are ± SEM.

Time 0 is the time of administration of

30 mg cisplatin per liter extremity vol-

ume.

35


Ta

ble

1D

istribu

tion

of clin

ical param

eters at 1 w

eek b

efore, an

d at 2

, 6 an

d 1

2 w

eeks after p

erfusio

n

Clin

ical

Gro

up

I an

d II

Gro

up

IG

rou

p II

Tim

e-1

we

ek

2 w

eeks

6 w

eeks

12 w

eek

-1 w

eek

2 w

eeks

6 w

eeks

12 w

eeks

-1 w

eek

2 w

ee

ks

6 w

ee

ks

12 w

eeks

Nu

mb

er o

f do

gs

N=

28

N=

24

N=

24

N=

17

N=

14

N=

11

N=

11

N=

9N

=14

N=

13

N=

13

N=

8

I Th

ree

leg

s 6

(21%

) 5

(21%

) 3

(13%

)2 (1

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)3 (2

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%)

_3

(21%

)5 (3

8%

)2 (1

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(25%

)

II Se

ve

re lim

p1

5 (5

4%

) 6

(25%

) 3

(13%

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)8 (5

8%

)5 (4

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_7 (5

0%

)1 (8

%)

3 (2

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5%

)

III Slig

ht lim

p 6

(21%

)11

(46%

) 8

(33%

)4 (2

4%

)3 (2

1%

)6 (5

5%

)6 (5

5%

)3 (3

3%

)3

(21%

)5 (3

8%

)2 (1

5%

)1

(13%

)

IV W

alk

s n

orm

ally

1 (4

%)

2 (8

%)

10 (4

1%

)9 (5

2%

)_

_4 (3

5%

)6 (6

7%

)1 (8

%)

2 (1

6%

)6 (4

6%

)3

(47%

)

36

Chapter 2

The clinical scores in the total group before and after treatment could be compared.

Before perfusion: 6 dogs (21%) walked on three legs; 15 dogs (54%) walked with a

severe limp; 6 dogs (21%) walked with a slight limp; and 2 dogs (4%) walked

normally. Two weeks after perfusion: 5 dogs (21%) walked on three legs; 6 dogs

(25%) walked with a severe limp; 11 dogs (46%) walked with a slight limp; and 2

dogs (8%) walked normally (Table 1).At 6 and 12 weeks after HILP therapy, the

improvement of walking with a severe limp towards a normal walking pattern

continued.

Radiological scores for the total group 2 weeks after perfusion: progression in 3

dogs (12%); stationary in 13 dogs (52%); and an improved X-ray was found in 9

dogs (36%). Radiological scores 6 weeks after perfusion: progression in 6 dogs (25%);

stationary in 3 dogs (12%) and an improved X-ray was found in 15 dogs (63%)

(Table 2). These scores illustrate a change from a stationary X-ray, 2 weeks after

perfusion toward an improved X-ray 6 weeks after perfusion.

The histological effect of cisplatin on the tumor was classified according to Huvos et

al.20 Biopsy scores for the total group two weeks after perfusion showed: no reaction,

Huvos I in 5 dogs (20%); moderate effect, Huvos II in 8 dogs (32%); good effect,

Huvos III in 7 dogs (28%); total necrosis, Huvos IV in 5 dogs (20%). Six weeks after

perfusion, biopsy scores were as follows: no reaction, Huvos I in 3 dogs (14%);

moderate effect, Huvos II in 12 dogs (57%); good effect, Huvos III in 6 dogs (29%);

total necrosis, Huvos IV in none of the dogs (Table 3). At 2 and at 6 weeks after

perfusion, the overall histological score is one of moderate effect according to Huvos

et al.20

After summation of the individual scores, there was a significant improvement

(p<0.001) in the clinical score in the total group 6 and 12 weeks after perfusion;

respectively; 2.04 before perfusion to 3.04 6 weeks and 3.18 at 12 weeks after

perfusion (Table 4). Radiological and histological scores only classify the response

to treatment; therefore, mean radiological and histological scores before and after

perfusion could not be compared. However, a comparison of the radiological and

histological scores between 2-6 weeks could be made. There was no significant

improvement or deterioration in radiological and histological mean scores between

2-6 weeks in the total group. Analysis of the distribution and the mean scores of all

three parameters demonstrate that additional hyperthermia of 1°C (group I versus

group II) did not improve the results of the measured parameters. Retrospective

analysis of survival time showed a median survival for all dogs of 115 days (Fig. 2).

Three dogs underwent a resection or amputation of the affected limb and survived

12, 24 and 43 months, respectively, after perfusion without evidence of disease.

37


Table 2 Distribution of radiological parameters at 2 and 6 weeks after perfusion

Radiological Group I and II Group I Group II

Time 2 weeks 6 weeks 2 weeks 6 weeks 2 weeks 6 weeks

Number of dogs N=25 N=24 N=12 * N=11 N=13 N=13

I progression 3 (12%) 6 (25%) 2 (18%) 3 (23%) 4 (31%)

II stationary 13 (52%) 3 (12%) 10 (83%) 1 (9%) 3 (23%) 2 (15%)

III improved 9 (36%) 15 (63%) 2 (17%) 8 (73%) 7 (54%) 7 (54%)

*The radiological score of the dog that died 1 week after perfusion from a myocardial infarction included

Table 3 Distribution of histological parameters at 2 and 6 weeks after perfusion

Histology Group I and II Group I Group II

Time 2 weeks 6 weeks 2 weeks 6 weeks 2 weeks 6 weeks

Number of dogs N=25 * N=21 N=12 * N=11 N=13 N=10

I no reaction 5 (20%) 3 (14%) 1 (8%) 1 (10%) 4 (30%) 2 (20%)

II moderate 8 (32%) 12 (57%) 2 (17%) 5 (45%) 6 (46%) 7 (70%)

III good 7 (28%) 6 (29%) 7 (58%) 5 (45%) 1 (10%)

IV necrosis 5 (20%) 2 (17%) 3 (24%)

*The histological score of the dog that died 1 week after perfusion from a myocardial infarction included

Table 4 Mean clinical, radiological and histological scores

Mean scores Clinical Radiological Histological

Time -1 wks 2 wks 6 wks 12 wks 2 wks 6 wks 2 wks 6 wks

Mean group I 1.93 2.55 3.18 3.67 * 2.17 2.55 2.83 2.36

Mean group II 2.14 2.31 2.92 2.63 2.31 2.23 2.15 1.90

Mean group I + II 2.04 2.42 3.04 ** 3.18 ** 2.24 2.38 2.48 2.14

*p < 0.05 group I versus group II; ** p < 0.001 compared with the pre-perfusion score

38

Chapter 2

Discussion

In the treatment of osteogenic sarcoma a distinction can be made between systemic

therapy and locoregional treatment. Systemic therapy is primarily concerned with

eradicating possible micrometastatic disease and its use was a major breakthrough in

the clinical treatment of osteosarcomas in the 1970s. About 60 % of patients with

resectable primary tumors and no metastases at diagnosis will be cured.

The primary objective in locoregional treatment is to prevent local recurrence and

allow limb salvage procedures in an attempt to preserve limb function. New surgical

techniques and the development of endoprosthetic materials, coupled with the systemic

neo adjuvant chemotherapy, have offered less radical surgery for 40 % - 80 % of

patients with osteosarcoma in the 1980s.6 Procedures that increase tumor necrosis of

the primary tumor, and with that reduction of viable tumor cells and tumor volume,

could contribute to limb preservation strategies. At first changes in systemic

chemotherapy regimens were investigated to achieve this goal. In 1978, cisplatin

exhibited activity in the treatment of osteogenic sarcoma.22,23 Since its first use,

cisplatin has been one of the most effective chemotherapeutic agents and has been

incorporated in most systemic treatment regimens for osteosarcoma. The potential

local tumor effect of systemically administered cisplatin, however, is limited due to

the nephrotoxicity and ototoxicity of cisplatin. Techniques that administer cisplatin

locally have been introduced to surmount these systemic side effect restrictions.

Powers et al. demonstrated the superiority of the intra-arterial administration of

cisplatin to the intravenous route in canine osteosarcoma.24 Jaffe et al. reported a 50

% response rate from intra-arterial infusion with cisplatin in osteosarcoma and

recommended it for use in inoperable tumors, to render them suitable for limb

salvage.25 On the other hand, Wile et al. demonstrated the superiority regional perfusion

with cisplatin to the intra-arterial or intravenous route in an experimental

pharmacokinetic study.26

Fig. 2 Survival of all dogs after

treatment with cisplatin hyperther-

mic isolated limb perfusion. Ex-

cluded are 3 dogs treated with

cisplatin perfusion, followed by

resection of the osteosarcoma

39


In a study of cisplatin pharmacokinetics during HILP in humans with recurrent

melanoma, cisplatin levels were 10-20 times higher than those found in systemic

treatment and about 5 times higher than those found in intra-arterial infusion.27 During

these perfusions, high total platinum concentrations in the limb were reached that

would be unacceptably toxic for systemic use. A substantial drug extraction occurred

with minimal leakage to the systemic circulation.

The aim of the present study was to investigate the short-term effect of HILP with

cisplatin in dogs with spontaneous osteosarcoma. All three parameters used to evaluate

the short-term effect showed a trend toward improvement. However, only the clinical

score reached statistical significance for the total group with 1.93 before perfusion,

2.42 (n.s.), 3.04 (p<0.001), and 3.18 (p<0.001) at 2, 6 and 12 weeks, respectively,

after perfusion. The mean radiological score was 2.24 and 2.38 at 2 and 6 weeks

after perfusion, i.e. 15 out of 24 (63%) dogs had an improved radiological picture 6

weeks after perfusion, compared with the picture before perfusion. The histological

results showed a moderate effect at 2 weeks (mean score 2.48) with a slightly lower

mean score at 6 weeks (2.14). This could be an indication that viable tumor cells are

growing out between 2 and 6 weeks after an initial favorable response to perfusion,

meaning that a limb salvage procedure should be planned 2 weeks after perfusion.

Surprisingly none of the dogs showed a complete histological response of the tumor

6 weeks after perfusion. Three dogs with total necrosis 2 weeks after perfusion, showed

viable tumor cells 6 weeks after perfusion. Sampling error could account for these

observations. Although high total platinum levels in the perfusate were reached, the

histological outcome were modest and comparable with those observed in the systemic

treatment of osteogenic sarcoma.

Although several authors report an enhanced toxicity of cisplatin combined with

hyperthermia,28,29 due to phenomena of enhanced blood flow, enhanced cellular drug

uptake, tissue extraction, DNA cross-linking and decreased DNA repair30-34 , no

additional therapeutic effect, according to the three parameters, could be demonstrated

by increasing the limb temperature by 1°C (group II). The mean clinical, radiological

and histological scores for both groups were comparable at 2 and 6 weeks. At 12

weeks however the second group deteriorated in clinical score significantly with

regard to the first group (p < 0.05). Elevated normal tissue damage, occurring at

higher perfusion temperatures could be a reasonable explanation for this observed

difference in clinical performance of the dogs.

Median survival time in our series for all dogs was 115 days, similar to survival

times in dogs that had amputation alone without any adjuvant chemotherapy.35 This

may not be a surprising, as it is estimated that 90 % of the dogs with osteosarcoma

already have micrometastatic disease predominantly in the lungs.36 An improvement

40

Chapter 2

of survival is only to be expected when the locoregional treatment is combined with

effective systemical therapy to eradicate micrometastatic disease. Mc Ewen et al.

improved the overall median survival time from 77 to 222 days (p<0.002) using

adjuvant treatment with liposome-encapsulated muramyl tripeptidephophatidyle-

thanolamine (liposome/MTP-PE) after amputation for osteosarcoma in dogs.36

Combining HILP for local treatment together with adjuvant liposome/MTP-PE as

the systemic component, may improve local control and increase disease free survival

in canine osteosarcoma.

The present study shows that a single HILP with cisplatin in dogs having extremity

osteosarcoma is feasible with acceptable locoregional toxicity, improved functional

outcome at 6 and 12 weeks and a steadily improving radiological picture. However,

the histological results were modest with none of the dogs showing a complete

response 6 weeks after perfusion. Results of recent publications and of our own

experience with a new perfusion modality, which combines tumor necrosis factor

(TNF), Interferon (IFN), and melphalan in patients with recurrent melanoma or soft

tissue sarcoma, are very promising.37,38 Since the endothelial cells are supposed to

play a key role in the working mechanism of TNF, osteosarcomas with a high extent

of tumor vessels, are of particular interest. Therefore the combination of TNF and

Interferon with cisplatin could theoretically induce more tumor necrosis in

osteosarcoma than could perfusion with cisplatin alone. A similar study designed to

investigate the additional effect of TNF with cisplatin in the treatment of canine

osteogenic sarcoma, is being initiated. If results are as good as they are in the treatment

of recurrent melanoma and soft tissue sarcoma a step forward could be made in the

locoregional treatment of osteosarcoma.

Conclusions

HILP with cisplatin is feasible in the local treatment of spontaneous osteosarcoma in

dogs with acceptable locoregional toxicity, improved functional outcome at 6 and 12

weeks, and a steadily improving radiologic picture. However the histological results

were modest with none of the dogs showing a complete response 6 weeks after

perfusion and no additional therapeutic effect, according to the three parameters,

could be demonstrated by increasing the perfusate temperature by 1°C. Therefore,

the search for the ideal perfusion agent with substantial contribution to the limb

sparing treatment in human osteosarcoma continues.

41


References

1 Mc Kenna RJ, Schwinn CD, Soong KY, Higinbotham NL. Sarcomata of the

osteogenic series: An analysis of 552 cases. J Bone Joint Surg 1966; 48A: 1-26.

2 Friedman MA, Carter SK. The therapy of osteogenic sarcoma: Current status and

thoughts for the future. J Surg Oncol 1972; 4: 482-510.

3 Rosen G, Tan C, Sanmaneechai A, et al. The rationale for multiple drug chemotherapy

in the treatment of osteogenic sarcoma. Cancer 1975; 35: 936-945.

4 Eilber F, Giuliano A, Eckardt J, et al. Adjuvant chemotherapy for osteosarcoma: A

randomized prospective trial. J Clin Oncol 1987; 5: 21-26.

5 Link MP, Goorin AM, Miser AW, et al. The effect of adjuvant chemotherapy on

relapse free survival in patient with osteosarcoma of an extremity. N Eng J Med

1986; 314: 1600-1606.

6 Meyer WH, Malawer MM. Osteosarcoma. Clinical features and evolving surgical

and chemotherapeutic strategies. Pediatr Clin North Am 1991; 38: 317-348.

7 Winkler K, Beron G, Delling G, et al. Neoadjuvant chemotherapy of osteosarcoma:

results of a randomized trial (COSS-82) with salvage chemotherapy based on

histological tumor response. J Clin Oncol 1988; 6: 329-337.

8 Hoekstra HJ, Naujocks T, Schraffordt Koops H, et al. Continuous leakage monitoring

during hyperthermic isolated regional perfusion of the lower limb: techniques and

results. Reg Cancer Treat 1992; 4: 301-304.

9 Klein ES, Ben-Ari GY. Isolation perfusion with cisplatin for malignant melanoma

of the limbs. Cancer 1987; 59: 1068-1071.

10 Roseman JM, Tench D, Bryant LR. The safe use of cisplatin in hyperthermic isolated

limb perfusion systems. Cancer 1985; 56: 742-744.




72: 1224-1229.

13 Brodey RS. The use of naturally occurring cancer in domestic animals for research

into human cancer: General considerations and a review of canine skeletal

osteosarcoma. Yale J Biol Med 1979; 52: 345-361.

14 Wolke RE, Nielsen SE. Site incidence of canine osteosarcoma. J Small Anim Pract

1966; 7: 489-492.

15 Thrall DE, Page RL, Dewhirst MW, et al. Temperature measurements in normal

and tumor tissue in dogs undergoinig whole body hyperthermia. Cancer Res 1986;

46: 6229-6235.

16 De Vries J, Hartel RM, Schraffordt Koops H, Oosterhuis JW. Dosage of cisplatin in

hyperthermic isolated regional perfusion. Surg Res Commun 1987; 2: 107-112.

17 Den Heeten GJ, Thijn CJ, Kamps WA, et al. The effect of chemotherapy on

osteosarcoma of the extremities as apparent from conventional roentgenograms.

Pediatr Radiol 1986; 16: 407-411.

18 Smith J, Heelan RT, Huvos AG, et al. Radiographic changes in primary osteogenic

sarcoma following intensive chemotherapy. Radiology 1982; 143: 355-360.

19 Coombs R, Halliday K. Biopsy techniques. In: Coombs R, Friedlaender G, eds.

Bone tumor management. Cornwall, Great Britain: Robert Hartnoll Ltd. 1987; 1

edn. 81-87.

42

Chapter 2

20 Huvos AG, Rosen G, Marcove RC. Primary osteogenic sarcoma. Pathologic aspects

in 20 patients after treatment with chemotherapy, en bloc resection and prosthetic

replacement. Arch Pathol Lab Med 1977; 101: 14-18.

21 Kaplan EL, Meier P. Nonparametric estimates from incomplete observations. J Am

Stat Assoc 1958; 53: 457-481.

22 Baum E, Greenberg L, Gaynon P, Krivit W, Hammond D. Use of cisplatinum

diammine dichloride (CPDD) in osteogenic sarcoma (OS) in children.

Proc.Am.Assoc.Cancer Res.and ASCO 1978; 19: 385 (Abstract).

23 Ochs JJ, Freeman AI, Douglass HO, et al. Cis-dichloro-diammine platinum (II) in

advanced osteogenic sarcoma. Cancer Treat Rep 1978; 62: 239-245.

24 Powers BE, Withrow SJ, Thrall DE, et al. Percent tumor necrosis as predictor of

treatment response in canine osteosarcoma. Cancer 1991; 67: 126-134.

25 Jaffe N, Knapp J, Chuang VP, et al. Osteosarcoma: Intraarterial treatment of the

primary tumor with cis-diamminodichloro-platinum (CDP): angiographic, pathologic

and pharmacologic studies. Cancer 1983; 51: 402-407.

26 Wile AG, Kar R, Cohen RA, Jakowatz JG, Opfell RW. The pharmacokinetics of

cisplatin in experimental regional chemotherapy. Cancer 1987; 59: 695-700.

27 Guchelaar HJ, Hoekstra HJ, De Vries EGE, et al. Cisplatin and platinum

pharmacokinetics during hyperthermic isolated limb perfusion for human tumors of

the extremities. Br J Cancer 1992; 65: 898-902.

28 Alberts DS, Peng YM, Chen G. Therapeutic synergism of hyperthermia and cisplatin

in a mouse tumor model. J Nat Cancer Inst 1980; 65: 455-460.

29 Fisher G, Hahn GM. Enhancement of cisplatin(II)diamine-chloride cytotoxicity by

hyperthermia. Natl Cancer Inst Monogr 1982; 61: 255-257.

30 Herman TS. Temperature dependence of adriamycin, cis-diammine-

dichloroplatinum, bleomycin and 1,3-bis-(2-chloroethyl)-1- nitrosurea cytotoxicity

in vitro. Cancer Res 1983; 43: 517-520.

31 Herman TS, Teicher BA, Chan V, Collins LS, Abrams MJ. Effect of heat on the

cytotoxicity and interaction with DNA of a series of platinum complexes. J Rad

Oncol Biol Phys 1989; 16: 443-449.

32 Meyn RG, Corry PM, Fletcher SE, Demetriades M. Thermal enhancement of DNA

damage in mammalian cells treated with cis-diamminedichloro-platinum(II). Cancer

Res 1980; 40: 1130-1139.

33 Riviere JE, Page RL, Dewhirst MW, Tyczkowska K, Thrall DE. The effect of

hyperthermia on cisplatin pharmacokinetics in normal dogs. Int J Hyperth 1986; 2:

351-358.

34 Wallner KE, Degregorio MW, Li GC. Hyperthermic potentation of cis-

diamminedichloroplatinum(II) cytotoxicity in chinese hamster ovary cells resistent

to the drug. Cancer Res 1986; 46: 6242-6245.

35 Thompson JP, Fugent MJ. Evaluation of survival times after limb amputation, with

and without subsequent administration of cisplatin, for treatment of appendicular

osteosarcoma in dogs: 30 cases (1979-1990). J Am Vet Med Assoc 1992; 200: 531-

533.

36 Mac Ewen EG, Kurzman JD, Rosenthal RC, et al. Therapy for osteosarcoma in

dogs with intravenous injection of liposome-encapsulated muramyl tripeptide. J

Nat Cancer Inst 1989; 81: 935-938.

43





10: 52-60.

38 Eggermont AMM, Lienard D, Schraffordt Koops H, Van Geel AN, Hoekstra HJ,

Lejeune FJ. Limb salvage by high dose tumor necrosis factor alpha (TNF) , gamma-

interferon (IFN) and melphalan isolated limb perfusion (ILP) in patients with

irresectable soft tissue sarcomas. Proc.Am.Soc.Clin.Oncol. 1992; 11: 1444 (Abstract).

45

Robert J. van Ginkel 1

Charles L.H. van Berlo 1

Peter C. Baas 1

Heimen Schraffordt Koops 1

Ries van Groningen á Stuling 1

Jan Elstrodt 2

Harald J. Hoekstra 1

Department of Surgical Oncology1 and Central Animal Laboratory2, University

Hospital Groningen, The Netherlands.

Sarcoma 1999; 3: 89-94.

Hyperthermic isolated limb perfusion with TNF and

cisplatin in the treatment of osteosarcoma of the

extremities: A feasibility study in healthy dogs

46

Chapter 3

Abstract

The feasibility of hyperthermic isolated limb perfusion (HILP) with tumor necrosis

factor-α (TNF) and cisplatin for the management of osteosarcoma was studied in the

canine model. During seven perfusions in six healthy mongrel dogs (weight 32±2 kg)

the technical aspects of HILP under mild hyperthermia (39-40o) were studied. In five

experiments HILP was performed with TNF alone (0.5 mg/L extremity volume), and

in two experiments TNF was combined with cisplatin (25 mg/L extremity volume).

During the perfusions physiological parameters were monitored and TNF and total

cisplatin concentrations were determined. Perfusion conditions (pH, PCO2, PO

2, flow

and pressure) remained within physiological ranges. Three dogs died within 24 hours

despite a sublethal systemical concentration of TNF that leaked from the perfusion

circuit. Three dogs were terminated; one dog after the second experiment in accor-

dance with Dutch ethical rules; one dog because it showed an invagination of the

small bowel resulting in an ileus; one dog because of necrosis of the perfused limb.

This feasibility study in healthy dogs demonstrated that HILP with TNF and cisplatin

was associated with a high mortality rate and therefore does not allow us to treat

dogs with spontaneous osteosarcoma with TNF and cisplatin HILP. Therefore, an

alternative model should be used in the search for the ideal combination of perfusion

agents for the limb sparing treatment in human osteosarcoma.

Introduction

Osteosarcoma is the most frequent primary malignant bone tumor in humans. Until

the 1970s the most common approach to the management of localized osteosarcoma

was surgical resection, amputation or radiation therapy.1 During the last decades a

definite role for neoadjuvant high dose methotrexate and cisplatin based

polychemotherapy was established.1-4 The potential local tumor effect of systemically

administered cisplatin, however, is limited due to the nephrotoxicity and ototoxicity

of cisplatin. Therefore an attempt was made to increase the local effect of cisplatin

without increasing systemic toxicity by using hyperthermic isolated regional limb

perfusion (HILP) with cisplatin in dogs with spontaneous osteosarcoma.5 These

studies showed an acceptable locoregional toxicity, improved functional outcome at

6 and 12 weeks, and a steadily improving radiological picture. However, the

histological results were modest, with none of the dogs showing a complete response

at 6 weeks after perfusion. The same experience was found in patients with sarcomas

of soft tissue and bone treated with cisplatin HILP.6 Results of recent publications

and of our own experience with a new perfusion modality, which combines tumor

necrosis factor-α (TNF) and melphalan in patients with recurrent melanoma or soft

tissue sarcoma, are very promising.7,8 However, in 6 of 8 evaluable patients with

47

Limb perfusion with TNF and cisplatin in healthy dogs

unresectable osteosarcoma of the lower limb treated with TNF and melphalan HILP,

histological evaluation revealed moderate results with ≥ 80% necrosis in 3 patients,

50%-60% necrosis in 2 patients and < 50% necrosis in one patient. After TNF and

melphalan HILP, limb sparing surgery was possible in 6 patients.9 As cisplatin is one

of the most active chemotherapeutics in the treatment of osteosarcoma, it seems

worthwhile to investigate the results of HILP with TNF and cisplatin. With the high

frequency of occurrence in dogs, canine osteosarcoma is a useful model for evaluation

of new treatment regimens in humans as rapid case accrual and rapid time to reach

measurable end points are possible.10 The canine osteosarcoma therefore appears to

be a valid model for studying the potential treatment of HILP with TNF and cisplatin

in the local treatment of osteosarcoma of the extremity in humans. To establish optimal

HILP conditions using TNF and cisplatin for local tumor control in dogs bearing

osteosarcoma, a feasibility study in healthy dogs was undertaken.


Dogs

During 7 experiments in 6 healthy mongrel dogs with a mean average weight of 32 ±2 kg and a mean age of 6 ± 1 years different aspects of HILP with TNF and cisplatin

were studied. Preoperatively, all dogs were thoroughly clinically evaluated at the

Central Animal Facility of the University of Groningen. The study was approved by

the Animal Welfare Committee of the Faculty of Medicine of the Groningen

University.

Anesthetics

The dogs fastened for 12 hours and were anaesthetized with thiopental (30mg/kg

BW, i.v.)(Pentothal, Abbott, Amstelveen, The Netherlands) and after muscle relaxation

with pancuroniumbromide (0.08 mg/kg BW, i.v.) (Pavulon, Organon, Oss, The

Netherlands), the dogs were ventilated (Ohmeda Modulus 2) with a mixture of O2

and isoflurane. The oxygen concentration in the gas mixture was continuously

measured by means of an oxygen analyzer (Ohmeda Modulus 2) and minute volumes

( 4-6 L/min) were adjusted to maintain an end-expiratory CO2 concentration of 4-5%

(Siemens CO2 - analyzer 930). The dogs were placed in the supine position on a

heated mattress to maintain their normal body temperature of 38 0C.11 During the

operations all dogs were given about 2 L of glucose 5% through a cephalic or internal

jugular vein. Central arterial pressure was recorded as well as an ECG and diuresis.

48

Chapter 3

Operation and Perfusion Techniques

During anaesthesia the volume of the extremity was measured using Archimedes

rule (1.7-2 L). The iliac vessels were exposed under sterile conditions and collateral

vessels were clipped. Cannulas were inserted into the artery (Bardic, 14-18 F) and

vein ( Bardic, 14-18 F). Both cannulas were connected to an extracorporeal circuit

consisting of an occlusive roller pump, a cardiotomy reservoir and a bubble oxygenator

with heat-exchanger. A nylon tourniquet was placed around the base of the extremity

using, a pin in the bone and bandage around the middle to complete the isolation of

the limb from the systemic circulation. The perfusate consisted of 350 ml 5% dextran

40 in glucose 5% (Isodex, Pharmacia AB, Uppsala, Sweden), 250 ml red blood cells

(canine blood donors), 250 ml plasma, 30 ml sodiumbicarbonate 8.4% and 0.5 ml

5000 IU/ml heparin (Thromboliquine, Organon B.V., Oss, The Netherlands). The

mixture of oxygen, air and carbondioxide through the oxygenator was adjusted to

maintain the blood gas values within the physiological range and when necessary,

bicarbonate was added to adjust the pH value.

All perfusions were performed under mild hyperthermic conditions (39-400 C) and

optimal physiologic conditions.12,13 Thermistor probes (Electrolaboriet, Copenhagen,

Denmark) were inserted into the subcutaneous tissues and into a muscle of the thigh

just above the knee for continuous monitoring of the temperatures during perfusion.

In the first 5 experiments TNF was the sole perfusion agent, in the last 2 experiments

TNF was combined with cisplatin. The dosage of TNF (0.5 mg/L extremity volume)

(Boehringer, Ingelheim, Germany) was calculated not to exceed ten times acceptable

systemic levels (Systemic: 10 µg/kg bodyweight).14 The dosage of cisplatin (25 mg/

L extremity volume) (Platinol 0.5 mg/ml, Bristol Myers Squibb, Weesp, The

Netherlands) used in the perfusion had been established in a previous study and was

based on a maximum tolerable dose of 30 mg/ L extremity volume.15 Cisplatin was

added to the circulated perfusate in 10 minutes. During perfusion, serum TNF and

total cisplatin levels were determined in the regional and systemic circulation at 0, 5,

15, 30, 45, 60, 75 and 90 minutes by ELISA and flameless atomic absorption

spectophotometry (FAAS), respectively. The perfusion time was 1 hour, followed by

washout of the extremity with 3L of Isodex. Tourniquet, cannulas and clips were

then removed and the incisions in the vessels repaired. Protamine hydrochloride

(Hoffman La Roche, Mijdrecht, The Netherlands) was administered, to neutralize

heparin, in a ratio of 1:1 to the initial dose of heparin. All dogs were closely observed

during at least 24 hours. No anti-inflammatory or analgesic drugs were administered

during follow-up. All dogs were followed for local and systemic side effects of TNF

and cisplatin perfusion, as well as survival.

49


Results

Table 1 shows the characteristics of the 7 experiments in 6 dogs. During the

experiments conditions for perfusions (pH, PCO2, PO

2) were kept within the

physiological ranges as in human perfusions. Figure 1 shows the flow, blood pressure,

perfusion pressures, weight gain or loss of the extra corporeal circuit and temperature

during 60 minutes of perfusion in the 7 experiments. In the first 5 experiments only

TNF was administered to the perfusion circuit. In the last two experiments cisplatin

was added. Figure 2 illustrates the TNF concentrations (mean ± SEM) in the perfused

limb as well as in the systemic circulation of the dog during perfusion and afterwards.

Peak TNF concentrations in the perfused limb were 650 ± 158 ng/ml and in the

systemic circulation of the dog 37 ± 15 ng/ml. The peak systemic concentrations in

the dog were in the same range as those of in TNF and melphalan HILP used in the

treatment of humans at our institute.16 Figure 3 shows the measured total cisplatin

values in the last two experiments. During the experiments we were not able to perform

any leakage monitoring by means of radionuclear detection techniques which are

used in the clinical perfusion setting. Therefore leakage was calculated afterwards

according to Stehlin with the amount of blood in the dogs estimated at 69 ml/kg

bodyweight.17 Calculated leakage values are summarized in Table 1.

Three dogs died within 24 hours: the first two during the TNF experiment, the third

after TNF and cisplatin perfusion. Postmortem examination of these animals did not

provide any macroscopic or microscopic evidence to explane the cause of death.

Three dogs were terminated; two due to treatment complications. One dog showed

an invagination of the small bowel resulting in an ileus and another was terminated 1

Table 1 Characteristics for the seven experiments in six dogs

Exp. Body Limb TNF Cisplatin Leakage Limb Follow-up

Nr. weight volume dose dose toxicity

(kg) (L) (mg) (mg) (%)

1 35 2.7 1.3 0 8.7 II Dead < 24 h

2 31.5 1.4 0.6 0 0.3 II Dead < 24 h

3 26.5 2.3 1.15 0 5.1 II Ileus, terminated < 1 wk

4 33.5 2.3 1.15 0 4.9 I Alive, experiment 1

5 31 1.9 1 0 6.1 n.a. Terminated, experiment 2

6 29.5 2.0 1 50 33.0 V Necrotic limb, terminated<1wk

7 28.5 1.8 0.8 50 10.8 II Dead < 24 h

Exp. Nr. = experiment number; n.a. = not applicable, dog 4 underwent 2 experiments; limb toxicity

according to Wieberdink 26; Grade I, no reaction, objectively and subjectively; Grade II, slight erythema,

edema or loss of sensation; Grade III, considerable erythema or edema with some blistering, slight

functional disturbances; Grade IV, extreme epidermolysis and/or obvious damage to the deep tissues

causing definite functional disturbances; Grade V, reaction that might necessitate amputation

50

Chapter 3

Fig. 1 Perfusion characteristics (flow, systemic blood pressure of the dog (BP); arterial

catheter pressure (P-art); venous catheter pressure (P-ven); extra corporeal circulation (ECC)

weight gain (+) or loss (-) and temperature of the perfused limb (oC)) in time during 60 min-

utes of perfusion in 7 experiments

51


week after TNF and cisplatin perfusion because of necrosis of the perfused limb. The

third dog was terminated after the second experiment in accordance with the Dutch

ethical rules.

Discussion

In the treatment of osteogenic sarcoma a distinction can be made between systemic

therapy and locoregional treatment. High dose methotrexate based systemic

chemotherapy is primarily administered in order to eradicate possible micrometastatic

disease and its use was a major breakthrough in the clinical treatment of osteosarcomas

in the 1970s.1,2 Today about 60% of patients with resectable primary tumors and no

metastases at the time of the initial diagnosis will be cured.1 The primary objective in

locoregional treatment is to prevent local recurrence and allow limb salvage procedures

in an attempt to preserve limb function. New surgical techniques and the development

of endoprosthetic materials, coupled with the systemic neoadjuvant chemotherapy,

Fig. 2 TNF levels in ng/ml (mean

± SEM) as obtained by human TNF

ELISA in the perfused limb, as well

as in the systemic circulation of the

dog

Fig. 3 Total cisplatin levels (tPt) in

mg/L (mean ± SEM) as obtained

by flameless atomic absorption

spectrophotometry in the perfused

limb, as well as in the systemic cir-

culation of the dog

52

Chapter 3

have offered less radical surgery for 40-80% of patients with osteosarcoma since the

1980s.1,18 Procedures that increase tumor necrosis of the primary tumor, with reduction

of viable tumor cells and tumor volume, could contribute to limb preservation

strategies. Since its first use, cisplatin has been one of the most effective

chemotherapeutic agents and has been incorporated in most systemic treatment

regimens for osteosarcoma. A recent attempt to overcome its nephrotoxic and ototoxic

limitations by administering cisplatin in HILP in the treatment of spontaneous canine

osteosarcoma was histologically modest.5 Promising results of recent publications

and our own experience with a new combination perfusion modality (TNF and

melphalan) for recurrent melanoma or soft tissue sarcoma, but moderate histological

results in patients with osteosarcoma, prompted us to investigate the combination of

TNF and cisplatin in HILP for osteosarcoma.7-9 Since the endothelial cells are

supposed to play a key role in the working mechanism of TNF, osteosarcomas with

a high extent of tumor vessels, are of particular interest.

Before application of TNF and cisplatin HILP in humans and client owned

osteosarcoma bearing dogs, the present feasibility study was performed in normal

healthy dogs. Despite sufficient experience in HILP in dogs as well as in humans, an

unexpected high mortality rate was encountered. Although there was no mortality

related to the operation, 3 dogs died within 24 hours after perfusion (50%). This

direct postoperative mortality could not be explained by a surplus of systemical

leakage of TNF. In the experiment, thedog with the highest leakage and, as a

consequence the highest systemical TNF concentrations, survived immediately

postoperatively, and the dog with the lowest leakage (lowest systemical TNF

concentrations) died within 24 hours after perfusion. No correlation between leakage

and mortality rate could be established. Maximal leakage encountered in these

experiments was 33%, this corresponds with 330 µg TNF given systemically per

dog; since the average dog weighs 33 kg, the dose of TNF that reaches the systemical

circulation of the dog is sublethal (10 µg/kg).14 Although only sublethal doses of

TNF leaked to the systemical circulation, the clinical picture resembled responses

observed in lethal doses (>100µg/kg), characterized by progressive hypotension, shock

and death within 24 hours.19 Due to the lack of facilities, we were not able to support

the dogs with intensive postoperative care, as is the case after human TNF HILP. In

part this could explain the observed direct postoperative mortality and supports the

need for intensive treatment after TNF HILP in the dog.

Three dogs survived the first days after perfusion, however, one dog developed an

ileus and was terminated within 1 week after perfusion. One dog that underwent two

experiments survived the first without morbid effects, but was terminated after the

second experiment according to the Dutch ethical rules. Leg toxicity consisted in

53


slight erythema and edema in all dogs except one in the cisplatin treated group. In

this dog, necrosis of the perfused limb was encountered, necessitating termination.

We have never observed necrosis of the perfused limb with the cisplatin dose used

(25 mg/L extremity volume) in experiments were cisplatin was the sole perfusion

agent.15 This observation may indicate that TNF might enhance the effect of cisplatin

to the local tissues of the perfused limb. The in vitro anticancer potential, and

overcoming cisplatin resistance with the combination of TNF and cisplatin in different

cell lines, has been established by others.20-22 Buell et al, demonstrated an increased

cellular cisplatin accumulation and DNA adduct formation as the possible cellular

basis for the augmented cisplatin cytotoxicity in the presence of TNF and

hyperthermia.23 Recently, Anda et al demonstrated that TNF selectively promoted

the in vitro permeability of the blood-brain barrier to CDDP without disrupting the

tight junctions.24 An improved penetration of cisplatin in the interstitial space due to

a higher permeability of the vascular wall, combined with an increased cellular

cisplatin accumulation and DNA adduct formation, could explain the observed

necrosis of the limb in this in vivo model with the cisplatin dose used, which was

previously non-toxic.

The observed mortality and morbidity that we encountered in this canine study was

in similar to the experience of Withrow and colleagues (unpublished observations).

The present results in normal elderly mongrel dogs indicate that treatment of dogs

with spontaneous osteosarcoma using TNF and cisplatin HILP is not appropriate.

Future research could focus on postoperative monitoring and care in dogs after TNF

HILP; perhaps a better alternative for testing the effect of TNF with cisplatin HILP,

is the use of the rat osteosarcoma model described by Manusama et al.,25 since rats

are much less susceptible to TNF than dogs.

54

Chapter 3

References

1 Ham SJ, Schraffordt Koops H, van der Graaf WT, et al. Historical, current and

future aspects of osteosarcoma treatment. Eur J Surg Oncol 1998; 24: 584-600.

2 Rosen G, Tan C, Sanmaneechai A, et al. The rationale for multiple drug chemotherapy

in the treatment of osteogenic sarcoma. Cancer 1975; 35: 936-945.

3 Link MP, Goorin AM, Miser AW, et al. The effect of adjuvant chemotherapy on

relapse free survival in patient with osteosarcoma of an extremity. N Eng J Med

1986; 314: 1600-1606.

4 Eilber F, Giuliano A, Eckardt J, et al. Adjuvant chemotherapy for osteosarcoma: A


5 van Ginkel RJ, Hoekstra HJ, Meutstege FJ, et al. Hyperthermic isolated regional

perfusion with cisplatin in the local treatment of spontaneous canine osteosarcoma:

assessment of short-term effects. J Surg Oncol 1995; 59: 169-176.

6 van Ginkel RJ, Schraffordt Koops H, de Vries EG, et al. Hyperthermic isolated limb

perfusion with cisplatin in four patients with sarcomas of soft tissue and bone. Eur

J Surg Oncol 1996; 22: 528-531.




10: 52-60.




J Clin Oncol 1996; 14: 2653-2665.

9 Bickels J, Manusama ER, Gutman M, et al. Isolated limb perfusion with tumor

necrosis factor alpha and melphalan for unresectable bone sarcomas of the lower

extremity. In: Manusama ER, ed. TNF-based isolated limb perfusion in the rat. The

Hague: Pasmans 1998; 1 edn. 105-117.

10 Withrow SJ, Powers BE, Straw RC, Wilkins RM. Comparative aspects of

osteosarcoma. Dog versus man. Clin Orthop 1991; 159-168.

11 Thrall DE, Page RL, Dewhirst MW, et al. Temperature measurements in normal

and tumor tissue in dogs undergoinig whole body hyperthermia. Cancer Res 1986;

46: 6229-6235.

12 Fontijne WP, Mook PH, Elstrodt JM, et al. Isolated hindlimb perfusion in dogs: the

effect of perfusion pressures on the oxygen supply (ptO2 histogram) to the skeletal

muscle. Surgery 1985; 97: 278-284.

13 Fontijne WP, De Vries J, Mook PH, et al. Improved tissue perfusion during pressure

regulated hyperthermic regional isolated perfusion in dogs. J Surg Oncol 1984; 26:

69-76.

14 Tracey KJ, Lowry SF, Fahey TJ3, et al. Cachectin/tumor necrosis factor induces

lethal shock and stress hormone responses in the dog. Surg Gynecol Obstet 1987;

164: 415-422.

15 De Vries J, Hartel RM, Schraffordt Koops H, Oosterhuis JW. Dosage of cisplatin in


55






17 Stehlin JS, Clark RL, White EC, et al. The leakage factor in regional perfusion with

chemotherapeutic agents. A M A Arch Surg 1960; 80: 934-945.

18 Meyer WH, Malawer MM. Osteosarcoma. Clinical features and evolving surgical

and chemotherapeutic strategies. Pediatr Clin North Am 1991; 38: 317-348.

19 Eichenholz PW, Eichacker PQ, Hoffman WD, et al. Tumor necrosis factor challenges

in canines: patterns of cardiovascular dysfunction. Am J Physiol 1992; 263: H668-

75.

20 Mutch DG, Powell CB, Kao MS, Collins JL. In vitro analysis of the anticancer

potential of tumor necrosis factor in combination with cisplatin. Gynecol Oncol

1989; 34: 328-333.

21 Mizutani Y, Bonavida B. Overcoming cis-diamminedichloroplatinum (II) resistance

of human ovarian tumor cells by combination treatment with cis-

diamminedichloroplatinum (II) and tumor necrosis factor-alpha. Cancer 1993; 72:

809-818.

22 Sleijfer S, Le TK, de Jong S, et al. Combined cytotoxic effects of tumor necrosis

factor-alpha with various cytotoxic agents in tumor cell lines that are drug resistant

due to mutated p53. J Immunother 1999; 22: 48-53.

23 Buell JF, Reed E, Lee KB, et al. Synergistic effect and possible mechanisms of

tumor necrosis factor and cisplatin cytotoxicity under moderate hyperthermia against

gastric cancer cells. Ann Surg Oncol 1997; 4: 141-148.

24 Anda T, Yamashita H, Khalid H, et al. Effect of tumor necrosis factor-alpha on the

permeability of bovine brain microvessel endothelial cell monolayers. Neurol Res

1997; 19: 369-376.

25 Manusama ER, Stavast J, Durante NM, Marquet RL, Eggermont AMM. Isolated

limb perfusion with TNF alpha and melphalan in a rat osteosarcoma model: a new

anti-tumor approach. Eur J Surg Oncol 1996; 22: 152-157.




57



Elisabeth G.E. de Vries2

Willemina M. Molenaar3

Donald R.A. Uges4

Harald J. Hoekstra1

Departments of Surgical Oncology1, Medical Oncology2, Pathology3, and

Pharmacy4 University Hospital Groningen, Groningen, The Netherlands.

European Journal of Surgical Oncology 1996; 22: 528-531.

Hyperthermic isolated limb perfusion with cisplatin

in four patients with sarcomas of soft tissue and

bone

58

Chapter 4

Abstract

The value of hyperthermic isolated limb perfusion (HILP) with cisplatin in the

management of locally advanced soft tissue sarcomas or metastatic bone sarcoma

was studied. Four patients were treated with HILP under mild hyperthermia

(39-40 °C) with 20-30 mg cisplatin / L perfused limb volume. Toxicity in the perfused

limbs was moderate, and the erythema and edema that occurred resolved

spontaneously within 7-14 days as did the slight motor and sensory neuropathy over

a longer period of time. Clinically, a reduction of pain was observed in all patients.

Two weeks after perfusion, tumor biopsies were taken to evaluate tumor response.

Two patients showed a pathological complete response, one patient showed >90%

necrosis and one patient showed no response. Currently patients are treated with

tumor necrosis factor and melphalan as perfusion agents. The above mentioned results

make the combination of tumor necrosis factor with cisplatin in the isolated limb

perfusion setting an interesting option.

Introduction

Malignant bone and soft tissue sarcomas are a heterogeneous group of lesions, which

all arise from tissue of mesenchymal origin. With an incidence of 3 per 100000, and

given that somatic soft tissue and skeleton comprise more than 75 % of the average

body weight, these cancers are rare. Most sarcomas of soft tissue and bone originate

in the extremities and are often quite large at the time of diagnosis. Limb saving

treatment of extremity sarcomas of soft tissue and bone is a multidisciplinary matter

and has avoided ablative surgical procedures in the majority of patients.1,2 Apart

from locoregional treatments, systemic adjuvant chemotherapy is now well established

for the treatment of osteosarcoma3, whereas in soft tissue sarcomas it is still a subject

of investigation.4 The main goal of the systemic treatment is the eradication of possible

micrometastatic disease with a possible favourable response on the primary tumor.

With hyperthermic isolated limb perfusion (HILP) it is possible to obtain a higher

local chemotherapy concentration in the perfused extremity than with systemically

administered chemotherapy.5 Cisplatin, discovered in 1965 by Rosenberg et al.6 is

one of the most active chemotherapeutic agents and it has been postulated that a high

dose of cisplatin could create significant necrosis of the primary tumor. Before

introducing cisplatin perfusions in the clinical treatment for sarcomas of soft tissue

and bone, a dose escalating and feasibility study in spontaneous canine osteogenic

sarcoma showed a maximum tolerable dose of 30 mg cisplatin / L extremity volume,

with improvements in clinical and X-ray parameters after treatment.7,8 The aim of

the present study was to investigate the feasibility and efficacy of HILP with cisplatin

in the locoregional tumor control of locally advanced soft tissue sarcomas or metastatic

59

Cisplatin perfusion in patients with sarcomas

bone sarcomas.

Patients and methods

Patients eligible for the study were suffering from histologically proven locally

advanced extremity sarcomas of soft tissue or bone. The primary tumor was locally

not resectable locally except when an amputation of the affected limb was performed.

Patients were treated with the intent of preserving the affected extremity.

Before perfusion the renal function of all patients was normal. Patients were

prehydrated with 2.5 L normal saline 12 hours preoperatively, and hydration was

maintained during the first 5 postoperative days, in order to protect against

nephrotoxicity. The perfusion technique employed is based on the technique developed

by Creech and Krementz9 and was performed during 60 min under mild hyperthermia

(39-40oC) and physiologically optimal conditions.10 Cisplatin (Platinol 0.5 mg/ml,

Bristol Myers SAE, Barcelona, Spain) was added to the perfusate over 10 min. In

this study the cisplatin dose, administered as part of a phase I-II dose finding study,

varied from 20-30 mg / L perfused limb volume.11 Leakage of cisplatin from the

perfused limb to the systemic circulation was checked by an isotope scanner placed

over the heart using 131I-albumin in the perfusate.12

All patients were followed up clinically by physical examination, chest X-rays and

routine blood chemistry for treatment related morbidity. In patients with a sarcoma

of bone, response to perfusion was scored on conventional X-rays in two directions.

Preperfusion X-rays were compared with 6 weeks postperfusion X-rays. Regression

of the tumor was defined as a decrease in tumor volume, increased ossification of

intra-osseous tumor osteoid, periosteal new bone, and soft tissue margins more densely

ossified, resulting in a more benign appearance of the tumor. One week after perfusion

an electromyogram was performed to investigate nerve toxicity of the cisplatin

perfusion. The local perfusion toxicity was graded according to the criteria described

by Wieberdink et al.11

Two weeks after perfusion, biopsies of the tumor were taken in three directions with

a 3.5 mm diameter Coombs bone biopsy system13 to evaluate the response of the

tumor to the perfusion treatment. The biopsies were histologically scored: little or no

effect of chemotherapy noted (score I), a partial response to chemotherapy with 50%

- 90% tumor necrosis noted and attributable to chemotherapy (score II), > 90% tumor

necrosis (score III), no viable tumor cells noted in any of the histological sections

(score IV).

Before and during perfusion, 10 ml perfusate samples were collected at 10 min

intervals to determine total platinum (tPt) and ultrafiltrated platinum (fPt) levels as

previously published.14 The study was approved by the local Medical Ethical

60

Chapter 4

Committee of the Groningen University Hospital and all patients gave informed

consent.

Results

Four patients entered the study. Three patients presented with metastasized lower

extremity sarcoma of bone, in two patients the primary tumor concerned an

osteosarcoma and in one patient a malignant fibrous histiocytoma. Both osteosarcoma

patients had multiple lung metastases and the patient with the malignant fibrous

histiocytoma of bone had multiple skeletal metastases at time of diagnosis. The fourth

patient presented with a localised recurrent malignant fibrous histiocytoma of the

soft tissues. She had first been treated with local excision followed by radiotherapy

(Table 1). All primary tumors were localised in the lower extremity.

Characteristics of the perfusion and cisplatin dose used in each patient are summarized

in Table 2. No technical perfusion related problems were encountered. After perfusion,

the total serum proteins and albumin levels decreased in all patients. The mean total

serum proteins decreased from 73.7±0.5 to 46.7±4.8 g/L on the first postoperative

day, and mean serum albumin from 45.3±1.9 to 29.3±4.6 g/L (p<0.05 paired Student’s

t-test). Serum albumin was corrected postoperatively with intravenous albumin

administration.

The acute treatment related toxicity consisted of a local edema and erythema (Grade

II toxicity) in three patients, and one patient had a considerable edema and erythema

of the skin with some blistering (Grade III toxicity). The erythema and edema resolved

spontaneously within 7-14 days as did the slight motor and sensory neuropathy over

a longer period of time. All patients experienced pain relief after perfusion. The X-

rays of the first osteosarcoma patient showed regression of the tumor and more than

90% necrosis was found in the tumor biopsies 2 weeks after perfusion. In the second

osteosarcoma patient, a 5% leakage of albumin to the systemical circulation occurred.

After the treatment, the leaked cisplatin brought about a measurable reduction of the

pulmonary metastases before systemic treatment with cisplatin was started and caused

temporarily renal function disturbances. Notably, the primary tumor did not respond

to the perfusion in terms of the pathological evaluation. The X-rays could not be

properly scored in this patient due to bone formation after a pathological fracture.

Both osteosarcoma patients received systemic chemotherapy after cisplatin perfusion

because of metastatic disease at time of diagnosis. The patient with a malignant

fibrous histiocytoma of bone showed progression of the tumor on X-ray, however,

tumor biopsies showed a complete response. In none of these three patients was the

affected limb amputated, and all three patients died from distant metastatic disease

after 6, 12 and 7 months. After an initial complete response clinically and

61


pathologically, a recurrence was found in the patient with a malignant fibrous

histiocytoma of the soft tissues. Subsequently a lower leg amputation was performed

and this patient was alive without evidence of disease 36+ months after amputation.

Discussion

HILP was first used to treat patients with melanoma, and the drug employed was the

alkylating agent melphalan.9 Although the overall remission in melanoma patients is

80%, 60% of patients fail to achieve a complete response. Drugs which might be

more effective or less toxic than melphalan in the treatment of melanoma by HILP

have therefore been sought. The first report by Aigner et al15, indicated that cisplatin

might be an useful alternative to melphalan in the treatment of melanoma. Cisplatin

is a non-cell-cycle dependent drug, which forms DNA-crosslinks and inhibits DNA

synthesis.16 Klein and Ben-Ari17 experienced no serious toxicity after HILP with

cisplatin. In the present study toxicity was moderate. In a previous experience of

HILP with cisplatin however, we found an unacceptable treatment related neuropathy.18

With cisplatin dosages in the same range as in the present study, the only difference

from this study was that six of seven patients with recurrent extremity melanoma

were treated with one or more melphalan perfusions with or without dactinomycin

prior to the cisplatin perfusion.

The pharmacokinetic data from the patients in this study showed extremely high

perfusate levels of Pt, up to 30 times higher than systemic levels which remained

acceptably low.14 Given that effective intra-arterial and systemic levels of Pt are in

the range of 5.5 mg/L, HILP produced 2 to 10 times higher levels. The pharmacokinetic

data further indicated that HILP with cisplatin produced high levels of Pt in the

tumor and surrounding muscle and fat14, which may harbour malignant cells that

later result in local recurrences.

Both malignant fibrous histiocytomas responded with total necrosis in the biopsy

material. Recently we showed that malignant fibrous histiocytoma of bone is a very

Table 1 Patients characteristics

No. Age Sex Primary tumor Metastatic disease

1 21 M Osteosarcoma Lungs

2 10 F Osteosarcoma Lungs

3 70 M Malignant fibrous histiocytoma of bone Bone

4 58 F Recurrent malignant fibrous histiocytoma No

of soft tissues

62

Chapter 4

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63


chemosensitive tumor.19 The malignant fibrous histiocytoma of soft tissue in the

present study however, recurred after 1 month. Fletcher et al. also reported a high

local recurrence rate of 66% (2 of 3 patients) in cisplatin perfusions for recurrent

malignant fibrous histiocytoma, indicating the difficulty of controlling local

recurrences of malignant fibrous histiocytoma with cisplatin perfusion.20

One osteosarcoma patient in the present study reacted with >90% necrosis of the

tumor, and in one patient the tumor showed no response after perfusion. These

moderate histological results in osteosarcomas are in accordance with our histological

data in canine experiments.8 The reason for this wide variation in response may be

exists in the sensitivity of the osteosarcoma cells, or that the pH changes in the tumor

occur during HILP, resulting in a less active form of cisplatin.16,21 Vaglini et al.22

found more favourable results with 95-100% necrosis of the tumor in eight of 11

evaluable osteosarcoma patients, 60-70% necrosis in two patients and 40% necrosis

in one patient after HILP with cisplatin combined with intra-arterial infusion of

cisplatin and systemic high-dose methotrexate.22. HILP with cisplatin was well

tolerated. The only significant complication was an extensive edema of the extremity

that spontaneously resolved in 2-3 weeks. Clinically, they observed a significant

reduction of pain, macroscopic reduction of tumor diameter, functional improvement

and rearrangement of the bone on the X-ray.

In the present study, no limb-salvage procedures were performed because of rapid

progression of systemic disease. In one osteosarcoma patient with a 5 % albumin

leakage, the accordingly leaked Pt brought about a measurable reduction of the

pulmonary metastasis, although the primary tumor did not respond. Di Filippo

demonstrated a systemical peak of Pt 3 hours after HILP due to the release of bound

Pt. Although this was encouraging, since a prolonged presence of Pt in the perfused

limb may contribute to lower recurrence rates, this indicated the need for adequate

and prolonged hyperhydration therapy after HILP with cisplatin, in order to prevend

nephro- and ototoxicity.

Following the treatment of the four patients described in this study, the Groningen

University Hospital now participates in a trial with a new perfusion modality, which

combines melphalan with biological response modifiers such as tumor necrosis factor

and interferon.23,24 As the endothelial cells are the main target cells of tumor necrosis

factor in these new perfusion schedules, treatment with this modality of sarcomas of

soft tissue and bone with a high extent of tumor vessels are of particular interest. A

trial which combines tumor necrosis factor with cisplatin as perfusion agents in the

treatment of sarcomas of soft tissue and bone could be clinically important.

64

Chapter 4

References

1. Suit HD. Local control and patient survival. Int J Radiat Oncol Biol Phys 1992; 23:

653-60.

2. Sadoski C, Suit HD, Rosenberg A, et al. Preoperative radiation, surgical margins,

and local control of extremity sarcomas of soft tissues. J Surg Oncol 1993; 52: 223-

30.

3. Eilber F, Giuliano A, Eckardt J, et al. Adjuvant chemotherapy for osteosarcoma: A


4. Casper ES, Gaynor JJ, Harrison LB, et al. Preoperative and postoperative adjuvant

combination chemotherapy for adults with high grade soft tissue sarcoma. Cancer

1994; 73: 1644-51.

5. Hoekstra HJ, Schraffordt Koops H, Molenaar WM, et al. Results of isolated regional

perfusion in the treatment of malignant soft tissue tumors of the extremities. Cancer

1987; 60: 1703-7.

6. Rosenberg B, Van Camp L, Krigas T. Inhibition of cell division in escherichia coli

by electrolysis products from platinum electrode. Nature 1965; 205: 157-64.

7. De Vries J, Hartel RM, Schraffordt Koops H, et al. Dosage of cisplatin in


8. van Ginkel RJ, Hoekstra HJ, Meutstege FJ, et al. Hyperthermic isolated regional

perfusion with cisplatin in the local treatment of spontaneous canine osteosarcoma:

assessment of short-term effects. J Surg Oncol 1995; 59: 169-76.

9. Creech O, Krementz ET, Ryan RF, et al. Chemotherapy of cancer: regional perfusion

utilizing an extra-corporeal circuit. Ann Surg 1958; 148: 616-32.

10. Fontijne WP, Mook PH, Schraffordt Koops H, et al. Improved tissue perfusion during

pressure regulated regional perfusion: a clinical study. Cancer 1985; 55: 1455-61.

11. Wieberdink J, Benckhuysen C, Braat RP, et al. Dosimetry in isolation perfusion of

the limb by assessment of perfused tissue volume and grading of toxic tissue reactions.

Eur J Cancer Clin Oncol 1982; 18: 905-10.

12. Hoekstra HJ, Naujocks T, Schraffordt Koops H, et al. Continuous leakage monitoring



13. Coombs R, Halliday K: Biopsy techniques. Coombs R, Friedlaender G (eds). In:

Bone tumor management (1st edn). Cornwall, Great Britain: Robert Hartnoll Ltd.

1987: 81-7.

14. Guchelaar HJ, Hoekstra HJ, De Vries EGE, et al. Cisplatin and platinum



15. Aigner K, Schwemmle K. Technic of isolated perfusion of the extremities. Experience

with 171 cases. Langenbecks Arch Chir 1983; 359: 113-22.

16. Rosenberg B. Fundamental studies with cisplatin. Cancer 1985; 55: 2303-l6.

17. Klein ES, Ben-Ari GY. Isolation perfusion with cisplatin for malignant melanoma

of the limbs. Cancer 1987; 59: 1068-71.

18. Hoekstra HJ, Schraffordt Koops H, De Vries EGE, et al. Toxicity of hyperthermic

isolated limb perfusion with cisplatin for recurrent melanoma of the lower extremity

after previous perfusion treatment. Cancer 1993; 72: 1224-9.

65


19. Ham SJ, Hoekstra HJ, van der Graaf WTA, et al. The value of high-dose methotrexate

based (HD-MTX) neoadjuvant chemotherapy in malignant fibrous histiocytoma

(MFH) of bone. J Clin Oncol 1996;

20. Fletcher WS, Pommier RF, Woltering EA, et al. Pharmacokinetics and results of

dose escalation in cis-platin hyperthermic isolation limb perfusion. Ann Surg Oncol

1994; 1: 236-43.

21. van der Zee J, Broekmeyer-Reurink MP, van den Berg AP, et al. Temperature

distribution and pH changes during hyperthermic regional isolation perfusion. Eur

J Cancer Clin Oncol 1989; 25: 1157-63.

22. Vaglini M, Belli F, Santinami M. Isolation perfusion of the lower limb with platinum.

World J Surg 1988; 12: 307-9.

23. Lienard D, Ewalenko P, Delmotte JJ, et al. High-dose recombinant tumor necrosis

factor alpha in combination with interferon gamma and melphalan in isolation

perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992; 10: 52-60.

24. Eggermont AMM, Lienard D, Schraffordt Koops H, et al. Limb salvage by high

dose tumor necrosis factor alpha (TNF) , gamma-interferon (IFN) and melphalan

isolated limb perfusion (ILP) in patients with irresectable soft tissue sarcomas. Proc

Am Soc Clin Oncol 1992; 11: 1444

67


Harald J. Hoekstra1

Alex M.M. Eggermont2

Elisabeth Pras3


Departments of Surgical Oncology1, Radiotherapy3, University Hospital

Groningen, Groningen, The Netherlands and Dr. Daniel den Hoed Cancer Center2,

Rotterdam, The Netherlands.

Archives of Surgery 1996; 131: 672-674.

Isolated limb perfusion of an irradiated foot with

TNF, interferon and melphalan

68

Chapter 5

Abstract

Hyperthermic isolated limb perfusion (HILP) with tumor necrosis factor alpha (TNF),

interferon gamma (IFN) and melphalan is a highly effective limbsaving treatment in

patients with irresectable soft tissue sarcoma or satellitosis and in-transit metastases

of melanoma. A 57-yr old woman presented with the second recurrence of a high

grade malignant fibrous histiocytoma of the right foot following previous local

resection plus curative adjuvant radiotherapy. The first recurrence of the lesion was

treated by HILP with cisplatin; the second recurrence was treated by HILP with

TNF, IFN and melphalan. The tumor and the area that had been irradiated showed a

bluish color a few hours after tumor necrosis factor perfusion. Nine days after TNF

perfusion a lower leg amputation had to be performed because of severe necrosis of

the foot.

Introduction

Recently Lienard and colleagues described the magnificent effect of hyperthermic

isolated limb perfusion (HILP) with recombinant tumor necrosis factor alfa (TNF),

recombinant interferon gamma (IFN) and melphalan in 23 patients with locally

advanced melanomas and soft tissue sarcomas of the extremities.1 The effect on the

tumors was striking: 19 (83%) complete responses and four (17%) partial responses

after a single perfusion with the triple-drug regimen. Local toxicity in the perfused

limb was minimal, 88% grade II and 12% grade III classified according to

Wieberdink.2 These figures are comparable with local tissue toxicity in patients treated

with melphalan as the single perfusion agent. The preliminary results of this study

suggested that high-dose TNF can be administrated safely by regional perfusion.

HILP of the limb with this triple drug regimen was started in 1991 at Groningen

University Hospital in the Netherlands, one of several institutions participating in a

multicenter study. The effect of this new combined modality therapy of isolated limb

perfusion and delayed surgery in a patient with a previous history of irradiation of

the foot is described.

Case report

In 1988 a 57-yr old woman presented with a 5 x 6 cm high grade malignant fibrous

histiocytoma on the lateral side of the right foot without distant metastases. She

refused a curative amputation of the lower leg. Therefore a marginal resection was

performed, followed by 60 Gy external beam radiotherapy, 40 Gy (2 Gy per day) on

the whole foot, and a 20 Gy boost on the tumor (Fig. 1). Two years after initial

treatment the tumor recurred locally without evidence of distant metastases. Again

she refused a lower leg amputation. In an attempted to render the tumor resectable,

69

Radiotherapy prior to HILP with TNF

HILP through the popliteal vessels with 100 mg cisplatin (30mg cisplatin per liter

limb volume) was performed. Histologic biopsy specimens of the tumor obtained 1

and 2 weeks after cisplatin perfusion showed no viable tumor cells, and a complete

remission was observed clinically.

In January 1991 the second local recurrence without distant metastases was observed,

again with persistent refusal by the patient for a curative amputation. During six

months the patient withdrew from follow-up but presented in June 1991 with a local

ulcerating tumor measuring 10 x 12 cm, still without metastatic disease (Fig. 2).

Because of the patients persistent refusal to undergo an amputation, a HILP with

TNF, IFN and melphalan was suggested and informed consent was obtained. One

and 2 days before HILP, a dose of 0,2 mg of IFN (Boehringer Ingelheim, Ingelheim,

Germany) was administered subcutaneously. A 90-minute mild hyperthermic (39°C

to 40°C), popliteal perfusion was performed with 0,2 mg of IFN, 4 mg of TNF

Fig. 1 Clinical appearance of the

patients right foot demonstrating the

radiation field and dosages of the ini-

tial treatment

Fig. 2 Clinical appearance of the

patients right foot demonstrating

the second recurrence

70

Chapter 5

(Boehringer Ingelheim), and 45 mg of melphalan (10 mg/L of limb volume)(Burroughs

Welcome, London, England). Leakage to the systemic circulation measured with131I labeled albumin as a tracer was 2.8 %.3 ECG, urine output, blood pressure,

venous and pulmonary pressures were recorded during and after perfusion until the

second postoperative day. A continuous infusion of dopamine at 2.8 mg/kg/min for

18 hours was given. Postoperatively the patient experienced fever and chills but no

hematological, hepatic or renal toxicity was observed.

A few hours after TNF perfusion, the entire right foot appeared bluish up to a definite

line at the ankle; the rest of the leg had a normal circulation. Two days after perfusion,

the tumor was black and necrotic in concordance with the 60-Gy total dose

Fig. 3 The right foot and ankle re-

gion 2 days after hyperthermic iso-

lated limb perfusion with TNF, IFN

and melphalan. The tumor is black

necrotic and the rest of the foot is

blue, sharply delineated at the edge

of the radiation field.

Fig 4 Left, A necrotic tumor tissue specimen after tumor necrosis factor perfusion (hema-

toxylin-eosin, x64) Right, The border area between nonirradiated normal skin (right) and

irradiated skin (left). The irradiated site demonstrates infiltration of both the dermis and the

epidermis with granulocytes and marked stasis with thrombosis of the microcirculation. In

the nonirradiated area, normal vascular structures are seen (hematoxylin-eosin, x64).

71


radiotherapy field, while the rest of the foot (40-Gy field) was blue and showed

some dry shrinkage of the skin (Fig. 3). Because of the severe necrosis, amputation

of the right foot below the knee had to be performed 9 days after TNF perfusion.

Histological findings were consistent with complete necrosis of the tumor (Fig. 4,

left). The amputation wound healed without complications, and the patient is alive

without evidence of disease 3 years after TNF perfusion and subsequent amputation.

Discussion

In 1975, Carswell et al. showed that tumor-necrotizing activity in the sera of animals

given injections of endotoxin was due to a host factor named tumor necrosis factor

(TNF).4 The mechanisms of the anti tumor activity of TNF however, are still not

elucidated and basis of further research. The results of intralesional or intravenous

administration of recombinant human TNF in mice with solid Meth A sarcoma of the

skin were recently described by van de Wiel and Bloksma.5 Treatment with TNF

caused red discoloration and necrosis of the central portion of the tumor within 24

hours. However, incubation of Meth A cells in the presence of TNF in vitro did not

affect their capacity to incorporate tritiated thymidine, indicating resistance of the

Meth A cells to TNF in vitro and supporting the thought that other mechanisms are

responsible for the observed discoloration and necrosis of the tumor in vivo.

Microscopic investigations of the tumors showed hyperemia, congestion, endothelial

damage and hemorrhage in the central part of the tumor, while just outside the tumor

edema and an infiltrate of polymorphonuclear cells was seen. Locally injected normal

skins with TNF showed moderate vascular effects without necrosis. This and other

investigations demonstrated that the vascular endothelial cells in particular are the

main target cells of this TNF induced antitumor effect.6,7 When incubated with TNF,

cultured endothelial monolayers show two phenomena. Stolpen et al. demonstrated

that TNF causes morphological changes of the endothelial cells, they become

elongated, overlap, rearrange their actin filaments and lose their stainable fibronectin

matrix.8 Suppression of anticoagulant mechanisms and the production of the

procoagulant cofactor tissue factor, is the second phenomenon9. These TNF-induced

changes are more prominent in areas with growing and/or migrating endothelial cells,

a situation that occurs within the tumor bed and explaining why the tumor vasculature

is more susceptible for TNF compared with normal vessels.10 A higher expression of

TNF receptors on the endothelial cell surface of dividing and growing endothelial

cells seem to be the cause of this high sensitivity for TNF.10,11 In summary; TNF

exposure emerges an altered endothelial cell phenotype, anticoagulant mechanisms

are suppressed and tissue factor is produced, leading to fibrin accumulation at the

endothelial cell surface 12 and thrombus formation in the tumor vessel, causing

72

Chapter 5

circulatory stasis and ischemia inside the tumor followed by necrosis of the tumor.

Besides this early vascular phenomenon, a latter in time occurring immune effect

with polymorphonuclear cell binding to the activated endothelium,7,13,14 and a direct

cytotoxic effect of TNF demonstrated in vitro against a variety of cell lines 15,16 are

two other mechanisms that could contribute in the anti-tumor effect of TNF.

In our case, not only the vascularization of the tumor was affected by TNF exposure

but also the microvascularization of the area that had been irradiated 3 years earlier.

Microscopical examination of the border area between irradiated and nonirradiated

areas revealed infiltration of both dermis and the epidermis with granulocytes, and

marked stasis with trombosis of the micro circulation of the irradiated area, causing

necrosis. These phenomena were absent from the perfused nonirradiated area (Fig.

4,right).

Late effects of normal tissues after radiotherapy are well known. Hopewell

demonstrated that arteries of the hamsters cheek pouch showed localized constrictions

after irradiation.17 These constrictions were caused by clones of dividing endothelial

cells and might be the predominant factor influencing the degeneration of the capillary

bed after radiotherapy.18 Evidence of this occlusive effect of vessels by proliferating

endothelial cells after radiation have also been reported by other investigators.19 Since

dividing and migrating endothelial cells are more sensitive to TNF than quiescent

endothelial cells, it is likely that not only the dividing and migrating endothelial cells

of the tumor bed but also the endothelial cells in the irradiated area of the foot in this

patient were activated by TNF, causing stasis and thrombosis of the microcirculation

in both areas. Recently Milas and coworkers also demonstrated a synergistic effect

between radiotherapy and TNF.20,21

One and a half year before TNF perfusion, this patient received HILP with cisplatin.

No literature is available describing the acute or long term effect of cisplatin on the

endothelial cells, however treatment with antineoplastic agents is associated with

vascular toxicity.22 With regard to cisplatin Vogelzang et al. described the relation

between hypomagnesemia and an increased risk of Raynaud´s phenomenon after

cisplatin, vinblastine and bleomycin treatment.23 Jackson et al. described a thrombotic

microangiopathic syndrome characterized by renal insufficiency, microangiopathic

hemolytic anemia and thrombocytopenia in patients treated with an identical

regimen.24 Histological examination of renal biopsy specimens showed marked

luminal narrowing of small arteries as a result of thrombus formation and subintimal

thickening. Whether cisplatin is the sole agent responsible for this vascular effect

remains unclear especially since it is known that bleomycin in this combination

chemotherapy has a prominent effect on endothelial cells.25 Analysis of cisplatin

perfusions data, performed at our clinic showed severe neurotoxicity, yet no signs of

73


vascular disturbances were found (i.e. Raynaud´s phenomenon).26 A synergistic effect

between cisplatin and radiotherapy is known when cisplatin is administered shortly

before or after radiotherapy,27 in this patient the interval between radiotherapy and

cisplatin perfusion lasted to long to make synergism likely.

Other radiation-related or radiation-independent factors may also be contributory,

i.e., the radiation dosage, time interval between radiation and TNF perfusion, and

the irradiated anatomical site. To distinguish the contribution of each of these different

factors experimental investigations should be performed. Awaiting the results of such

experiments, we would like to alert surgeons and radiation oncologists to the possible

complications that may occur after TNF perfusion, when the perfused limb has already

been irradiated.

74

Chapter 5

References




10: 52-60.




3 Hoekstra HJ, Naujocks T, Schraffordt Koops H, et al. Continuous leakage monitoring



4 Carswell EA, Old LJ, Kassel RL. An endotoxin induced serum factor that causes

necrosis of tumors. Proc Natl Acad Sci USA 1975; 72: 3666-3670.

5 Van de Wiel PA, Bloksma N, Kuper CF, Hofhuis FM, Willers JM. Macroscopic and

microscopic early effects of tumor necrosis factor on murine Meth A sarcoma, and

relation to curative activity. J Pathol 1989; 157: 65-73.

6 Van de Wiel PA, Pieters RH, van der Pijl A, Bloksma N. Synergic action between

tumor necrosis factor and endotoxins or poly(A.U) on cultured bovine endothelial

cells. Cancer Immunol Immunother 1989; 29: 23-28.

7 Palladino MAJR, Shalaby MR, Kramer SM, et al. Characterization of the antitumor

activities of human tumor necrosis factor-? and the comparison with other cytokines:

induction of tumor-specific immunity. J Immunol 1987; 138 : 4023-4032.

8 Stolpen AH, Guinan EC, Fiers W, Pober JS. Recombinant tumor necrosis factor and

immune interferon act singly and in combination to reorganize human vascular

endothelial cell monolayers. Am J Pathol 1986; 123: 16-24.

9 Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by

tumor necrosis factor. J Exp Med 1986; 163: 740-745.

10 Gerlach H, Lieberman H, Bach R, et al. Enhanced responsiveness of endothelium

in the growing/motile state to tumor necrosis factor/cachectin [published erratum

appears in J Exp Med 1989 Nov 1;170(5):1793]. J Exp Med 1989; 170: 913-931.

11 Espevik TP, Brockhaus M, Loetscher H, Nonstad U, Shalaby R. Characterization of

binding and biological effects of monoclonal antibodies against a human tumor

necrosis factor receptor. J Exp Med 1990; 171: 415-426.

12 Nawroth P, Handley D, Matsueda G, et al. Tumor necrosis factor/cachectin-induced

intravascular fibrin formation in meth A fibrosarcomas. J Exp Med 1988; 168: 637-

647.

13 Gamble JR, Harlan JM, Klebanoff SJ, Vadas MA. Stimulation of the adherence of

neutrophils to umbilical vein endothelium by human recombinant tumor necrosis

factor. Proc Natl Acad Sci U S A 1985; 82: 8667-8671.

14 Renard N, Lienard D, Lespagnard L, et al. Early endothelium activation and

polymorphonuclear cell invasion precede specific necrosis of human melanoma and

sarcoma treated by intravascular high-dose tumor necrosis factor alpha (TNF). Int J

Cancer 1994; 57: 656-663.

15 Helson L, Green S, Carswell E, Old LJ. Effect of tumor necrosis factor on cultured

human melanoma cells. Nature 1975; 258: 731-732.

75


16 Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Palladino MA. Recombinant human

tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells

in vitro. Science 1985; 230: 943-945.

17 Hopewell JW. Early and late changes in the functional vascularity of the hamster

cheek pouch after local x-irradiation. Radiat Res 1975; 63: 157-164.

18 Hopewell JW. Letter: The late vascular effects of radiation. Br J Radiol 1974; 47:

157-158.

19 Fajardo LF, Stewart JR. Capillary injury preceding radiation-induced myocardial

fibrosis. Radiology 1971; 101: 429-433.

20 Nishiguchi I, Willingham V, Milas L. Tumor necrosis factor as an adjunct to

fractionated radiotherapy in the treatment of murine tumors. Int J Radiat Oncol Biol

Phys 1990; 18: 555-558.

21 Sersa G, Willingham V, Milas L. Anti-tumor effects of tumor necrosis factor alone

or combined with radiotherapy. Int J Cancer 1988; 42: 129-134.

22 Doll DC, Ringenberg QS, Yarbro JW. Vascular toxicity associated with antineoplastic

agents. J Clin Oncol 1986; 4: 1405-1417.

23 Vogelzang NJ, Torkelson JL, Kennedy BJ. Hypomagnesemia, renal dysfunction,

and Raynaud’s phenomenon in patients treated with cisplatin, vinblastine, and

bleomycin. Cancer 1985; 56: 2765-2770.

24 Jackson AM, Rose BD, Graff LG, et al. Thrombotic microangiopathy and renal

failure associated with antineoplastic chemotherapy. Ann Intern Med 1984; 101:

41-44.

25 Nicolson GL, Custead SE. Effects of chemotherapeutic drugs on platelet and

metastatic tumor cell-endothelial cell interactions as a model for assessing vascular

endothelial integrity. Cancer Res 1985; 45: 331-336.




72: 1224-1229.

27 Vokes EE. Interactions of chemotherapy and radiation. Semin Oncol 1993; 20: 70-

79.

77


Harald J. Hoekstra1

Jan Pruim2

Omgo E. Nieweg1,3


Anne M.J. Paans2

Anton T.M. Willemsen2

Wim Vaalburg2


Department of Surgical Oncology1, PET Center2 and Department of Pathology4,

University Hospital Groningen, The Netherlands and Department of Surgery3, The

Netherlands Cancer Institute, Amsterdam, The Netherlands.

Journal of Nuclear Medicine 1996; 37: 984-990.

FDG-PET to evaluate response to hyperthermic

isolated limb perfusion for locally advanced soft-

tissue sarcoma

78

Chapter 6

Abstract

We investigated FDG-PET in patients undergoing hyperthermic isolated limb

perfusion (HILP) with TNF, IFN and melphalan for locally advanced soft-tissue

sarcoma of the extremities. Twenty patients (11 women, 9 men; aged 18-80 yrs, mean

age 49 yrs) were studied. FDG-PET studies were performed before, 2 and 8 weeks

after HILP. After the final PET study, the tumor was resected and pathologically

graded. Patients with a pathologically complete response (pCR) showed no viable

tumor after treatment. Those with a pathologically partial response (pPR) showed

various amounts of viable tumor in the resected tumor specimens. Seven patients

showed a pCR (35%) and 12 patients showed a pPR (60%). In one patient,

pathological examination was not performed (5%). The pre-perfusion glucose

consumption in the pCR group was significantly higher than in the pPR group

(p<0.05). Visual analysis of the PET images after perfusion showed a rim of increased

FDG uptake around a core of absent FDG uptake in 12 patients. The rim signal

contained a fibrous pseudocapsule with inflammatory tissue in the pCR group, but

viable tumor tissue was seen in the pPR group. The glucose consumption in the pCR

group at 2 and 8 weeks after perfusion had decreased significantly (p<0.05) compared

with the glucose consumption in the pPR group. Based on the pretreatment glucose

consumption in soft-tissue sarcomas, one could predict the probability of a patient

achieving a complete pathologically response after TNF HILP. FDG-PET indicated

the pathologic tumor response to HILP, although the lack of specificity of FDG, in

terms of differentiation between an inflammatory response and viable tumor tissue,

hampered the discrimination between pCR and pPR.

Introduction

Malignant soft-tissue sarcomas are a heterogeneous group of lesions that all arise

from tissue of mesenchymal origin and are characterized by aggressive local growth

and hematogenic metastases. They account for 1% of all malignant tumors and have

an incidence rate of 2 per 100.000. About 60% of these tumors occur in the extremities

and are often quite large at diagnosis.1 Limb-saving treatment of extremity soft-tissue

sarcomas is a multidisciplinary matter, with surgery and radiotherapy as the usual

treatment protocol.2,3 This combination therapy has avoided ablative surgical

procedures in the majority of patients.

The majority of locally advanced extremity soft-tissue sarcomas are treated by

amputation. Intra-arterial chemotherapy with adriamycin, combined with preoperative

radiotherapy, surgery and postoperative radiotherapy is effective in the treatment of

locally advanced soft-tissue sarcoma, but significant morbidity does occur.4 Recently

Eilber et al. reported a complete response rate of 49% and a limb-saving rate of 98%

79

FDG-PET to evaluate response to TNF perfusion

with neo-adjuvant chemotherapy and radiation for high-grade extremity soft-tissue

sarcoma with low treatment morbidity.5 Hyperthermic isolated limb perfusion (HILP)

also proved to be of value in the treatment for locally advanced extremity soft-tissue

sarcoma.6-8 With HILP, chemotherapeutic tissue concentrations may be up to 20 times

higher than can be attained with systemic administration.9 The introduction of

recombinant tumor necrosis factor-alpha (TNF), interferon-gamma (IFN) and

melphalan in regional perfusion represents a promising new development.10 With

this perfusion regimen, a complete response rate of 55% and a partial response rate

of 40% can be reached in the treatment of locally advanced soft-tissue sarcoma of

the extremities with a limb-saving rate of 90%.11 Since 1991, this perfusion strategy

has been used at our institution for these types of soft-tissue sarcomas.

PET enables visualization and quantification of metabolic processes in vivo. Fluorine-

18-2-fluoro-2-deoxy-D-glucose (FDG) has proven to be of value in the visualization

of various types of tumors.12,13 The use of FDG is based on Warburg´s observation of

increased glycolysis in cancer cells. The citric acid cycle, which is more efficient in

adenosine tri-phosphate generation, is suppressed.14 As a result, cancer cells

accumulate the glucose analog FDG which is trapped intracellularly as FDG

phosphate. FDG-PET can visualize soft-tissue sarcomas, indicate the malignancy

grade and detect locally recurrent disease.15-17 Various clinical reports suggest the

feasibility of FDG-PET to assess tumor response to radiotherapy and chemotherapy.18-20 This particular application of PET as a noninvasive technique to evaluate the

outcome of such often aggravating and expensive therapy may have a significant

effect on patient management. Ineffective treatment could be adjusted or discontinued

in an early stage and effective treatment could be continued with confidence.

The perfusion protocol provides us with histology before and after regional

chemotherapy. The tumor responses to this regional drug treatment are variable. This

clinical setting creates an opportunity to investigate the value of a noninvasive

diagnostic technique in the determination of tumor response to chemotherapy. The

aim of the present study was to investigate FDG-PET in patients undergoing HILP

for locally advanced soft-tissue sarcoma and to correlate PET findings with histology

before and after treatment.


Patients

Twenty (11 women, 9 men, aged 18-80 yrs, mean age 49 yrs) patients with biopsy-

proven soft-tissue sarcomas were entered in the study. Informed consent was obtained

from each patient. The diagnosis of the tumors was determined in a standard fashion

and graded according to Coindre.21,22 Thirteen patients presented with a newly

80

Chapter 6

diagnosed soft-tissue sarcoma (65%) and seven patients with a local recurrence (35%),

that had been previously treated with surgery alone. Nineteen tumors were located in

the lower limb (95%), and one patient (5%) had a sarcoma located in the right elbow.

All tumors were considered primarily irresectable because of size, their multicentricity

in the limb or fixation to the neurovascular bundle or bone. Median tumor size was

8.5 cm (range 2-30 cm). To render the tumors resectable for limb salvage, patients

were treated with HILP.

Treatment protocol

HILP is based on the technique developed by Creech and Krementz.23 Briefly, after

ligation of all collateral vessels and heparinization of the patient with 3.3 mg heparin/

kg bodyweight (Thromboliquine, Organon BV, Oss, the Netherlands), the axillary,

iliac, femoral or popliteal vessels were cannulated and connected to an extracorporeal

circuit. The perfused limb was wrapped in a thermal blanket to reduce heat loss and

a tourniquet was applied at the root of the extremity to minimize leakage of the

perfusate into the systemic circulation. Perfusion was performed during 90 min under

mild hyperthermia (39-40oC) and physiologically optimal conditions.24 At the start

of perfusion, 3 mg (upper extremity) or 4 mg (lower extremity) TNF (Boehringer,

Ingelheim, Germany) were injected as a bolus into the arterial line. Melphalan

(Burroughs Wellcome, London, England) was administered 30 min later, 10 mg/L

extremity volume (leg) or 13 mg/L extremity volume (arm).25 Since all perfusions

were performed in a Phase II clinical trial, the initial 13 patients in the PET study

also received a dose of 0.2 mg INF (Boehringer, Ingelheim, Germany) subcutaneously

1 and 2 days before perfusion, followed by 0.2 mg INF injected into the arterial line

at the start of perfusion. The final seven patients in the PET study did not receive the

INF. This alteration in treatment schedule was due to the decision of the trial

commission to investigate the additional effect of INF in the perfusion regiment

while the PET study was still in progress.

All perfusions were performed with a bubble oxygenator roller pump and heat

exchanger. The perfusate was oxygenated by a mixture of O2 and CO

2 and consisted

of 350 ml 5% dextran 40 in glucose 5% (Isodex, Pharmacia AB, Uppsala, Sweden),

500 ml blood (250 ml red blood cells, 250 ml plasma), 30 ml 8.4% NaHCO3, 0.5 ml

5000 IU/ml heparin. After 90 min of perfusion, the limb was flushed with 2 liters

dextran 40 in glucose 5% (Isodex, Pharmacia AB, Uppsala, Sweden) and 500 ml

blood (250 ml red blood cells, 250 ml plasma), catheters were removed, the circulation

restored and the heparin antagonized with protamine chloride (Hoffman La Roche,

Mijdrecht, the Netherlands). A lateral fasciotomy of the anterior compartment of the

lower leg or arm was performed to prevent a compartment syndrome.26 Approximately

81


8 weeks after perfusion (median 61 days, range 43-106 days) the residual tumor

masses were excised and pathologically examined.

Pathological examination

The tumor was measured in three dimensions and the percentage of necrosis estimated.

Representative tumor sections were taken, encompassing macroscopically different

tumor areas, including necrosis. Generally, one section per centimeter largest diameter

with a minimum of three was taken. Based on an integration of gross and microscopic

findings, a final estimate of the percentages of viable and necrotic or regressive tumor

was made. If possible, macroscopic examination and tissue sampling were performed

based on the latest PET images. The results were classified as either pathologically

complete response (pCR) or pathologically partial response (pPR), when remaining

viable tumor was observed.

PET imaging

Patients were scheduled for three PET studies: shortly before perfusion (median 14

days, range 1-30 days), two weeks after perfusion (median 13 days, range 7-27 days)

and shortly before resection of residual tumor tissue (median 55 days, range 42-77

days after perfusion). FDG was routinely produced by a robotic system following

the procedure as described by Hamacher27 with a radiochemical purity of more than

98%. PET sessions were performed using a Siemens ECAT 951/31 PET-camera

(Siemens/CTI, Knoxville, USA).

All patients fasted for at least 6 hours before the investigation. Serum glucose levels

were measured before each PET session and were found to be normal. A 20-gauge

needle was inserted into the radial artery under local anesthesia. In the contralateral

arm, an intravenous canula was inserted in the cephalic vein for the injection of the

FDG. The patients were positioned supine in the camera, with the tumor in the field

of view based on physical examination.

After attenuation scanning using 68Ge/68Ga source, 370 MBq (10mCi) FDG were

administered intravenously over 1 min. Dynamic images were acquired from the

time of injection after a dynamic protocol (five 1-min, five 2-min, five 3-min, two 5-

min, two 10-min, for a total duration of 60 min). Simultaneously, 2-ml blood samples

were taken from the arterial canula (time points 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10,

15, 25, 35, 45 and 55 min post-injection). The blood samples were centrifuged and

plasma activity was assessed using a well counter that was cross-calibrated with the

positron camera. Whole-body images were obtained after dynamic scanning. Total

time for the imaging procedure was approximately 2.5 hours.

82

Chapter 6

Data analysis

Images were displayed in coronal, sagittal and transaxial projections on a computer

display applying standard ECAT software (Siemens/CTI, Knoxville, USA) and

interpreted independently by two experienced physicians. Before perfusion, the tumor

location was first defined in all relevant tomographic planes of the study. Each tumor

was outlined automatically with a threshold technique that defines its contours at a

manually chosen percentage of the maximum number of counts per pixel. The level

of the threshold was chosen with the purpose to match the size of the region of

interest with the tumor size as outlined by MRI or CT. For each patient, a fixed

percentage (median 40, range 30-60) was used in all planes. All pixels above the

threshold were used for the calculation. An average time-activity curve as well as the

total volume of the lesion was obtained. Combining the averaged time-activity data

with the plasma input data, the average metabolic rate of glucose consumption

(MRglc) in µmol/100g tumor tissue /min was calculated using Patlak analysis,

assuming a lumped constant of 0.42.28,29 After perfusion, this threshold technique

could not be used since large areas of the tumors became inactive. The MRglc after

perfusion was therefore calculated by placing multiple regions of interest (ROI) over

the original tumor in all relevant planes of the study. Consequently, the necrotic parts

of the tumor that originated after perfusion were incorporated in this calculation.

The MRglc in the active parts of the tumor after perfusion was calculated separately

with the ROI technique. The change in MRglc after perfusion was related to the pre-

perfusional value and expressed as a percentage of basal value.

Visual evaluation of the PET studies was performed by quantifying the degree of

viable (active areas on the PET studies) and necrotic tumor (inactive areas) as a

percentage before and after perfusion.

Statistical analysis

Statistical procedures included a two-factor experiment with repeated measures on

one factor to compare glucose consumption between measures and groups. Analyses

were performed on datasets corrected for missing data according to Winer.30 Posthoc

comparison was made with Student t-tests. A p-value < 0.05 was considered

significant. SPSS/PC+

statistical software was used.

Results

The tumor characteristics, PET results and pathological response for each patient are

summarized in Tables 1 and 2. Pathological examination of the residual tumor mass

showed no viable tumor in seven patients (pCR 35%). In twelve patients, variable

amounts of viable tumor were found (pPR 60%). The pPR group also included one

83


A B C

Ta

ble

1T

um

or ch

aracteristics for each

patien

t.

Pat.

His

tolo

gy

Gra

de

Nu

mb

er L

arg

est

Pe

rfusio

n a

gen

ts

Nr.

of

dia

mete

r

lesio

ns

(MR

I)

1R

ha

bdom

yosarc

om

aP

rimary

31

10 c

mT

NF, IF

N, M

elp

ha

lan

2D

ediffe

rentia

ted m

yxoid

liposarc

om

aP

rimary

31

20 c

mT

NF, IF

N, M

elp

ha

lan

3M

yxo

id lip

osarc

om

aR

ecurre

nt

12

15 c

mT

NF, IF

N, M

elp

hala

n

4P

erip

hera

l neuro

ecto

derm

al tu

mor

Prim

ary

33

8 c

mT

NF, M

elp

hala

n

5M

alig

nant fib

rous h

istio

cyto

ma

Prim

ary

31

5 c

mT

NF, M

elp

hala

n

6M

alig

nant fib

rous h

istio

cyto

ma

Recurre

nt

31

4 c

mT

NF, M

elp

hala

n

7S

yn

ovia

lsa

rco

ma

Prim

ary

31

8 c

mT

NF, M

elp

hala

n

8M

yxo

id c

hondro

sarc

om

aP

rimary

21

8 c

mT

NF, IF

N, M

elp

hala

n

9M

alig

nant fib

rous h

istio

cyto

ma

Prim

ary

21

19 c

mT

NF, IF

N, M

elp

ha

lan

10

Ma

lignant fib

rous h

istio

cyto

ma

Recurre

nt

124 *

2 c

mT

NF, IF

N, M

elp

hala

n

11M

alig

nant s

chw

annom

aR

ecurre

nt

37

5 c

mT

NF, M

elp

hala

n

12

Fib

rosarc

om

aP

rimary

31

23 c

mT

NF, IF

N, M

elp

hala

n

13

Syn

ovio

sa

rco

ma

Prim

ary

31

9 c

mT

NF, IF

N, M

elp

hala

n

14

Myxoid

liposarc

om

aR

ecurre

nt

12

8 c

mT

NF, IF

N, M

elp

hala

n

15

Dediffe

rentia

ted lip

osarc

om

aP

rimary

21

17 c

mT

NF, IF

N, M

elp

ha

lan

16

Le

iom

yo

sa

rco

ma

Recurre

nt

31

12 c

mT

NF, IF

N, M

elp

hala

n

17

An

gio

sarc

om

aP

rimary

31

30 c

mT

NF, M

elp

ha

lan

18

Malig

nant s

chw

annom

aR

ecurre

nt

21

8 c

mT

NF, IF

N, M

elp

hala

n

19

We

ll diffe

rentia

ted lip

osarc

om

aP

rimary

11

29 c

mT

NF, IF

N, M

elp

ha

lan

20

Ma

lignant fib

rous h

istio

cyto

ma

Prim

ary

34

5 c

mT

NF, M

elp

hala

n

* M

ultip

le small lesio

ns o

f the lo

wer leg

(0.5

- 2 cm

); TN

F =

tum

or n

ecrosis facto

r; IFN

= in

terferon

84

Chapter 6

Ta

ble

2P

ET

res

ult

s an

d p

atholo

gic

al r

esponse

for

each

pat

ient

V

isu

al evalu

ati

on

of

the

Meta

bo

lic r

ate

of

glu

co

se (µ m

ol/

10

0g

/min

)

P

ath

olo

gic

al

ev

alu

ati

on

P

ET

stu

die

s

Pat.

B

efo

re H

ILP

A

fter

HIL

P B

efo

re H

ILP

2

weeks a

fter

HIL

P 8

weeks a

fter

HIL

P R

esp

on

se %

via

ble

M

acro

/ m

icro

sco

pic

C

orr

esp

on

den

ce

no

.

% o

f tu

mo

r

%

of

tum

or

Tu

mo

rTu

mo

rR

imC

ore

Tu

mo

rR

imC

ore

tum

or

vie

w o

f P

ET

wit

h h

isto

log

y

acti

ve *

acti

ve *

11

00

< 1

03

6.7

3.7

8.4

0.6

n.q

.n

.q.

n.q

.p

CR

0rim

: p

se

ud

oca

psu

le+

core

: necro

sis

21

00

< 1

04

1.8

n.p

.n

.p.

n.p

.2

.76

.30

.4p

CR

0rim

: p

se

ud

oca

psu

le+

core

: necro

sis

3 8

080

3.6

n.q

.n.q

.n.q

.5.6

Absent

Absent

pC

R0

regre

ssiv

e tum

ort

issue

-

41

00

< 1

03

3.2

6.2

7.9

1.8

5.4

7.2

1.5

pC

R0

rim

: p

se

ud

oca

psu

le+

core

: necro

sis

51

00

< 1

03

6.3

5.0

6.5

1.4

4.3

6.1

0.9

pC

R0

rim

: p

se

ud

oca

psu

le+

core

: necro

sis

61

00

< 1

01

3.3

12

.8A

bse

nt

Ab

se

nt

0.9

Ab

se

nt

Ab

se

nt

pC

R0

reg

ressiv

e t

um

ort

issu

e+

71

00

< 1

01

8.0

4.5

5.9

1.7

7.2

7.4

1.6

pC

R0

rim

: p

se

ud

oca

psu

le+

core

: necro

sis

8 5

05

06

.44

.66

.31

.23

.95

.20

.9p

PR

< 1

0rim

: a

rea

s o

f via

ble

tu

mo

r-

core

: necro

sis

91

00

< 1

01

3.0

4.4

9.1

0.5

4.8

7.4

0.3

pP

R<

10

rim

: a

rea

s o

f via

ble

tu

mo

r

core

: necro

sis

10

100

< 1

04.8

7.0

Absent

Absent

n.p

.n.p

.n.p

.pP

R<

10

are

as o

f via

ble

tum

or

+

11100

< 1

08.0

n.q

.n.q

.n.q

.5.9

Absent

Absent

pP

R<

10

are

as o

f via

ble

tu

mor

+

12

100

< 1

0n.q

.n.p

.n.p

.n.p

.5.8

9.0

1.5

pP

R<

10

rim

: are

as o

f via

ble

tum

or

+

core

: necro

sis

13

100

< 1

012.5

5.0

8.1

1.2

3.6

6.9

1.0

pP

R<

20

rim

: are

as o

f via

ble

tum

or

+

core

: necro

sis

14

50

50

5.0

n.p

.n.p

.n.p

.4.2

Absent

Absent

pP

R50

are

as o

f via

ble

tum

or

+

15

50

50

12.5

9.0

Abse

nt

Absent

10.0

Absent

Absent

pP

R50

are

as o

f via

ble

tu

mor

+

16

50

50

22.5

5.7

10.1

1.0

n.p

.n.p

.n.p

.pP

R50

rim

: are

as o

f via

ble

tum

or

+

core

: necro

sis

17

30

30

3.3

2.9

13.4

1.2

7.1

26.1

1.4

pP

R50

rim

: are

as o

f via

ble

tum

or

+

core

: necro

sis

18

80

40

25.7

n.p

.n.p

.n.p

.11.3

17.3

2.4

pP

R50

rim

: are

as o

f via

ble

tum

or

+

core

: necro

sis

19

100

100

4.3

5.8

Abse

nt

Absent

4.3

Absent

Absent

pP

R100

via

ble

tum

or

+

20

80

< 2

024.2

4.4

8.4

3.0

n.p

.n.p

.n.p

.n.p

.n.p

.n.p

.n.p

.

* =

per

centa

ge

of

tum

or

volu

me

acti

ve

on P

ET

stu

dy;

pC

R =

pat

holo

gic

ally

com

ple

te r

esponse

; pP

R =

pat

holo

gic

ally

par

tial

res

ponse

; n.p

. =

not

per

form

ed;

n.q

. =

no

qu

anti

fica

tio

n

85


Fig 1 Transversal image of a malignant fibrous histiocytoma of the lower leg in Patient 5.

Before perfusion (A) the tumor is clearly depicted as a homogeneous mass with a glucose

uptake of 36.3 µmol/100g tissue/min. Two (B) and 8 weeks after perfusion (C), the glucose

uptake in the tumor decreased to 5.0 and 4.3, respectively. The center of the tumor became

inactive surrounded by an active rim. Pathological examination revealed complete response.

The rim signal corresponded with a fibrotic vascular pseudocapsule with inflammatory tissue

surrounding a core of necrosis.

patient with no change in the percentage of viable tumor after treatment (clinically

this patient showed no response as well). In one patient (5%) the residual tumor was

not excised because of progressive distant disease. This patient died three months

after perfusion and data from this patient are excluded from the remainder of the

analyses.

Forty-nine of the scheduled 60 PET studies were completed (82%). Seven PET studies

were not performed due to patient-related problems. Technical problems prevented

quantification of PET data in four studies. Before perfusion, all tumors were easily

visualized on the baseline FDG-PET images. Twelve patients showed a homogeneous

active tumor on the preperfusion PET study, whereas eight patients also showed

inactive parts in the tumors before perfusion. Visual analysis of the PET images at 2

and 8 weeks after perfusion showed a rim of increased FDG uptake around a core of

absent FDG uptake in 12 patients (5 of 7 pCR, 7 of 12 pPR). The active rim

corresponded in the pCR patients with a fibrotic vascular pseudocapsule with reactive

inflammatory tissue, surrounding a core of absent FDG uptake representing necrosis

(Fig. 1). In patients with pPR, the active rim was found to contain both viable tumor

and an inflammatory response. Thus, the rim signal could correspond with either

viable tumor or a pseudocapsule with an inflammatory reaction. In seven patients

the tumor was visualized after perfusion as a homogeneous mass without the rim-

core configuration (2 of 7 pCR, 5 of 12 pPR). After perfusion, the amount of active

parts in the tumor declined significantly in 11 patients, corresponding with no or less

A B C

86

Chapter 6

than 20 % viable tumor tissue in the pathological specimens in each of these patients.

In two patients (Patients 3, 8) who also showed a good pathological response, the

PET study did not confirm this result. On histological examination, regressive tumor

tissue with an inflammatory reaction was found in Patient 3 and areas of viable tumor

accompanied by inflammatory tissue were found in Patient 8. The PET studies

correctly indicated moderate pathological outcome in six patients. Overall, 17 of 19

responses were correctly indicated by FDG-PET (89%), but the discrimination

between no and small amounts of viable tumor could not be made.

Pre-perfusion glucose consumption in the patients who ultimately had pCR was

significantly higher (p<0.05) than the pPR group (Fig. 2). At 2 and 8 weeks

postperfusion the MRglc in the pCR group had decreased significantly (p<0.05) in

contrast to the MRglc in the pPR group (Fig. 2). The most substantial decrease in

MRglc occurred within 2 weeks after perfusion. Figure 3 shows the percentage of

basal value of the tumor after perfusion. Patients in the pCR group showed a trend

towards a more reduced percentage of basal values than the pPR patients.

Three different histopathological groups could be distinguished after perfusion:

necrotic tissue, represented by the core MRglc of the pCR and pPR group, viable

tumor in combination with an inflammatory response, represented by the rim MRglc

of the pPR group and inflammatory with pseudocapsular tissue, represented by the

rim MRglc of the pCR group. The average MRglc in necrotic tissue was significantly

lower (p<0.05) than the values in tumor and inflammatory tissue, which were in the

same range (Fig. 4).

Fig. 2 MRglc of the tumor with

S.D. before, 2 and 8 weeks after

perfusion. Before perfusion, the

MRglc in the pCR group was sig-

nificantly higher than in the pPR

group (p<0.05). Two and 8 weeks

after perfusion, the MRglc in the

pCR group decreased significantly

(p < 0.05) in contrast to the pPR

group. HILP = hyperthermic iso-

lated limb perfusion.

87


Fig. 3 Percentage of basal value of the

tumor for each patient, 2 and 8 weeks

after perfusion. pCR = pathologically

complete response; pPR = pathologi-

cally partial response; HILP = hyper-

thermic isolated limb perfusion

Fig. 4 MRglc in necrotic, tumor and in-

flammatory tissue. MRglc in necrotic

tissue was significantly lower (p<0.05)

than that in tumor and inflammatory tis-

sue. The latter two could not be discrimi-

nated based on MRglc

Discussion

PET has made it possible to study biochemical changes of cancer tissue and to study

the effect of treatment on metabolism in vivo. The present study demonstrates

substantial decrements in the glucose metabolism of soft-tissue sarcomas with a

pathologically complete response after perfusion with TNF. These changes were

already evident within 2 weeks. In patients with a pPR, this decrease was less

pronounced. An active rim with an inactive core was seen in 13 out of 20 patients

after perfusion. Pathological examination showed that areas of absent intratumoral

FDG uptake were consistent with necrotic tissue. The rim signal represented either

viable tumor or a fibrous pseudocapsule with inflammatory tissue. Unfortunately,

FDG-PET could not discriminate a complete response from a partial response due to

the overlap in glucose metabolism between viable tumor and inflammatory tissue.

An explanation for the observed rim-core pattern can be found in the working

mechanism of TNF. Briefly; TNF exposure invokes an altered endothelial cell

phenotype, anticoagulant mechanisms are suppressed and tissue factor is produced,

which leads to fibrin accumulation at the endothelial cell surface and thrombus

88

Chapter 6

formation in the tumor vessels, causing circulatory stasis and ischemia inside the

tumor followed by necrosis of the tumor cells adjacent to the occluded vessels.31

Necrotic tissue is unable to accumulate FDG and represents the core on the PET

image. The central necrosis elicits an inflammatory response with the formation of a

fibrous pseudocapsule. This is reflected by the rim on the PET image in the pCR

group. On the other hand, peripheral tumor cells may obtain enough nutrients from

the surrounding environment to survive. This is reflected by the rim signal in patients

with pPR. Jones et al. also found an active rim with FDG-PET after neo-adjuvant

chemotherapy of soft-tissue sarcomas. In their patients, the rim signal did not signify

viable tumor but only a fibrous pseudocapsule.32 FDG accumulation in active

inflammatory lesions is in concordance with the observation of Tahara et al. who

found an increased glucose uptake in abdominal abscesses.33 Kubota et al. also found

a high accumulation in macrophages and granulation tissue in a microautoradiographic

study.34 They state that one should consider not only the tumor cells as FDG uptake

source, but also the non-neoplastic cellular elements, that may accompany tumor

growth or necrosis. These phenomena will occur particularly in tumors subjected to

treatment. The fact that both viable tumor and inflammatory tissue accumulate FDG

is one of the major limitations of FDG as the radiopharmaceutical for cancer treatment

evaluation.

One pCR patient showed an elevated MRglc 8 weeks after perfusion, in another pCR

patient, the MRglc did not decrease 2 weeks after perfusion. These observations

could be explained by the inflammatory cell invasion in the tumor. Beside the early

vascular phenomenon, a subsequent immune effect with polymorphonuclear cell

binding to the activated endothelium is another mechanism contributing to the anti-

tumor effect of TNF.35-37 This homing of inflammatory cells in the tumor may be

responsible for a high MRglc after perfusion in these two patients. This is in

concordance with the observation that FDG uptake was diffusely increased in the

remainder of the perfused leg. This phenomenon is thought to be caused by the diffuse

inflammatory reaction that follows perfusion.

Quantitative analysis demonstrated that the pre-perfusion MRglc in the pCR group

was significantly higher than in the pPR group. Thus, high MRglc appears to predict

a good response to TNF perfusion. Since glucose uptake in soft-tissue sarcoma

correlates well with the malignancy grade of the tumor, high grade tumors could be

more susceptible to TNF perfusion.16,17 In 17 of 19 (89%) patients the visual evaluation

of the PET studies corresponded well with the pathological response. In two patients

with a good pathological response, the PET study did not confirm this. In both patients

areas of inflammatory tissue were found on histological examination corresponding

with active areas on the PET scan and therefore resulted in an overestimation of

89


active tumor on the PET scans. Although visual evaluation gave a good indication of

the pathological outcome, the use of FDG-PET in routine clinical monitoring of

response of soft-tissue sarcomas to isolated limb perfusion is hampered by this overlap

between malignant tumor and inflammatory tissue.

Several other investigators have studied whether FDG-PET can be used to monitor

treatment for cancer. FDG uptake was found to decrease as early as 5 days after the

start of systemic therapy for breast cancer. 20,38 A change in FDG uptake was found to

better predict the ultimate outcome than change in tumor size. Decrease in FDG

uptake was more prominent in patients who responded favorably to radiotherapy or

chemotherapy for head and neck cancer compared to non-responding patients.18,39

Similar findings have been reported in other types of tumors and using a variety of

therapeutic schedules.19,40-43 These studies have in common that post-treatment PET

data were correlated with findings of physical examination, radiographic studies or,

at best, fine needle aspiration of the tumor mass, following generally accepted

guidelines.44 In none of these studies have the PET findings been verified by rigorous

microscopic examination of the whole tumor as the gold standard as we have done in

the present study. Our approach appears worthwhile, since change in tumor volume

and viability are not very well correlated. A palpable mass that remains after treatment

may consist of necrosis and fibrosis without viable tumor. On the other hand, viable

tumor may still remain when a palpable tumor that is visible on radiographic images

disappears after treatment. If one wants to investigate whether PET signifies an

improvement over radiographic techniques in the evaluation of treatment, it seems

less appropriate to use those same radiographic techniques as the reference standard.

Our results should be interpreted with caution. Our patient population was limited in

that it was a heterogeneous group of soft-tissue sarcomas and only large tumors were

included (median 8.5 cm). Additional data are needed on FDG-PET in more patients

with other tumor types treated with other drugs. Other PET tracers, such as labeled

aminoacids and 11C-thymidine, may be more suitable to distinguish between tumor

and inflammatory response.

Conclusion

The present study demonstrated that FDG-PET indicates the pathologic tumor

response to chemotherapy in an investigational setting used with isolated limb

perfusion for locally advanced soft-tissue sarcomas. The discrimination between viable

tumor and inflammatory tissue after perfusion treatment, however was hampered by

the limited specificity of FDG. A search for more specific tracers to monitor pathologic

tumor response is needed.

90

Chapter 6

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2 Suit HD. Local control and patient survival. Int J Radiat Oncol Biol Phys 1992; 23:

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3 Sadoski C, Suit HD, Rosenberg A, Mankin H, Efird J. Preoperative radiation, surgical

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17 Nieweg OE, Pruim J, van Ginkel RJ, et al. Fluorine-18-fluorodeoxyglucose PET

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18 Minn H, Paul R, Ahonen A. Evaluation of treatment response to radiotherapy in

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1521-1525.

19 Ichiya Y, Kuwabara Y, Otsuka M, et al. Assessment of response to cancer therapy

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Med 1991; 32: 1655-1660.

20 Wahl RL, Zasadny K, Helvie M, et al. Metabolic monitoring of breast cancer

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1988: 2 edn.

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27 Hamacher K, Coenen HH, Stöcklin G. Efficient stereospecific synthesis of no-carrier-

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28 Patlak CS, Blasberg RG, Fenstermacher JD. Graphical evaluation of blood-to-brain

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29 Patlak CS, Blasberg RG. Graphical evaluation of blood-to-brain transfer constants

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31 Nawroth P, Handley D, Matsueda G, et al. Tumor necrosis factor/cachectin-induced

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647.

32 Jones DN, Brizel DM, Charles HC, et al. Monitoring of response to neoadjuvant

therapy of soft tissue and musculoskeletal sarcomas using F18-FDG-PET.

J.Nucl.Med. 1994; 38P (Abstract).

33 Tahara T, Ichiya Y, Kuwabara Y, et al. High [18F]-fluorodeoxyglucose uptake in

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34 Kubota R, Yamada S, Kubota K, et al. Intratumoral distribution of fluorine-18-

fluorodeoxyglucose in vivo: high accumulation in macrophages and granulation

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35 Palladino MAJR, Shalaby MR, Kramer SM, et al. Characterization of the antitumor

activities of human tumor necrosis factor-? and the comparison with other cytokines:

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36 Gamble JR, Harlan JM, Klebanoff SJ, Vadas MA. Stimulation of the adherence of

neutrophils to umbilical vein endothelium by human recombinant tumor necrosis

factor. Proc Natl Acad Sci U S A 1985; 82: 8667-8671.

37 Renard N, Lienard D, Lespagnard L, et al. Early endothelium activation and

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sarcoma treated by intravascular high-dose tumor necrosis factor alpha (TNF). Int J

Cancer 1994; 57: 656-663.

38 Nieweg OE, Wong WH, Singletary SE, Hortobagyi GN, Kim EE. Positron emission

tomography of glucose metabolism in breast cancer. Potential for tumor detection,

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39 Haberkorn U, Strauss LG, Dimitrakopoulou A, et al. Fluorodeoxyglucose imaging

of advanced head and neck cancer after chemotherapy. J Nucl Med 1993; 34: 12-17.

40 Haberkorn U, Strauss LG, Dimitrakopoulou A, et al. PET studies of

fluorodeoxyglucose metabolism in patients with recurrent colorectal tumors receiving

radiotherapy. J Nucl Med 1991; 32: 1485-1490.

41 Strauss LG, Dimitrakopoulou A, Haberkorn U, Ostertag H, Helus F, Lorenz WJ.

PET studies with FDG in patients with metastatic melanoma prior and after therapy.

Eur.J.Nucl.Med. 1991; 18: 685 (Abstract).

42 Abe Y, Matsuzawa T, Fujiwara T, et al. Clinical assessment of therapeutic effects on

cancer using 18F-2- fluoro-2-deoxy-D-glucose and positron emission tomography:

preliminary study of lung cancer. Int J Radiat Oncol Biol Phys 1990; 19: 1005-

1010.

43 Bares R, Horstmann K, Altehoefer C, et al. FDG-PET to assess local effects of

radiation or chemotherapy in patients with malignant lymphoma. J.Nucl.Med. 1991;

32: 918 (Abstract).

44 Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer

treatment. Cancer 1981; 47: 207-214.

93


Annemieke C. Kole2

Omgo E. Nieweg1,3


Jan Pruim2


Wim Vaalburg2

Harald J. Hoekstra1

Department of Surgical Oncology1, PET Center2 and Department of Pathology4,

University Groningen Hospital, The Netherlands and Department of Surgery3, The

Netherlands Cancer Institute, Amsterdam, The Netherlands.

Journal of Nuclear Medicine 1999; 40: 262-267.

L-[1-11C]-Tyrosine PET to evaluate response to

hyperthermic isolated limb perfusion for locally

advanced soft-tissue sarcoma & skin cancer

94

Chapter 7

Abstract

PET with L-[1-11C]-tyrosine (TYR) was investigated in patients undergoing

hyperthermic isolated limb perfusion (HILP) with recombinant tumor necrosis factor

alpha (TNF) and melphalan for locally advanced soft-tissue sarcoma and skin cancer

of the lower limb. Seventeen patients (5 women, 12 men; age range 24-75 y; mean

age 52 y) were studied. TYR-PET studies were performed before HILP, and 2 and 8

weeks afterwards. The protein synthesis rates (PSR) in nanomoles per milliliter per

minute were calculated. After the final PET study, the tumor was resected and

pathologically examined. Patients with a pathologically complete response (pCR)

showed no viable tumor after treatment. Those with a pathologically partial response

(pPR) showed various amounts of viable tumor in the resected tumor specimens. Six

patients showed a pCR (35%) and 11 patients showed a pPR (65%). All tumors were

depicted as a hot spot on the PET study before HILP. The PSR in the pCR group at 2

and 8 weeks after perfusion had decreased significantly (p<0.05) compared to the

PSR before HILP. A significant difference was found in PSR between the pCR and

pPR group at 2 as well as at 8 week (p<0.05). Median PSR in nonviable tumor tissue

was 0.62 and ranged from 0.22 to 0.91. With a threshold PSR of 0.91, sensitivity and

specificity of TYR-PET were 82% and 100%, respectively. The predictive value of a

PSR > 0.91 for having viable tumor tissue after HILP was 100%, whereas the

predictive value of a PSR ≤0.91 for having nonviable tumor tissue after HILP was

75%. On pathological examination the 2 patients in the pPR group with a

PSR < 0.91 showed microscopic islets of tumor cells surrounded by extensive necrosis.

Based on the calculated PSR after HILP, TYR-PET gave a good indication of the

pathological outcome. Inflammatory tissue after treatment did not interfere with viable

tumor tissue on the images, suggesting that it may be worthwhile to pursue TYR-PET

in other therapy evaluation settings.

Introduction

Different metabolic processes such as glycolysis, protein synthesis, uptake of

disaccharides and transamination, are enhanced in tumors when compared to normal

tissues. PET enables visualization and quantification of metabolic processes in vivo.18Fluorine-labeled 2-fluoro-2-deoxy-D-glucose (FDG) is the most commonly used

radiopharmaceutical for PET and has proven to be of value to visualize various types

of solid tumors, to indicate the malignancy grade and to detect locally recurrent

disease.1-4 Various clinical reports suggest the feasibility of FDG-PET to assess tumor

response to radiotherapy and chemotherapy.5-7 A limitation of FDG-PET in therapy

evaluation is the inability to differentiate between viable tumor tissue and

inflammatory tissue.8-10 Therefore, there is a need for alternatives. Ishiwata et al.11

95

TYR-PET to evaluate response to TNF perfusion

have shown that the uptake of amino acids is high in tumor tissue due to an increased

protein synthesis rate (PSR). Amino acids play a minor role in the metabolism of

inflammatory cells, mainly neutrophils, compared to FDG. Most amino acid PET

studies have been performed with L-[methyl-11C]-methionine (MET).12-14 MET reflects

amino acid uptake rather than protein synthesis because it is involved in other

metabolic pathways such as transmethylation and polyamine synthesis.15,16 This

complicated metabolism of methionine has made it impossible to create a precise

metabolic model. Carboxyl-labeled amino acids, such as L-[1-11C]-tyrosine (TYR),

L-[1-11C]-methionine and L-[1-11C]-leucine, appear to be more appropriate compounds

to determine protein synthesis in tumors.16,17 The main metabolite of these amino

acids is 11CO2, which is rapidly cleared from tissue and exhaled and does not contribute

to the PET-measured 11C radioactivity in tumor tissue.

A model was developed to determine the PSR in tumor tissue using TYR.18 Initial

results in patients with brain tumors have been published recently.19 Kole et al. 20

reported a high uptake of TYR and, as a consequence, a high PSR in various types of

malignancies and low uptake in benign lesions. PET with TYR may be of value in

the assessment of the response of a malignant tumor to therapy, because a decrease

in tissue viability may result in a decrease in PSR.

Hyperthermic isolated limb perfusion (HILP) with recombinant tumor necrosis factor

alpha (TNF) and melphalan can usually prevent amputation in patients with locally

advanced soft-tissue sarcoma or extensive local regional melanoma.21,22 The aim of

this study was to investigate PET with TYR in patients undergoing HILP for locally

advanced soft-tissue sarcoma and skin cancer and to correlate PET findings with

histology before and after treatment.


Patients

Seventeen patients (5 women, 12 men; aged 24-75 yrs; mean age 52 yrs) with biopsy-

proven soft-tissue sarcoma or melanoma participated in the study approved by the

Medical Ethical Committee of the institute. Informed consent was obtained from

each patient. Ten patients presented with a newly diagnosed soft-tissue sarcoma, 2

patients presented with a local recurrence of a soft-tissue sarcoma previously treated

with surgery alone, 4 patients presented with a melanoma and 1 patient presented

with a squamous cell carcinoma. All tumors were located in the lower limb. The

diagnosis of the tumors was determined in a standard fashion and soft-tissue sarcomas

were graded according to Coindre et al.23 All tumors were considered primarily

irresectable because of size, multicentricity in the limb, or fixation to the neurovascular

bundle or bone. Median tumor size was 10 cm (range 3-25 cm). Patients and tumor

96

Chapter 7

characteristics are summarized in Table 1.

Methods

The perfusion technique used at the Groningen University Hospital is based on the

technique developed by Creech et al.24 and has been described in detail previously.22

Briefly, after cannulation of the vessels of the perfused limb, a tourniquet is placed at

the base to prevent systemic leakage. The limb is perfused with 4 mg TNF (Boehringer,

Ingelheim, Germany) administered directly intra-arterially, followed 30 min later by

10 mg/L volume melphalan (Burroughs Wellcome, London, England). Perfusion is

carried out for 90 min under hyperthermic conditions (39-40°C). Preventive measures

to cope with the expected side effects caused by leakage consist of fluid loading and

administration of vasoactive amines. After HILP, patients are mechanically ventilated

until they are hemodynamically stable and receive intensive care management as

described by Zwaveling et al.25 Approximately 8 weeks after perfusion (median 66

Table 1 Tumor characteristics

Pat. Histology Site Grade Number Largest

no. of diameter

lesions ( MRI)

1 Melanoma Recurrent Lower leg n.a. 2 3.5 cm

2 Squamous cell carcinoma Primary Foot n.a. 1 6.0 cm

3 Clear cell carcinoma Primary Lower leg 3 1 10.0 cm

4 Melanoma Recurrent Lower leg n.a. 3 4.0 cm

5 Leiomyosarcoma Primary Lower leg 3 1 12.5 cm

6 Melanoma Primary Lower leg n.a. 1 7.0 cm

7 Synoviosarcoma Primary Popliteal fossa 2 1 4.0 cm

8 Fibrosarcoma Primary Knee 1 1 3.0 cm

9 Synoviosarcoma Recurrent Lower leg 2 1 9.0 cm

10 Haemangiopericytoma Primary Popliteal fossa 2 1 15.0 cm

11 Malignant fibrous histiocytoma Primary Thigh 3 1 23.0 cm

12 Angiosarcoma Primary Lower leg 3 1 12.5 cm

13 Extra-osseous osteosarcoma Primary Thigh 3 1 25.0 cm

14 Myxoid liposarcoma Primary Thigh 1 1 8.0 cm

15 Myxoid liposarcoma Primary Popliteal fossa 2 1 11.0 cm

16 Malignant fibrous histiocytoma Primary Thigh 2 1 10.0 cm

17 Melanoma Primary Toe n.a. 5 3.0 cm

n.a. = not applicable

97


days, range 27-125 days) the residual tumor masses were excised and pathologically

examined. The tumor remnants were measured in three dimensions and the percentage

of necrosis estimated. Representative tumor sections were taken, encompassing

macroscopically different tumor areas including necrosis. As a general rule, one section

per centimeter largest diameter with a minimum of three was taken. Based on an

integration of gross and microscopic findings, a final estimate of the percentages of

viable and necrotic or regressive tumor was made. If possible, macroscopic

examination and tissue sampling was performed based on the latest PET images.

The results were classified as either pathologically complete response (pCR) or

pathologically partial response (pPR), when remaining viable tumor was noted.

PET imaging

Patients were scheduled for three PET studies: shortly before perfusion (n = 17,

median 10 days, range 1 - 23 days), 2 weeks after perfusion (n = 14, median 16 days,

range 12 - 23 days) and shortly before resection of residual tumor tissue (n = 15,

median 55 days, range 47 - 68 days after perfusion). TYR was produced by a modified

microwave-induced Bücherer-Strecker synthesis26 , with a radiochemical purity of

more than 99%. PET sessions were performed using an ECAT 951/31 PET camera

(Siemens/CTI, Knoxville, USA).

All patients fasted for at least 8 hours before the investigation. Serum tyrosine levels

were measured before each PET session and were found to be normal (mean 0.053

mmol/L, range 0.028 - 0.1 mmol/L). A 20-gauge needle was inserted into the radial

artery under local anesthesia. In the contralateral arm, an intravenous canula was

inserted in the cephalic vein for the injection of TYR. The patients were positioned

supine in the camera, with the tumor in the field of view based on physical

examination.

After attenuation scanning using a 68Ge/68Ga source, we administered a mean dose of

322 MBq (range 126 - 381 MBq) TYR intravenously over 1 min. Dynamic images

were acquired from the time of injection following a dynamic protocol (ten 0.5 min,

three 5 min, three 10 min) for a total duration of 50 min. Simultaneously, 2-ml blood

samples were taken from the arterial canula (time points 0.25, 0.5, 0.75, 1.0, 1.25,

1.5, 1.75, 2.25, 2.45, 3.75, 4.75, 7.5, 12.5, 17.5, 25, 35, and 45 min postinjection).

The blood samples were centrifuged and plasma activity of TYR, the 11C-labeled

CO2 and protein levels were measured by high-performance liquid chromatography

(HPLC). The duration of the imaging procedure was approximately 2.5 h.

98

Chapter 7

Data analysis

Images were displayed in coronal, sagittal and transaxial projections on a computer

display using standard ECAT software and were interpreted independently by two

experienced physicians. To determine tumor PSR, one must first define the tumor in

all relevant tomographic planes of the study. Usually this is done by placing regions

of interest (ROIs) in each plane, matching the size of the tumor as outlined by MRI.

The tissue time-activity curves obtained from these ROIs can be averaged and the

average PSR can be calculated. Because this technique is rather laborious, an

alternative method was developed at our institute. By using the same activity threshold

as the one used to define the ROI, we selected all voxels in the study above this

threshold. For each analysis, a fixed percentage of 95% was used. The corresponding

activity was summed and the average time-activity curve and the total volume were

obtained. The advantage of this approach is that the analysis of the whole tumor is

performed quickly and simply and the results are identical to those of the ROI method.

Parts of the tumor that do not accumulate TYR are ignored by this method. By

combining this averaged time-activity data with the plasma input data (corrected for11CO

2 and 11C-proteins), we calculated the average PSR in nanomoles per milliliter

tumor tissue per minute using the modified Patlak analysis as described previously.18

The PSR in contralateral normal tissue was calculated using a ROI technique. A

tumor-to-nontumor ratio (T/N ratio) was calculated from the PSR in tumor tissue

and the PSR in contralateral normal tissue. The change in PSR after perfusion was

related to the preperfusional value and was expressed as a percentage of basal value.


The statistical procedures included a two-factor experiment with repeated measures

on one factor to compare PSR between measures and groups. Analyses were performed

on data sets corrected for missing data according to Winer.27 Post hoc comparison

was made with Student´s t-tests. A p value < 0.05 was considered significant. Analysis

was carried out by SPSS.

Results

PET results and pathological response for each patient are summarized in Table 2.

Pathological examination of the residual tumor mass showed no viable tumor in 6

patients (pCR 35%), 3 of whom had a melanoma. In 11 patients, variable amounts of

viable tumor were found at pathological examination (pPR 65%). Forty-six of the

scheduled 51 PET studies were completed (90%). Five PET studies were not

performed due to patient-related problems. All tumors were depicted as a hot spot on

the PET study before HILP with variable degrees of TYR accumulating parts (T/N

99


Ta

ble

2P

ET

results an

d p

atholo

gical resp

onse

Pro

tein

sy

nth

es

is ra

te (n

mo

l/ml/m

in) P

ath

olo

gic

al e

va

lua

tion

Pa

t. Be

fore

HIL

P 2

we

ek

s a

fter H

ILP

8 w

ee

ks

afte

r HIL

P R

es

po

ns

e %

Via

ble

Ma

cro

/mic

ro-s

co

pic

vie

w

no

. Tu

mo

r Co

ntra

lat T

/N ra

tio T

um

or C

on

trala

t T/N

ratio

Tu

mo

r Co

ntra

lat T

/N ra

tio tu

mo

r

12.4

50.1

417.5

0.1

70.1

41

.20.2

20.4

10.5

pC

R0

Rim

with

infla

mm

ato

ry tis

sue

23.5

10.1

523.4

1.0

20.1

66

.40.9

10.2

04.6

pC

R0

Infla

mm

ato

ry tis

sue

32

.53

0.4

35

.9n

.p.

n.p

.n

.p.

0.5

00

.31

1.6

pC

R0

Infla

mm

ato

ry tis

su

e

42.0

60

.40

5.2

0.7

40.2

62

.9n

.p.

n.p

.n

.p.

pC

R0

Infla

mm

ato

ry tis

su

e

52.1

30.4

15.2

0.7

10.2

33

.10.8

50.2

04.3

pC

R0

Rim

with

infla

mm

ato

ry tis

sue

61.3

10.4

03

.30.6

20.2

72.3

0.3

50.2

11

.7pC

R0

Infla

mm

ato

ry tis

sue

72.0

60.2

48

.61.3

90.4

33.2

0.4

80.2

91

.7pP

R<

10

Mic

roscopic

isle

ts o

f via

ble

tum

or

81.7

60.2

57

.00.6

40.2

62.5

0.8

00.2

13

.8pP

R<

10

Mic

roscopic

isle

ts o

f via

ble

tum

or

90.9

20.3

32

.8n.p

.n.p

.n.p

.0.9

90.3

23.1

pP

R<

10

Mic

rosco

pic

isle

ts o

f via

ble

tum

or

10

2.0

00.2

38.7

2.3

00.3

07

.71.4

60.2

26.6

pP

R<

10

Are

as o

f via

ble

tum

or

11

2.3

00.2

68.9

1.2

60.1

87

.02.3

10.1

614.4

pP

R<

10

Are

as o

f via

ble

tum

or

12

5.2

70.2

521.1

2.7

90.1

815.5

n.p

.n

.p.

n.p

.pP

R<

10

Are

as o

f via

ble

tum

or

13

1.6

80

.17

9.9

n.p

.n

.p.

n.p

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10.4

88

.2pP

R<

20

Rim

with

via

ble

tum

or

14

0.7

30.2

43.4

1.6

10.4

73

.41.8

30.4

83.8

pP

R<

50

Are

as o

f via

ble

tum

or

15

0.6

40.2

42.7

1.3

50.3

34

.12.1

30.6

03.6

pP

R<

50

Are

as o

f via

ble

tum

or

16

2.9

00.4

66.3

1.4

70.4

33

.42.4

80.3

18.0

pP

R<

50

Are

as o

f via

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tum

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17

1.6

10.2

46.7

1.2

40.0

913.8

0.9

40.0

811

.8pP

R<

50

Via

ble

tum

or

Contra

lat =

contra

late

ral n

orm

al tis

sue; p

CR

= p

ath

olo

gic

ally

com

ple

te re

sponse; p

PR

= p

ath

olo

gic

ally

partia

l response; n

.p. =

not p

erfo

rmed

100

Chapter 7

ratio > 1.00 in all patients). Preperfusion PSR in the patients who ultimately went on

to have a pCR was not significantly different from the PSR in the pPR group

(Fig.1). Analysis of the PET images at 2 and 8 weeks after perfusion showed a decrease

of TYR accumulating parts in all pCR patients.

Fig. 1 Protein synthesis rate (PSR) of

tumor with S.D. before, 2 and 8 weeks

after perfusion. Two and 8 weeks after

perfusion, PSR in pathologically com-

plete response (pCR) group decreased

significantly (p<0.05) in contrast with

PSR in pathologically partial response

(pPR) group. Significant difference

was found in PSR between the pCR and

pPR groups at 2 and 8 weeks (p<0.05).

HILP = hyperthermic isolated limb

perfusion

The PSR in the pCR group had decreased significantly at 2 and 8 weeks after perfusion

compared with preperfusional values (p<0.05) in contrast to the PSR in the pPR

group. A significant difference was found in PSR between the pCR and pPR group at

2 as well as at 8 weeks (p<0.05). The most substantial decrease in PSR occurred

within 2 weeks after perfusion. Figure 2 illustrates the succeeding PET studies in

patient 11. After an initial decrease in PSR at 2 wk, a renewed outgrow of the tumor

was observed at 8 weeks after perfusion. Necrosis within the tumor was visualized

as a cold spot. In this patient, TYR-PET indicated the need for an early resection of

the tumor as perfusion did not seem to have the desired result. Pathological

examination revealed areas of viable tumor that encompassed less than 10% of the

total tumor volume.

The median PSR in contralateral muscle tissue was 0.28 and ranged from 0.08 to

0.60. PSR in tumor tissue was higher than in the corresponding contralateral normal

tissue (p<0.05). Median PSR in nonviable tumor tissue was 0.62 and ranged from

0.22 to 0.91. With a threshold PSR of 0.91, the highest value obtained from nonviable

tumor tissue, the sensitivity and specificity of TYR-PET after HILP treatment were

82% and 100%, respectively. The predictive value of a PSR > 0.91 for having viable

tumor after HILP was 100%, whereas the predictive value of a PSR ≤ 0.91 for having

nonviable tumor tissue after HILP was 75% (Fig. 3). The two patients in the pPR

group with a PSR < 0.91, patients 8 and 9, showed microscopic islets of tumor cells

surrounded by extensive necrosis on pathological examination. With a threshold PSR

of 0.48, the lowest value obtained from viable tumor tissue, the sensitivity and

101


specificity of TYR-PET after HILP treatment were 100% and 33%, respectively.

The predictive values for viable and nonviable tumor tissue after HILP were 73%

and 100%, respectively.

Figure 4 shows the percentage of basal value of the tumor after perfusion. All patients

in the pCR group showed a reduction of PSR whereas some pPR patients showed a

reduction and others an increase in PSR after perfusion. The reduction in the pCR

group was significant at 2 and 8 weeks after perfusion. However, based on a certain

percentage of reduction of basal value, no assumption could be made as to whether

or not the individual patient showed a pCR or a pPR.

Two different histopathological groups could be distinguished after perfusion:

nonviable tumor tissue, corresponding with inflammatory tissue, and viable tumor

tissue. Figure 5 shows the PSR in these two different histopathological groups

Fig 2. Transversel PET image of a patient with a malignant fibrous histiocytoma of the thigh

(patient 11). Before perfusion (A) the tumor is clearly depicted as a heterogeneous mass with

a PSR of 2.30 nmol/ml/min. After initial reduction in PSR (1.26) at 2 weeks after perfusion

(B), the malignant fibrous histiocytoma showed renewed growth (PSR 2.31) at 8 weeks after

perfusion (C). The gray scale equates a particular hue to a particular PSR in nmol/ml/min.

PA viable PA Nonviable Total

PET Viable 9 0 9

PET Nonviable 2 6 8

Total 11 6 17

Fig 3. Cross tabulation table with threshold PSR of 0.91. Sensitivity and specificity of TYR-

PET were 82% and 100%, respectively. Predictive value for viable tumor after HILP was

100%, whereas predictive value for nonviable tumor tissue after HILP was 75%. Two patients

in the pPR group with PSR<0.91, showed microscopic islets of tumor cells surrounded by

extensive necrosis on pathological examination. PA = pathology

A B C

102

Chapter 7

compared with the PSR in normal contralateral muscle. The average PSR in

inflammatory tissue was significantly lower than the PSR values in viable tumor

tissue (p<0.05).

Discussion

PET has made it possible to study biochemical changes of cancer tissue and to study

the effect of treatment on metabolism in vivo. This study demonstrates a significant

decline in the protein metabolism of locally advanced soft-tissue sarcomas and skin

cancer with a pathologically complete response after HILP with TNF and melphalan.

These changes were already evident within 2 weeks. In patients with a pPR, this

decrease was not significant. These findings are similar to the results of a previous

study performed in the same manner with FDG.9 With TYR-PET, however, there

was a significant difference at 2 and at 8 weeks in PSR between the pCR and pPR

groups, a finding that was not observed with FDG-PET. That previous finding may

have been caused by the fact that FDG is also accumulated by inflammatory tissue,

resulting in an overlap in glucose metabolism between viable tumor and inflammatory

tissue. In this study, TYR was also accumulated by inflammatory tissue that existed

Fig. 4 Percentage of basal value of tu-

mor for all patients 2 and 8 weeks after

perfusion. pCR = pathologically com-

plete response; pPR = pathologically

partial response; HILP = hyperthermic

isolated limb perfusion

Fig. 5 Protein synthesis rate (PSR) in

viable tumor, inflammatory and con-

tralateral normal tissue. PSR in viable

tumor tissue was significantly higher

than inflammatory tissue (p<0.05)

103


after HILP but was accumulated significantly less than in viable tumor tissue. Because

TYR can better discriminate between viable tumor tissue and inflammatory tissue

than FDG, TYR is a more reliable technique to evaluate treatment response. The

major question for clinicians is the exact timing for the surgical resection after HILP

because the tumoricidal effect of HILP seems to be time related. If the threshold PSR

of 0.91 was exceeded 8 weeks after HILP, we could with certainty predict that viable

tumor was still present and surgical resection of the tumor remnants was indicated.

However, when resection was omitted with a PSR less than 0.91, there was a 25%

chance of leaving microscopic islets of tumor tissue. The resolution of the PET camera

may be the limiting factor in detecting these microscopic islets of viable tumor,

although it remains questionable if these small amounts of tumor tissue surrounded

by avascular necrosis can lead to a local recurrence. Instead of surgical resection,

these patients could possibly also be treated with external beam radiotherapy, and

monitored closely for development of a local recurrence. Leaving residual tumor

mass was safe when PSR was lower than 0.48 after HILP.

Before perfusion, there was no significant difference in PSR between the patients in

the pCR and pPR group. This was in contrast with the results of the FDG study

where we found a significant difference in glucose consumption before perfusion

between both groups.9 So TYR can not be used to predict the likelihood of a response

to HILP. For FDG we also found a correlation between tumor malignancy grade and

the level of glucose metabolism.3 This is not the case for the protein synthesis of the

different grades of soft-tissue sarcomas in this study but the number of patients is

small. This difference between FDG and TYR may be explained by the fact that

FDG is trapped inside the cell as a result of an increased level of glucose transporters

on the cell membrane.28 FDG accumulates as it reaches its end in its metabolic pathway

as FDG-6-phosphate; the more glucose transports there are on the cell membrane,

the more FDG is incorporated in the cell, corresponding with a high malignancy

grade. TYR, not hampered by an anorganic isotope, continues its metabolic pathway

and is not accumulated in the cell.

Combining the results of this study with the results of our previous FDG study, it is

tempting to state that FDG-PET should be performed before HILP to identify patients

who will most likely benefit from this treatment and TYR-PET should be performed

8 weeks after HILP to evaluate the outcome of the therapy. However, our results

should be interpreted with caution since this patient population is a small group of

heterogeneous soft-tissue sarcomas and skin cancers and only large tumors were

included. Additional data are needed on TYR-PET in more patients with other tumor

types treated with other chemotherapeutic agents and pathological examination as

the gold standard.

104

Chapter 7

Conclusion

This study demonstrates that TYR-PET indicates the pathologic tumor response to

chemotherapy in an investigational setting used with HILP with TNF and melphalan

for locally advanced tumors. Based on the calculated PSR after perfusion, a good

indication was found towards the pathological outcome. Inflammatory tissue after

treatment did not interfere with viable tumor on the images, suggesting that it may be

worthwhile to pursue TYR-PET in other therapy evaluation settings.

References

1 Conti PS, Lilien DL, Hawley K, et al. PET and [18F]-FDG in oncology: a clinical

update. Nucl Med Biol 1996; 23: 717-735.

2 Rigo P, Paulus P, Kaschten BJ, et al. Oncological applications of positron emission

tomography with fluorine-18 fluorodeoxyglucose. Eur J Nucl Med 1996; 23: 1641-

1674.

3 Nieweg OE, Pruim J, van Ginkel RJ, et al. Fluorine-18-fluorodeoxyglucose PET

imaging of soft-tissue sarcoma. J Nucl Med 1996; 37: 257-261.

4 Kole AC, Nieweg OE, van Ginkel RJ, et al. Detection of local recurrence of soft-

tissue sarcoma with positron emission tomography using [18F]fluorodeoxyglucose.

Ann Surg Oncol 1997; 4: 57-63.

5 Findlay M, Young H, Cunningham D, et al. Noninvasive monitoring of tumor

metabolism using fluorodeoxyglucose and positron emission tomography in colorectal

cancer liver metastases: correlation with tumor response to fluorouracil [see

comments]. J Clin Oncol 1996; 14: 700-708.

6 Jones DN, McCowage GB, Sostman HD, et al. Monitoring of neoadjuvant therapy

response of soft-tissue and musculoskeletal sarcoma using fluorine-18-FDG-PET.

Blood 1996; 37: 1438-1444.

7 Lindholm P, Leskinen Kallio S, Grenman R, et al. Evaluation of response to

radiotherapy in head and neck cancer by positron emission tomography and

[11C]methionine. Int J Radiat Oncol Biol Phys 1995; 32: 787-794.

8 Kubota R, Yamada S, Kubota K, et al. Intratumoral distribution of fluorine-18-

fluorodeoxyglucose in vivo: high accumulation in macrophages and granulation

tissues studied by microautoradiography. J Nucl Med 1992; 33: 1972-1980.

9 van Ginkel RJ, Hoekstra HJ, Pruim J, et al. FDG-PET to evaluate response to

hyperthermic isolated limb perfusion for locally advanced soft-tissue sarcoma. J

Nucl Med 1996; 37: 984-990.

10 Lewis PJ, Salama A. Uptake of fluorine-18-fluorodeoxyglucose in sarcoidosis. J

Nucl Med 1994; 35: 1647-1649.

11 Ishiwata K, Vaalburg W, Elsinga PH, Paans AM, Woldring MG. Metabolic studies

with L-[1-14C]tyrosine for the investigation of a kinetic model to measure protein


12 Schober O, Meyer GJ, Duden C, et al. [Amino acid uptake in brain tumors using

positron emission tomography as an indicator for evaluating metabolic activity and

malignancy]. ROFO Fortschr Geb Rontgenstr Nuklearmed 1987; 147: 503-509.

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13 Derlon JM, Bourdet C, Bustany P, et al. [11C]L-methionine uptake in gliomas.

Neurosurgery 1989; 25: 720-728.

14 Lilja A, Lundqvist H, Olsson Y, et al. Positron emission tomography and computed

tomography in differential diagnosis between recurrent or residual glioma and

treatment-induced brain lesions. Acta Radiol 1989; 30: 121-128.

15 Daemen BJ, Elsinga PH, Ishiwata K, Paans AM, Vaalburg W. A comparative PET

study using different 11C-labelled amino acids in Walker 256 carcinosarcoma-bearing

rats. Int J Rad Appl Instrum [B] 1991; 18: 197-204.

16 Ishiwata K, Vaalburg W, Elsinga PH, Paans AM, Woldring MG. Comparison of L-

[1-11C]methionine and L-methyl-[11C]methionine for measuring in vivo protein


17 Bolster JM, Vaalburg W, Paans AM, et al. Carbon-11 labelled tyrosine to study

tumor metabolism by positron emission tomography (PET). Eur J Nucl Med 1986;

12: 321-324.

18 Willemsen ATM, van Waarde A, Paans AM, et al. In vivo protein synthesis rate

determination in primary or recurrent brain tumors using L-[1-11C]-tyrosine and

PET. J Nucl Med 1995; 36: 411-419.

19 Pruim J, Willemsen AT, Molenaar WM, et al. Brain tumors: L-[1-C-11]tyrosine

PET for visualization and quantification of protein synthesis rate. Radiology 1995;

197: 221-226.

20 Kole AC, Pruim J, Nieweg OE, et al. PET with L-[1-carbon-11]-tyrosine to visualize

tumors and measure protein synthesis rates. J Nucl Med 1997; 38: 191-195.




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26 Giron C, Luurtsema G, Vos MG, et al. Microwave-induced preparation of 11C-

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27 Winer BJ. Statistical principles in experimental design. New York: McGraw Hill,

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107


Piet C. Limburg2

D. Albertus Piers3


Harald J. Hoekstra1

Department of Surgical Oncology1, Internal Medicine2 and Nuclear Medicine3,

University Hospital Groningen, The Netherlands.

Annals of Surgical Oncology 2002; 9: 355-363.

Value of continuous leakage monitoring with radio-

active Iodine-131 labeled human serum albumin

during hyperthermic isolated limb perfusion with

TNF and melphalan

108

Chapter 8

Abstract

The aim of this study was to analyze the value of continuous leakage monitoring with

radioactive Iodine-131 labeled human serum albumin (RISA) in patients treated with

hyperthermic isolated limb perfusion (HILP) with tumor necrosis factor alpha (TNF)

and melphalan. Forty-eight patients with melanoma (n = 14) or soft tissue sarcoma

(n = 34) of an extremity underwent 51 perfusions. Perfusion was performed at the

iliac level in 22 cases, at the popliteal level in 16 cases, at the femoral level in 7

cases and in 6 cases at the axillary level. Leakage rates, perfusion circuit and systemic

levels of TNF, interleukin-6, C-reactive protein (CRP) were determined, as were

systemic hematological and metabolic profiles and tumor response. The mean

isotopically measured leakage was 2.9 % (95% confidence interval 2.0 – 3.8%, range

0-15.5%). Systemic leakage was ≤2% in 28 perfusions (55%) and >2% in 23

perfusions (45%). The correlation between the maximal monitored leakage and

maximal systemic TNF levels was 0.7114 (p < 0.0001). The area under the curve

(AUC) for TNF in the perfusion circuit, indicating the exposure of the perfused limb

to TNF, was 18.7% lower in the >2% leakage group (p=0.0457). No significant

differences in tumor response were found between groups. AUC for systemic TNF,

indicating the exposure of the patient to TNF, was 18.1 times higher in the >2%

leakage group (p<0.0001) resulting in a significant decrease in leucocyte and platelet

count, hyperbilirubinemia, hypocholesterolemia and proteinemia. No beneficial effect

of the systemically leaked TNF and melphalan was seen on the occurrence of distant

metastasis during follow-up. There was a significant difference between perfusions

performed at the iliac and femoral levels compared with leakage values at the popliteal

level, p < 0.0001 and 0.0159 respectively. A good correlation between RISA leakage

measurement and TNF exposure during and after HILP with TNF and melphalan

was demonstrated. RISA leakage measurement serves as a good guide for the

effectiveness of isolation during perfusion. If leakage exceeds the 2% limit during

perfusion, less exposure of the tumor bearing limb to TNF, increased exposure of the

patient systemic circulation to TNF, and more systemic side effects can be expected.

Introduction

Creech and Krementz developed isolated limb perfusion with chemotherapy for the

treatment of extremity melanoma in humans in 1958.1 Stehlin et al modified the

technique in 1969 to include hyperthermia.2 Since then, hyperthermic isolated limb

perfusion (HILP) with different chemotherapeutic agents has been used by several

institutes worldwide for the treatment of advanced extremity melanoma and soft

tissue sarcoma. Recently, an international study comparing local excision and adjuvant

109

Leakage during isolated limb perfusion with TNF

HILP with melphalan with wide excision only revealed a trend for a longer disease-

free interval after HILP with melphalan but no benefit from HILP in terms of time to

distant metastasis or survival.3 With the conclusion that prophylactic HILP with

melphalan could not be recommended as an adjunct to standard surgery in high-risk

primary limb melanoma, the indication for HILP is currently restricted to advanced

melanoma and primarily irresectable soft tissue sarcoma. For these indications the

addition of tumor necrosis factor alpha (TNF) to melphalan seems promising.4,5

With the introduction of TNF, monitoring of leakage of the isolated circuit into the

systemic circulation has been mandatory since TNF levels in the perfusion circuit

are approximately 10 times the maximum tolerated systemic levels.6 If significant

leakage occurs during HILP, the resultant TNF induced systemic inflammatory

response syndrome (SIRS) could be fatal.7 Different methods for measurement of

leakage are used. In the early days, Stehlin et al determined the amount of radioactive

Iodine-131 labeled human serum albumin (RISA) through the use of blood samples

from the systemic circulation, and calculated the leakage factor (LF).8 Although

determination of blood samples takes time and is discontinuous, it is frequently used

by other groups.9,10 To overcome these disadvantages, Stehlin and associates were

the first to describe a method of continuous external leakage monitoring with RISA.11

Because of safety regulations, nuclear medicine techniques are not always allowed

in the operation zoom. Another method, the measurement of Evans blue concentration

in plasma by means of a spectral photometer, overcomes this problem.12 Two other

groups introduced the use of handheld gamma detectors for leakage measurements;

however, a great dependency was observed on the distance and angle from the source

with this system.13,14

Since 1991, patients with advanced melanoma or soft tissue sarcoma of the limbs,

have been treated at the Groningen University Hospital by HILP with TNF, melphalan

with or without interferon gamma (IFN) as perfusion agents, followed by delayed

surgical excision. The aim of this study was to analyze the value of continuous leakage

monitoring with RISA in patients treated with TNF perfusion with respect to systemic

levels of TNF, interleukin (IL)-6, C-reactive protein (CRP) as well as hematological

and metabolic profiles and tumor response.

Patients and methods

Forty-eight patients with melanoma (n = 14) or soft tissue sarcoma (n = 34) of the

extremity underwent 51 perfusions with a combination of TNF and melphalan, with

or without IFN. Twenty-one males and 27 females, with a median age of 54 years

(range 18-80 years) were treated. Perfusion was performed at the iliac level in 22

cases (43%), at the popliteal level in 16 cases (31%), at the femoral level in 7 cases

110

Chapter 8

(14%), and in 6 cases (12%) at the axillary level. All patients were treated after

informed consent was obtained according to institutional guidelines.

Perfusion Technique

The perfusion technique employed at the Groningen University Hospital is based on

the technique developed by Creech et al.1 and has been described in detail previously.15

Briefly, after ligation of all collateral vessels and heparinization of the patient with

3.3 mg heparin/kg bodyweight (Thromboliquine, Organon BV, Oss, the Netherlands),

the axillary, iliac (internal iliac artery is temporarily closed), femoral, or popliteal

vessels were cannulated and connected to an extracorporal circuit. The perfused limb

was wrapped in a thermal blanket to reduce heat loss. To prevent collateral circulation

in subcutaneous tissue and muscle, an occluding rubber bandage was twisted around

the root of the extremity and fixed around a pin inserted into the head of the humerus

(axillary perfusion) or iliac crest (iliac perfusion). An inflating tourniquet was used

in femoral or popliteal perfusions. Perfusion was performed during 90 min under

mild hyperthermia (39-40°C) and physiologically optimal conditions.16 At the start

of perfusion, 3 mg (upper extremity) or 4 mg (lower extremity) TNF (Boehringer,

Ingelheim, Germany) was injected as a bolus into the arterial line. Eighteen patients

also received a dose of 0.2 mg INF (Boehringer, Ingelheim, Germany) subcutaneously

1 and 2 days before perfusion, followed by 0.2 mg INF injected into the arterial line

at the start of perfusion. Melphalan (L-phenylalanine mustard, Glaxo-Wellcome,

London, England) was administered 30 min later, as 10 mg/L extremity volume (leg)

or 13 mg/L extremity volume (arm).17 The volume of the limb was determined before

surgery by immersion.

All perfusions were performed with a bubble oxygenator roller pump and heat

exchanger. The perfusate was oxygenated by a mixture of O2 and CO

2 and consisted

of 350 ml 5% dextran 40 in glucose 5% (Isodex, Pharmacia AB, Uppsala, Sweden),

500 ml blood (250 ml red blood cells, 250 ml plasma), 30 ml of 8.4% NaHCO3, 0.5

ml 5000 IU/ml heparin. The perfusions were flow regulated on the basis of the arterial

and venous pressure measured at one end of the double lumen catheter used. After 90

min of perfusion, the limb was flushed with 2 L dextran 40 in glucose 5% (Isodex)

and 500 ml blood (250 ml red blood cells, 250 ml plasma), catheters were removed,

the circulation restored and the heparin antagonized with protamine chloride (Hoffman

La Roche, Mijdrecht, the Netherlands). A lateral fasciotomy of the anterior

compartment of the lower leg or arm was performed to prevent a compartment

syndrome.18

111


Leakage Measurement

Any leakage into the systemic circulation was continuously monitored with radioactive

tracers. A small calibration dose of RISA (0.5 MBq) and a dose of radioactive

Technetium-99m labeled human serum albumin (RTcSA; 10 MBq) were administered

into the systemic circulation, after surgical isolation of the extremity was

accomplished. The thyroid was saturated 1 day before the operation by oral

administration of iodine (15 drops of Lugol solution twice daily). A 10 times higher

dose of RISA (5 MBq) was injected into the perfusion circuit when perfusion was

stable. The 364-keV gamma rays emerging from the RISA and the 140-keV gamma

rays emerging from the RTcSA are measured with a NaI detector, which was placed

in a flat field lead lined collimator that was mounted on an articulating mobile stand.

This stand permits easy positioning of the detector, after it is covered with a sterile

bag, above the heart of the patient. Careful attention is paid to ensure that the field of

view of the detector did not cover parts of the HILP circuit. The detector signals

generated by the photomultiplier tube were directed to an amplifier and then to a

single-channel analyzer, allowing online data processing by a personal computer.

The count rate of the 0.5-MBq RISA determined a baseline count level, corrected for

room background. The 10-MBq RTcSA served to check the volume dilution caused

by to fluid infusions or displacement of the NaI detector during the registration period.

Leakage from the perfused limb to the systemic circulation resulted in an increase of

the baseline count level. This increase, corrected for the blood volume ratio and the

radioactivity ratio in both compartments, was a direct measure for the percentage of

leakage and was continuously registered during the whole procedure. Stehlin was

the first to describe the LF based on the following equation:

where cpmsystemic

is the systemic count rate observed during perfusion, cpmbaseline

is

the systemic count rate at the beginning of perfusion, Dsystemic

is the dose injected into

the patient’s systemic circulation, and Dperfusion

is the dose injected into the perfusion

circuit Vtotal

is the total blood volume (perfusion circuit + patient’s systemic

circulation), Vsystemic

is the blood volume of the patient’s systemic circulation.11

Blood sampling procedure and assays

A baseline blood sample from the patients systemic circulation was taken from an

indwelling radial artery cannula before the start of the operation, and at 5, 30, 60,

LF = (cpmsystemic - cpmbaseline)

< > Dsystemic

< > Vtotal

< > 100%

cpmbaseline Dperfusion Vsystemic

112

Chapter 8

and 89 min after the start of perfusion. Samples from the perfusion circuit were also

taken at the same time intervals. After restoration of the circulation in the perfused

limb, systemic samples were taken at 1, 5, 10, 30 and 60 min after removal of the

arterial clamps, hourly thereafter for at least 8 hours and finally the next morning.

Venous blood samples to study the hematological and metabolic profiles of urea

nitrogen, creatinine, bilirubin, alkaline phosphatase, aspartate aminotransferase,

alanine aminotransferase, gammaglutamyltranspeptidase, protein, cholesterol, lactic

dehydrogenase (LDH) with its iso-enzymes, creatine phosphokinase and myoglobin

were taken a day before perfusion, at the day of perfusion, and every day after

perfusion until day 7. A final blood sample was taken one month after perfusion.

Blood samples (3 ml) were collected in EDTA vacutainer tubes, and kept on melting

ice during transport to a centrifuge. Samples were centrifuged for 10 min at 3000

rpm at 0°C and the separated plasma kept at –80°C until analysis.

TNF levels were determined by specific immunoradiometric assay (Medgenix

Diagnostics, Soesterberg, the Netherlands). Samples were processed according to

the guidelines of the manufacturer. IL-6 and CRP levels were measured by in-house

sandwich ELISA’s as described previously19, by using commercial reagents for IL-6

(CLB, Amsterdam, the Netherlands; detection limit 10 ng/L) and for CRP (DAKO,

Glostrup, Denmark; normal level <2.3 mg/L).

Assessment of tumor response

Responses were assessed by standardized World Health Organization criteria.20

Complete response (CR) was defined as the disappearance of all measurable disease

in the limb for longer than 4 weeks, partial response (PR) as regression of the tumor

size by >50% for longer than 4 weeks, and no change (NC) as regression of <50% of

the tumor in the limb or progression of <25% for longer than 4 weeks. To analyze

whether or not a high systemic leakage was of influence in the occurrence of distant

metastasis subanalyses of this parameter in a group of patients with grade II and III

soft tissue sarcomas was performed.


Values are expressed as mean ± SEM. Comparison between mean values of different

groups was performed with the unpaired or in case of measuring the same variable in

the same patient at different time points, with the paired Student’s t-test. Areas under

the curve (AUC) were determined by the trapezoid rule. Survival curves were

calculated according to the Kaplan Meier method and log rank test.21 Values of

p ≤ .05 were considered to be statistically significant. Graph Pad Prismâ version 2.0

for Windows (GraphPad, San Diego, CA) statistical software was used.

113


Results

Systemic leakage

For the 51 perfusions, the mean isotopically measured leakage was 2.9 % (95%

confidence interval, 2.0 – 3.8%, range 0-15.5%). After 60 minutes of perfusion in

the patient with the highest leakage (15.5%), it was noted that the rubber bandage

twisted around the root of the extremity was ruptured. Since this was the cause of the

high leakage and perfusion was not completed, the data from this patient are excluded

from the remainder of the analyses. Systemic leakage was ≤ 2% in 28 perfusions

(55%) and >2% in 23 perfusions (45%). In the latter group, 11 perfusions (22%) led

to systemic leakage of >5%. In addition, analysis of different parameters between

the group of patients with ≤ 2% leakage and the group of patients with >2% leakage,

was made. Figure 1 shows the measured leakage at different perfusion levels. There

was a significant difference between perfusions performed at the iliac and femoral

levels compared with leakage values at the popliteal level, (p < 0.0001 and 0.0159

respectively). There was no leakage encountered in patients with axillary perfusions.

Fig. 1 Scatter diagram of leakage at dif-

ferent perfusion levels, the uninterrupted

line represents mean values. A significant

difference was observed between perfu-

sions performed at the iliac and femoral

levels compared with leakage values at

the popliteal level, (p < 0.0001 and

0.0159 respectively)

Perfusion circuit levels

At 5 minutes, mean TNF levels in the perfusion circuit were 6798 ± 528 ng/ml

(Fig. 2). During perfusion, a significant drop in TNF levels in the perfusion circuit

occurred with a significant lower concentration of TNF in the perfused limb in patients

with >2% leakage at 30 (p = 0.0201), 60 (p = 0.0337) and 89 minutes (p = 0.002).

The calculated mean AUC, indicating the exposure of the perfused limb to TNF, was

18.7% less in the >2% leakage group (p=0.0457). IL-6 levels in the perfusate, as one

of the most important proinflammatory cytokines, progressively increased from 30

minutes until the end of the perfusion, reaching 4.2 ± 1.1 ng/ml in the ≤2% leakage

114

Chapter 8

Fig. 2 Tumor necresis factor (TNF)

levels in the perfusion circuit (mean ±SEM). A significant decrease in TNF

levels occurred with significant lower

concentration of TNF in the perfused

limb in patients with >2% leakage at

30 (p = 0.0201), 60 (p = 0.0337) and

89 minutes (p = 0.002). Mean area un-

der the curve, indicating the exposure

of the perfused limb to TNF, was 18.7%

less in the >2% leakage group

group and 11.7 ± 3.5 ng/ml in the >2% leakage group (p=0.0455). CRP levels in the

perfusion circuit remained at the detection level, and no significant differences were

observed between the leakage groups.

Systemic levels

Systemic TNF levels in patients with >2% leakage were already significantly higher

at 5 min after TNF injection compared with the group of patients with ≤2% leakage.

Peak systemic TNF values of 116.5 ± 28.9 ng/ml were reached in the >2% leakage

group at the end of perfusion, compared with 11.8 ± 3.4 ng/ml in the ≤2% leakage

group (p < 0.0001) (Fig. 3). The calculated mean systemic AUC, indicating the

exposure of the patient to TNF, was 18.1 times higher in the >2% leakage group

(p<0.0001). Ten minutes after release of the tourniquet we observed a significant

systemic peak level of TNF in the ≤2% group possibly caused by the TNF still present

in the perfused limb after the washout procedure (p=0.026). To calculate the correlation

between maximum systemic TNF levels and the maximum monitored leakage using

RISA measured during perfusion, Pearson’s correlation (two-tailed) was used. Figure

4 illustrates the observed correlation with r = 0.7114 and p < 0.0001. A strong

correlation was also found between the maximal observed leakage and maximum

IL-6 concentration measured in the postoperative period (r = 0.7737, P<0.0001).

IL-6 levels appeared in the systemic circulation 30 minutes after the start of the

perfusion and maximal levels were reached 2 hours after HILP (19.5 ± 5.8 ng/ml

≤2% leakage versus 77.7 ± 20.8 ng/ml >2% leakage; p=0.0089). The AUC of IL-6

was 4.7 times higher in the >2%leakage group compared with ≤2% leakage group

(p<0.0243).

115


CRP started to increase 6 hours after HILP and reached its maximal value 2 days

after perfusion (185.8 ± 25.5 mg/L ≤2% leakage versus 226.7 ± 32.7 mg/L, >2%

leakage; not significant). The AUC of CRP between both groups however was not

significantly different.

Hematological and metabolic parameters

Leukocyte counts increased from 7.7 ± 0.3x109/L to 13.0 ± 0.6x109/L one day after

perfusion. Five, 6, and 7 days after perfusion a significant difference between the

two leakage groups was observed (Fig. 5). Platelet counts decreased from 303.6 ±13.4x109/L before perfusion to 124.3 ± 10.7x109/L 4 days after perfusion.

Fig. 3 Tumor necrosis factor (TNF) lev-

els in the systemic circulation of the pa-

tient (mean ± SEM). A significant dif-

ference was found between the >2%

leakage group and the ≤2% leakage

group is starting 5 min after TNF injec-

tion until the second postoperative day

(p<0.05). The mean systemic area un-

der the curve, indicating the exposure

of the patient to TNF, was 18-times

higher in the >2% leakage group

Fig. 4 Pearson’s correlation (two-

tailed) between maximal systemic TNF

levels measured during perfusion and

maximal monitored leakage using

RISA (r = 0.7114 and P<0.0001)

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Chapter 8

The low platelet levels persisted longer in the >2% leakage group. Kidney function

was well preserved in all patients, although urea nitrogen and creatinine levels in the

>2% leakage group were significantly higher during the first 5 days after perfusion;

these levels however remained within normal limits. Liver function tests showed an

increase in bilirubin values from 10.5 ± 0.9 µmol/L to 44.8 ± 11.3 µmol/L, 4 days

after perfusion in the >2% leakage group, with significant differences compared with

the ≤2% leakage group (Fig. 6). Figure 6 illustrates the decrease in protein levels and

cholesterol levels after perfusion, with significant differences between both leakage

groups. Alkaline phosphatase increased from 86.1 ± 6.5 U/L to 159.4 ± 32.8 U/L,

aspartate aminotransferase increased from 22.8 ± 1.5 U/L to a maximum of 62.1 ±13.4 U/L on the fifth day after perfusion, alanine aminotransferase increased from

21.9 ± 2.6 U/L to a maximum of 80.3 ± 11.6 U/L on the sixth day after perfusion, and

gammaglutamyltransferase increased from 37.7 ± 8.7 U/L to a maximum of 120.1 ±18.6 U/L on the sixth day after perfusion. LDH increased from 224.8 ± 9.0 U/L to a

maximum of 417.3 ± 19.1 U/L on the second day after perfusion. LDH iso-enzymes

1 and 2 showed a decrease, whereas LDH iso-enzymes 4 and 5 increased one day

after perfusion. LDH iso-enzyme 3 remained at the same level. Creatine phospho-

kinase levels increased from 28.3 ± 2.4 U/L to a maximum of 496.4 ± 197.6 U/L on

Fig. 5 White blood cell count (WBC)

and platelets (PLT) levels from before

perfusion to 30 days after perfusion

(mean ± SEM). * indicates a significant

difference between both leakage groups

(p < 0.05)

117


the second day after perfusion. Myoglobin levels increased from 30.2 ± 2.4 µg/L to

a maximum of 422.8 ± 99.7 µg/L one day after perfusion. None of these variables

showed a significant difference between both leakage groups.

Tumor Response

In the ≤2% leakage group, 14 patients showed a CR and the same number of patients

a PR. In the >2% leakage group, 11 patients showed a CR, 10 patients a PR and 2

patients had NC. No significant differences in response to TNF HILP were found

between the groups. No significant difference was observed in the occurrence of

distant metastasis or survival in the subanalyses of a group of patients with grade II

or III soft tissue sarcoma and >2% leakage (Fig. 7).

Fig. 6 Bilirubin, protein and cholesterol

levels from before perfusion to 30 days

after perfusion (mean ± SEM). * indicates

a significant difference between both leak-

age groups (p < 0.05)

118

Chapter 8

Discussion

The purpose of continuous leakage monitoring with RISA during HILP is to indicate

the amount of chemotherapeutic agent that is leaking from the perfusion circuit into

the patient’s systemic circulation. When leakage occurs, measures to reduce leakage

should be available. During perfusion there is a dynamic balance between two pressure

compartments: the patients systemic vasculature and the isolated circuit. The pressure

of the former compartment can be influenced by adjusting the systemic blood pressure,

whereas that of the latter can be affected by alterations in the extracorporeal flow

rate. Thus, to decrease leakage, the anesthesiologist can increase the patient’s blood

pressure or the surgeon can reduce the flow rate in the heart-lung machine. Different

methods for measurement of leakage have been used. The previously described method

with RISA is the most frequently used; however, a MEDLINE search to find any

articles calculating the correlation between systemic melphalan levels and leakage

in case of HILP with melphalan only, produced no results. The first report on TNF

levels after HILP with TNF, from Gérain et al. in 1992, demonstrated no significant

correlation between leakage and cytokine levels at any time, raising questions about

the value of the leakage measurement procedure.6

The aim of this study was to investigate whether or not the RISA leakage

measurements during HILP with TNF used in the Groningen University Hospital are

accurate in predicting systemic TNF levels. We observed a good correlation between

maximal systemic TNF levels and the maximum monitored leakage (r = 0.7114;

p < 0.0001). We were surprised to find that the correlation between maximal leakage

and maximal IL-6 concentration measured in the postoperative period was higher

than the correlation between maximal leakage and maximal TNF levels (r = 0.7737

versus r = 0.7114). IL-6 levels occurred in response to TNF, with a high correlation

between maximal levels of both cytokines (r = 8097). Stam et al. also found a strict

correlation between the degree of leakage estimated by isotope monitoring and the

Fig. 7 Absence of distant metastasis in

patients with grade II or III soft tissue sar-

coma. No significant difference was found

between the >2% leakage group and the

≤2% leakage group

119


measured maximal systemic TNF levels in the same treatment setting (r = 0.7886,

p=0.0067; calculation based on their data).22 They also found a sharper relation

between systemic IL-6 curves and duration of exposure to high TNF levels in patients

with high leakage compared with a group of patients with no leakage. A significant

difference in leakage was found between the iliac/femoral perfusion levels and

popliteal perfusion level. This corresponds with the study of Klaase et al., who assessed

six variables for their influence on systemic leakage. The level of isolation and the

diameter of the venous cannula emerged as significant factors.23 In our study we

could not find a significant role for the diameter of the venous cannula (data not

shown). The importance of the perfusion level could be partly explained by the

different type of isolation technique used, namely, a rubber band tourniquet at the

iliacal level versus a inflatable pressure regulated band at the popliteal level.

In the analysis of our data, we distinguished two leakage groups, with a cutoff point

at 2%. Two percent represents approximately the measurement fault of the RISA

procedure. TNF levels in the perfusion circuit were about 7000 ng/ml, approximately

50-times higher than peak systemic levels. A significantly lower concentration of

TNF in the perfused limb in patients with >2% leakage was demonstrated resulting

in a decreased AUC, indicating an 18.7% lower exposure of the perfused limb to

TNF in the >2% leakage group. This decrease in TNF exposure, however, did not

result in a significant reduction of tumor response between the groups. This result

supports the initiation of TNF dose reduction studies. Thom et al. observed the same

decreased TNF perfusion circuit levels in patients with ≥1% leakage.24 The Rotterdam

perfusion group did not demonstrate a significant difference in perfusion circuit TNF

levels between a high and low-leakage group, possibly because of a limited number

of samples available.22

TNF levels in the systemic circulation of the patients were approximetely 100 ng/ml

in the >2% leakage group at the end of perfusion, compared with 10 ng/ml in the

≤2% leakage group. In patients with ≤2% leakage, systemic TNF exposure was 18.1

times less as calculated by the AUC. On the basis of the hypothesis that micro

metastatic disease is attacked by the leaked TNF and melphalan, a higher systemical

exposure of TNF could have its effect on the occurence of distant metastasis during

follow-up. However, subanalysis of the occurence of distant metastasis or survival

in a group of patients with grade II or III soft tissue sarcomas did not reveal this

phenomenon. IL-6, as one of the most important proinflammatory cytokines, appeared

in the systemic circulation 30 minutes after the start of the perfusion with maximum

levels reached 2 hours after HILP. CRP levels started to increase 6 hours after HILP

and reached its maximum 2 days after perfusion. A three wave pattern was seen; the

first wave caused by the systemically leaked TNF that generated a second wave of

120

Chapter 8

IL-6 some hours after perfusion, followed by a third wave of CRP that lasts for

several days.

TNF leakage was associated with a decrease in leucocyte and platelet count, with

significantly lower values in the >2% leakage group. Representing cytolytic liver

toxicity, a significantly hyperbilirubinemia, hypocholesterolemia and proteinemia

was observed in the >2% leakage group. A increase in the activity of the fraction of

LDH iso-enzymes 4 and 5 after perfusion was partly related to hepatotoxicity and

partly to muscle damage. No significant difference between both leakage groups

was found for creatine phosphokinase levels or myoglobin levels although both

parameters showed a significant rise after HILP. The same results were obtained by

Sorkin et al. who diminished TNF leakage after flow rate reduction during TNF

HILP.25 Analysis of our own flow data in relation to systemic leakage revealed a

weak negative correlation of r = -0.2910 with p = 0.0448 with a mean flow of 455 ±172 ml/min in our perfusions.

Like others, we also found a significant systemic TNF peak in patients with low

leakage after restoration of the circulation of the perfused limb.22,26 Despite a washout

procedure with 2 L of Isodex, TNF in the limb reaches the systemic circulation. A

corresponding rise in RISA was also observed.27 Therefore, today a more extensive

washout with 6 L and massage of the perfused limb is recommended in TNF perfusions

to reduce TNF release.

In a previous study we described the clinical features of HILP with TNF characterized

by a short- lived sepsis-like syndrome.28 This best called SIRS, was seen in all patients

and accompanied by fever, increase in cardiac output, a decrease in systemic vascular

resistance, and the need for fluid resuscitation and inotropes. Perfusion with melphalan

as the sole perfusion agent did not trigger these effects. Detailed analysis showed

positive correlations between maximum TNF concentrations and systemic vascular

resistance and cardiac index. The National Cancer Institute perfusion group

demonstrated the relation between the vascular response with the need for vasopressor

support and systemic TNF levels in patients with TNF leakage as well.24 Lejeune

also demonstrated severe toxicity in patients with leaks of >5%.4,6 Vrouwenraets et

al. reported an absence of severe systemic toxicity of TNF in patients without systemic

leakage.26 Stam et al. observed only a mild postoperative toxicity in the event of

significant leakage during perfusion.22 This was easily managed on the intensive

care unit with fluid substitution and, in some cases, vasopressors. On the basis of

their data, they rightly plead for renewed study of the potential use of TNF

systemically. Currently, SIRS is only seldom seen since the majority of the institutions

performing HILP with TNF and melphalan are experienced and are using a more

extensive washout procedure. One could ask oneself if leakage measurements during

121


HILP are still worthwhile when side effects of TNF leakage are so easily dealt with.

In this study we demonstrated a good correlation between RISA leakage measurement

and TNF exposure during and after HILP with TNF and melphalan. RISA leakage

measurement serves as a good guide for the effectiveness of isolation during perfusion.

If leakage exceeds the 2% limit during perfusion, less exposure of the tumor bearing

limb to TNF, an increased exposure of the patient’s systemic circulation to TNF, and

more systemic side effects can be expected. Because leakage >2% did not influence

the tumor response, further dose-reduction studies of TNF in the HILP setting are

warranted.

122

Chapter 8

References



2 Stehlin JS. Hyperthermic perfusion with chemotherapy for cancers of the extremities.

Surg Gynecol Obstet 1969; 129: 305-308.

3 Schraffordt Koops H, Vaglini M, Suciu S, Kroon BB, Thompson JF, Gohl J, et al.

Prophylactic isolated limb perfusion for localized, high-risk limb melanoma: results

of a multicenter randomized phase III trial. European Organization for Research

and Treatment of Cancer Malignant Melanoma Cooperative Group Protocol 18832,

the World Health Organization Melanoma Program Trial 15, and the North American

Perfusion Group Southwest Oncology Group-8593. J Clin Oncol 1998; 16: 2906-

2912.




10: 52-60.

5 Eggermont AMM, Schraffordt Koops H, Lienard D, Kroon BBR, Van Geel AN,

Hoekstra HJ, et al. Isolated limb perfusion with high-dose tumor necrosis factor-

alfa in combination with interferon-gamma and melphalan for nonresectable

extremity soft tissue sarcomas: a multicenter trial. J Clin Oncol 1996; 14: 2653-

2665.

6 Gerain J, Lienard D, Ewalenko P, Lejeune FJ. High serum levels of TNF-alpha after

its administration for isolation perfusion of the limb. Cytokine 1992; 4: 585-591.

7 Eggimann P, Chiolero R, Chassot PG, Lienard D, Gerain J, Lejeune FJ. Systemic

and hemodynamic effects of recombinant tumor necrosis factor alpha in isolation

perfusion of the limbs. Chest 1995; 107: 1074-1082.

8 Stehlin JS, Clark RL, White EC, Healey JE, Dewey WC, Beerstecher S. The leakage

factor in regional perfusion with chemotherapeutic agents. A M A Arch Surg 1960;

80: 934-945.

9 Alexander C, Omlor G, Berberich R, Gross G, Feifel G. Rapid measurement of

blood leakage during regional chemotherapy. Eur J Nucl Med 1993; 20: 187-191.

10 Hafstrom L, Hugander A, Jonsson PE, Westling H, Ehrsson H. Blood leakage and

melphalan leakage from the perfusion circuit during regional hyperthermic perfusion

for malignant melanoma. Cancer Treat Rep 1984; 68: 867-872.

11 Stehlin JS, Clark RL, Dewey WC. Continuous monitoring of leakage during regional

perfusion. Arch Surg 1961; 83: 943-950.

12 Ghussen F, Nagel K, Sturz I, Isselhard W. A modified dye dilution method to estimate

leakage during regional isolated perfusion of the extremity. Res Exp Med (Berl)

1982; 180: 179-187.

13 Sardi A, Minton JP, Mojzisik C, Nieroda CA, Ferrara PJ, Hinkle GH, et al. The use

of a hand-held gamma detector improves the safety of isolated limb perfusion. J

Surg Oncol 1989; 41: 172-176.

14 Sandrock D, Horst F, Gatzemeier W, Ghorbani M, Rauschecker HF, Munz DL, et

al. Leakage measurement during selective limb perfusion using a gamma probe.

Eur J Nucl Med 1996; 23: 534-538.

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15 Schraffordt Koops H, Oldhoff J, Oosterhuis JW, Beekhuis H. Isolated regional

perfusion in malignant melanoma of the extremities. World J Surg 1987; 11: 527-

533.

16 Fontijne WP, Mook PH, Schraffordt Koops H, Oldhoff J, Wildevuur CR. Improved

tissue perfusion during pressure regulated regional perfusion: a clinical study. Cancer

1985; 55: 1455-1461.






19 Maring JK, Klompmaker IJ, Zwaveling JH, van Der M, Limburg PC, Slooff MJ.

Endotoxins and cytokines during liver transplantation: changes in plasma levels

and effects on clinical outcome. Liver Transpl 2000; 6: 480-488.

20 World Health Organization. Handbook for Reporting Results of Cancer Treatment.

WHO Offset Publication no 48 Geneva, Switzerland, 1979 World Health

Organization.

21 Kaplan EL, Meier P. Nonparametric estimates from incomplete observations. J Am

Stat Assoc 1958; 53: 457-481.

22 Stam TC, Swaak AJ, de Vries MR, ten Hagen TL, Eggermont AM. Systemic toxicity

and cytokine/acute phase protein levels in patients after isolated limb perfusion with

tumor necrosis factor-alpha complicated by high leakage. Ann Surg Oncol 2000; 7:

268-275.

23 Klaase JM, Kroon BB, Van Geel AN, Eggermont AMM, Franklin HR. Systemic

leakage during isolated limb perfusion for melanoma. Br J Surg 1993; 80: 1124-

1126.

24 Thom AK, Alexander HR, Andrich MP, Barker WC, Rosenberg SA, Fraker DL.

Cytokine levels and systemic toxicity in patients undergoing isolated limb perfusion

with high-dose tumor necrosis factor, interferon gamma, and melphalan. J Clin Oncol

1995; 13: 264-273.

25 Sorkin P, Abu Abid S, Lev D, Gutman M, Aderka D, Halpern P, et al. Systemic

leakage and side effects of tumor necrosis factor alpha administered via isolated

limb perfusion can be manipulated by flow rate adjustment. Arch Surg 1995; 130:

1079-1084.

26 Vrouenraets BC, Kroon BB, Ogilvie AC, Van Geel AN, Nieweg OE, Swaak AJ, et

al. Absence of severe systemic toxicity after leakage-controlled isolated limb

perfusion with tumor necrosis factor-alpha and melphalan. Ann Surg Oncol 1999; 6:

405-412.

27 Barker WC, Andrich MP, Alexander HR, Fraker DL. Continuous intraoperative

external monitoring of perfusate leak using iodine-131 human serum albumin during

isolated perfusion of the liver and limbs. Eur J Nucl Med 1995; 22: 1242-1248.

28 Zwaveling JH, Maring JK, Clarke FL, van Ginkel RJ, Limburg PC, Hoekstra HJ, et

al. High plasma tumor necrosis factor (TNF)-alpha concentrations and a sepsis-like

syndrome in patients undergoing hyperthermic isolated limb perfusion with

recombinant TNF-alpha, interferon- gamma, and melphalan. Crit Care Med 1996;

24: 765-770.

125

Summary and conclusions

Samenvatting en conclusies

126

Chapter 9

Summary

Chapter 1

Using technology to support extracorporeal circulation developed for cardiac surgery

in the 1950s, the surgical oncologists Creech, Krementz, Ryan and Winblad of the

Tulane University in New Orleans developed the technique of isolated limb perfusion

(ILP). In this procedure the blood circulation of a tumor bearing limb is isolated

from the circulation of the rest of the body by clamping the major artery and vein and

tightening a tourniquet around the root of the limb. The artery and vein are

subsequently connected to a heart-lung machine and a cytotoxic drug is administered

through this isolated circuit. Key point in ILP is that the dose of chemotherapeutics

used, can be 15-20 fold the maximum systemic tolerated dose since vital organs are

isolated from the perfusion circuit. Cavaliere and co-workers investigated the addition

of hyperthermia in the treatment of cancer and, as this appeared to augment the anti-

tumor effects of melphalan, in doing so they laid the basis for hyperthermic isolated

limp perfusion (HILP). The original patient population treated with HILP was a

subgroup of melanoma patients with extensive local recurrence in the arm or leg.

Later on also patients with soft tissue sarcomas of the extremities were treated.

Throughout the years different chemotherapeutic agents were used in HILP, all with

variable results.

William Coley, a surgeon who lived and worked in New York City during the second

half of the 19th century, was the first to investigate the phenomenon of tumor necrosis,

occurring in patients suffering from severe infections. By administering preparations

of gram-positive and gram-negative bacteria or their products to patients with

inoperable neoplastic diseases, Coley hoped to bring about an involution of the tumor.

The side effects of Coley’s regimen were unacceptable, however, and his treatment

ultimately fell into disrepute. The phenomenon that bacteria were capable of producing

a tumor necrotisizing factor stimulated other investigators. In 1975 Old and co-workers

identified a factor produced in mice pretreated with Bacillus Calmette-Guérin (BCG)

and subsequently challenged with lipopolysaccharide (LPS). This factor was able to

cause hemorrhagic necrosis of the meth A sarcoma, grown in the skin of a recipient

animal. The factor was dubbed “tumor necrosis factor” (TNF). A lot of articles

published both in scientific literature and in popular press claimed, that this molecule

would prove to be a revolutionary tool in the battle against cancer. However, phase I

and II clinical trials of systemic TNF were marked by a disappointing overall response

rate of 1-2% and a dose-limiting toxicity of hypotension. This dose-limiting toxicity

in patients kept the peak intravascular level achievable in humans 100-fold lower

than the level needed to have an anti-tumor effect in a mouse model. Because it

127


seemed impossible to achieve effective systemic concentrations of TNF in patients,

TNF was ideally suited for use in HILP where levels up to 10 to 20 times the

systemically tolerated dose could be achieved. Ferdy Lejeune and Danielle Lienard,

surgical oncologists working in Brussels at the time, were the first to observe the

dramatic effect of tumor necrosis in humans using HILP with a combination of TNF,

interferon (IFN) and melphalan.

The second part of the introduction describes the technique of positron emission

tomography (PET). This is a non invasive, diagnostic imaging technique for measuring

the metabolic activity of cells in the human body with the aid of short-lived positron

emitting radiopharmaceuticals. Not only is it possible to visualize the metabolic

processes of a tumor but it is also possible to quantify the metabolic processes. PET

was used to evaluate tumor metabolism and with that tumor response before and

after HILP with TNF, IFN and melphalan.

Chapter 2

Osteosarcoma is the most frequent occurring primary malignant bone tumor in human.

During the past few decades, the use and further development of systemic neo adjuvant

chemotherapy, e.g., including high-dose methotrexate (HD-MTX) and cisplatin,

appears to have a definite influence on the disease free and overall survival for patients

with osteosarcoma. However, the potential local tumor effect of this systemically

administered chemotherapy is not always favorable. To increase the effect of cisplatin

on locoregional osteosarcoma, the short term effect of HILP with cisplatin (30 mg/L

extremity volume) was studied in 28 dogs with spontaneous osteogenic sarcoma

using clinical, radiological, and histological parameters. Thirty days postoperatively

mortality was 14.3 %. Total platinum levels at the start of perfusion were

28.2 ± 14.3 mg/L. A significant improvement (p<0.001) in the clinical score was

observed in the overall group at 6 and 12 weeks after perfusion. The radiological

parameter showed a stationary X-ray 2 weeks after perfusion and an improved X-ray

6 weeks after perfusion. Overall histological scores showed a moderate effect

according to the Huvos classification. No additional therapeutic effect, according to

the three parameters, could be demonstrated by increasing the perfusate temperature

by 1°C. HILP with cisplatin is feasible in the local treatment of spontaneous

osteosarcoma in dogs with acceptable locoregional toxicity. However, the histological

results were modest, with none of the dogs showing a complete response 6 weeks

after perfusion. Therefore the search for the ideal perfusion agent with substantial

contribution to the limb sparing treatment in human osteosarcoma, continues.

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Chapter 9

Chapter 3

With the introduction of HILP with TNF, IFN and melphalan the question was raised

as to whether the combination of TNF with cisplatin in HILP could improve the

histological results after perfusion in dogs with spontaneous osteosarcoma. Before

starting with perfusion in dogs with osteosarcoma the feasibility of TNF perfusion

with and without cisplatin in healthy dogs was studied. During seven perfusions in

six mongrel dogs (weight 32±2 kg) the technical aspects of HILP under mild

hyperthermia (39-40o) were studied. In five experiments HILP was performed with

TNF alone (0.5 mg/L extremity volume), and in two experiments TNF was combined

with cisplatin (25 mg/L extremity volume). During the perfusions physiological

parameters were monitored and TNF and total cisplatin concentrations were

determined. Perfusion conditions (pH, PCO2, PO

2, flow and pressure) remained within

physiological ranges. Three dogs died within 24 hours despite a sublethal systemical

concentration of TNF that leaked from the perfusion circuit. Three dogs were

terminated; one dog after the second experiment in accordance with the Dutch ethical

rules; one dog because it showed an invagination of the small bowel resulting in an

ileus; one dog because of necrosis of the perfused limb. This feasibility study in

healthy dogs demonstrated that HILP with TNF and cisplatin was associated with a

high mortality rate and therefore does not allow us to treat dogs with spontaneous

osteosarcoma with TNF and cisplatin HILP.

Chapter 4

In order to study the value of HILP with cisplatin in the management of locally

advanced soft tissue sarcomas or metastatic bone sarcoma in humans, four patients

were treated in this manner under mild hyperthermia. Toxicity in the perfused limbs

was moderate, and the erythema and edema that occurred resolved spontaneously

within 7-14 days as did the slight motor and sensory neuropathy over a longer period

of time. Clinically, a reduction of pain was observed in all patients. Two weeks after

perfusion, tumor biopsies were taken to evaluate the response. Two patients showed

a pathological complete response, one patient showed >90% necrosis and one patient

showed no response. The histological results of this study were modest and with the

introduction of TNF in combination with melphalan as perfusion agents, no further

research with cisplatin as a perfusion agent was done.

Chapter 5

The first patient treated at the University Hospital Groningen with the perfusion

regimen of Lejeune, TNF with IFN and melphalan, had been treated with local

resection and adjuvant external beam radiotherapy 3 years earlier. Radiotherapy

129


consisted of 40 Gy given 2 Gy per day in 4 weeks on the whole foot and a 20 Gy as

a boost on the tumor bed. The first recurrence of the lesion was treated by HILP with

cisplatin. After a second recurrence of the malignant fibrous histiocytoma and the

patient refusing curative amputation, was treated with HILP with 4 mg TNF, 0.2 mg

IFN and 45 mg melphalan. Already some hours after TNF perfusion, not only the

tumor on the foot showed a bluish color, but also the area that had been irradiated

three years ago. Nine days after TNF perfusion a lower leg amputation had to be

performed because of severe necrosis of the foot. Histology showed complete necrosis

of the tumor and marked thrombosis of the smaller vessels of the foot. TNF did not

only have a damaging influence on the endothelial cells of the tumor, but also on the

endothelial cells that developed after high dose irradiation therapy. An explanation

of the observed dramatic effect was described and the case served to alert other

surgeons in the field of TNF perfusions in treating patients with a history of irradiation

therapy.

Chapter 6

In order to study the glucose metabolism of soft tissue sarcomas before and after

HILP with TNF, IFN and melphalan, a FDG-PET study was performed in 20 patients

before to, 2 and 8 weeks after HILP. After the final PET study, the tumor was resected

and pathologically graded. Patients with a pathologically complete response (pCR)


(pPR) showed various amounts of viable tumor in the resected tumor specimens.

Seven patients showed a pCR (35%) and 12 patients showed a pPR (60%). In one

patient, pathological examination was not performed (5%). The pre-perfusion glucose

consumption in the pCR group was significantly higher than in the pPR group

(p<0.05). Visual analysis of the PET images after perfusion showed a rim of increased

FDG uptake around a core of absent FDG uptake in 12 patients. The rim signal

contained a fibrous pseudocapsule with inflammatory tissue in the pCR group, but

viable tumor tissue was seen in the pPR group. The glucose consumption in the pCR

group at 2 and 8 weeks after perfusion had decreased significantly (p<0.05) compared

with the glucose consumption in the pPR group. Based on the pretreatment glucose

consumption in soft-tissue sarcomas one could predict the probability of a patient

achieving a complete pathologically response after TNF HILP. FDG-PET indicated

the pathologic tumor response to HILP, although the lack of specificity of FDG, in

terms of differentiation between an inflammatory response and viable tumor tissue,

hampered the discrimination between pCR and pPR.

130

Chapter 9

Chapter 7

After we had studied the glucose metabolism of tumors before and after HILP and

were confronted with the inability of FDG to discriminate between viable tumor

tissue and inflammatory tissue we decided to study the protein metabolism of tumors.

L-[1-11C]-tyrosine (TYR) was used as a tracer to study the protein metabolism before

and after HILP. Seventeen patients (5 women, 12 men; age range 24-75 y; mean age

52 y) were studied. TYR-PET studies were performed before HILP, and 2 and 8

weeks afterwards. The protein synthesis rates (PSRs) in nanomoles per milliliter per

minute were calculated. After the final PET study, the tumor was resected and

pathologically examined. Patients with a pathologically complete response (pCR)


(pPR) showed various amounts of viable tumor in the resected tumor specimens. Six

patients showed a pCR (35%) and 11 patients showed a pPR (65%). All tumors were

depicted as a hot spot on the PET study before HILP. The PSR in the pCR group at 2

and 8 weeks after perfusion had decreased significantly (p<0.05) compared to the

PSR before HILP. A significant difference was found in PSR between the pCR and

pPR group at 2 as well as at 8 weeks (p<0.05). Median PSR in nonviable tumor

tissue was 0.62 and ranged from 0.22 to 0.91. With a threshold PSR of 0.91, sensitivity

and specificity of TYR-PET were 82% and 100%, respectively. The predictive value

of a PSR > 0.91 for having viable tumor tissue after HILP was 100%, whereas the

predictive value of a PSR ≤ 0.91 for having nonviable tumor tissue after HILP was

75%. On pathological examination the 2 patients in the pPR group with a PSR < 0.91

showed microscopic islets of tumor cells surrounded by extensive necrosis.

Inflammatory tissue after treatment did not interfere with viable tumor tissue on the

images. Combining the results of the FDG and TYR-PET studies, we concluded that

FDG-PET predicted the probability of a patient achieving a pathological complete

response after perfusion and TYR-PET gave a good indication of the pathological

outcome.

Chapter 8

With the introduction of TNF, the monitoring of leakage of the isolated circuit into

the systemic circulation is mandatory since TNF levels in the perfusion circuit are

approximately 10 times the maximum tolerated systemic levels. If significant leakage

occurs during HILP the resultant TNF induced systemic inflammatory response

syndrome (SIRS) could be fatal. The aim of this study was to analyze the value of

continuous leakage monitoring with radioactive Iodine-131 labeled human serum

albumin (RISA) in patients treated with HILP with TNF and melphalan. Forty-eight

patients with melanoma (n = 14) or soft tissue sarcoma (n = 34) of an extremity

131


underwent 51 perfusions. Perfusion was performed at the iliac level in 22 cases, at

the popliteal level in 16 cases, at the femoral level in 7 cases and in 6 cases at the

axillary level. Leakage rates, perfusion circuit and systemic levels of TNF, interleukin-

6, C-reactive protein (CRP) were determined, as were systemic hematological and

metabolic profiles and tumor response. The mean isotopically measured leakage was

2.9 % (95% confidence interval 2.0 – 3.8%, range 0-15.5%). Systemic leakage was

≤2% in 28 perfusions (55%) and >2% in 23 perfusions (45%). The correlation between

the maximal monitored leakage and the maximal systemic TNF levels was 0.7114

(p < 0.0001). The area under the curve (AUC) for TNF in the perfusion circuit,

indicating the exposure of the perfused limb to TNF, was 18.7% lower in the >2%

leakage group (p=0.0457). No significant differences in tumor response were found

between groups. AUC for systemic TNF, indicating the exposure of the patient to

TNF, was 18.1 times higher in the >2% leakage group (p<0.0001) resulting in a

significant decrease in leukocyte and platelet count, hyperbilirubinemia,

hypocholesterolemia and proteinemia. No beneficial effect of the systemically leaked

TNF and melphalan was seen on the occurrence of distant metastasis during follow-

up. There was a significant difference between perfusions performed at the iliac and

femoral levels compared with leakage values at the popliteal level, p < 0.0001 and

0.0159 respectively. A good correlation between RISA leakage measurement and

TNF exposure during and after HILP with TNF and melphalan was demonstrated.

RISA leakage measurement serves as a good guide for the effectiveness of isolation

during perfusion. If leakage exceeds the 2% limit during perfusion, less exposure of

the tumor bearing limb to TNF, increased exposure of the patient systemic circulation

to TNF, and more systemic side effects can be expected.

Conclusions

1. HILP with cisplatin in dogs with spontaneous osteosarcoma can be done safely

with improvement of clinical and radiological parameters although histological

results were modest.

2. Since we encountered an unacceptable mortality and morbidity rate in HILP

with TNF and cisplatin in healthy dogs, an experiment in dogs with spontaneous

osteosarcoma was not initiated.

3. HILP with cisplatin in patients with sarcomas of soft tissue and bone resulted in

a reduction of pain after treatment. However, the histological outcome was

moderate.

4. HILP with TNF, IFN and melphalan does not only have an effect on the

vasculature of a tumor, but can also elicit an activation of the vasculature

originated after irradiation therapy.

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Chapter 9

5. By studying the glucose metabolism of a tumor before HILP with TNF and

melphalan with the aid of FDG-PET, an assumption can be made about the

reaction of the tumor to TNF HILP.

6. In order to evaluate the result of HILP with TNF and melphalan one should

perform a TYR-PET study to measure the protein metabolism of the tumor after

HILP.

7. In order to determine leakage of TNF from the isolated circuit of the extremity

to the systemical circulation of the patient, continuous recording with radioactive

Iodine-131 labeled human serum albumin is an appropriate procedure.

133


Samenvatting

Hoofdstuk 1

De Amerikaanse artsen Creech, Krementz, Ryan en Winbald pasten in de jaren vijftig

voor het eerst de techniek van geïsoleerde ledemaat perfusie (ILP) toe. Dankzij de

uitvinding van de hartlong machine, enige jaren tevoren ontwikkeld ten behoeve van

de hartchirurgie, konden zij de bloedsomloop van een arm of been tijdens een operatie

isoleren van de circulatie van de rest van het lichaam. Eenmaal aangesloten op een

hartlong machine was het mogelijk om aan dit geïsoleerde circuit een zeer hoge

dosis celdodende chemotherapeutica toe te voegen. Tumoren aan armen en benen

konden zo worden behandeld zonder dat vitale organen in de rest van het lichaam

van de patiënt werden blootgesteld aan chemotherapeutica.

Cavaliere en zijn medewerkers bestudeerden in de jaren zeventig het additionele

tumordodende effect van warmte, en legden daarmee de basis voor hypertherme

geïsoleerde ledemaat perfusie (HILP). Aanvankelijk vooral toegepast bij patiënten

met een melanoom (een kwaadaardige huidtumor) werd HILP later ook ingezet bij

de behandeling van tumoren van de weke delen (spieren, bindweefsel en bot). Door

de jaren heen zijn verschillende chemotherapeutica gebruikt met wisselend succes.

William Coley, een New Yorkse chirurg die aan het eind van de negentiende eeuw

leefde, ontdekte dat een extract van bacteriën, ingespoten in tumoren, in enkele

gevallen een regressie van de tumor opleverde. Soms was het middel echter erger

dan de kwaal en overleden patienten aan deze behandeling. Overtuigd van de gedachte

dat bacteriën een factor tegen tumoren konden produceren diende Old en zijn

medewerkers in 1975 endotoxine (een bestanddeel van de celwand van sommige

bacteriën) aan muizen toe. Het bloed van deze met endotoxine behandelde muizen

bleek bij andere muizen een ineenschrompelen van de tumor te veroorzaken. De met

endotoxine behandelde muizen moesten dus een factor in het bloed gemaakt hebben

die tumoren bij de tweede groep muizen deed verdwijnen. Deze tumordodende factor

kreeg de illustere naam ‘tumor necrosis factor’, kortweg TNF. Aanvankelijk waren

de verwachtingen over de kanker genezende werking van TNF hooggespannen. Toen

TNF echter na DNA recombinant technieken voor klinische trials beschikbaar kwam,

bleek het al in een zeer lage dosering ernstige bijwerkingen te hebben. Patiënten

toonden het beeld van een ernstige infectie met symptomen van lage bloeddruk,

koorts, verminderde hartwerking en een onvoldoende doorbloeding van de organen.

Een effect op de tumoren van deze patiënten werd nauwelijks waargenomen. TNF

leek dus niet alleen een tumordodend effect te hebben, maar eveneens een centrale

rol te spelen bij ontstekingsreacties. Aangezien ILP een 10 tot 20 keer zo hoge dosering

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Chapter 9

van chemotherapie mogelijk maakt in een geïsoleerde extremiteit, kwamen twee

Belgische chirurgen, Lejeune en Lienard, op het idee om TNF in de ILP setting te

gebruiken. Met TNF alleen bereikten zij onvoldoende effect, maar in combinatie

met melphalan waren de resultaten opzienbarend. Voor het eerst werd het effect van

tumornecrose, zoals dat eerder bij muizen werd gezien, ook waargenomen bij mensen.

Het tweede deel van de introductie geeft een overzicht van het werkingsmechanisme

van de techniek ‘positron emissie tomografie’ (PET). Met behulp van positron

uitzendende ‘tracers’ kan het metabolisme van tumoren niet alleen zichtbaar worden

gemaakt, maar ook in maat en getal worden uitgedrukt. Deze nieuwe techniek werd

gebruikt om de veranderingen in het tumormetabolisme te bestuderen voor en na

behandeling met HILP.

Hoofdstuk 2

Het osteosarcoom is de meest voorkomende bottumor bij de mens en komt met name

bij jonge mensen voor. Begin jaren zeventig werd een grote stap voorwaarts gezet in

de behandeling van deze tumoren door de introductie van systemische neoadjuvant

chemotherapie met hoge doseringen methotrexaat en cisplatinum. Evenwel, met deze

systemische chemotherapie leken vooral (micro)metastasen in de longen behandeld

te worden die bepalend waren voor de prognose; het lokale effect op de primaire

tumor was niet altijd optimaal. De vraag was of dit lokale effect verhoogd kon worden

door patiënten met een osteosarcoom van arm of been eerst te behandelen met HILP

en cisplatinum als chemotherapeuticum waarvoor het osteosarcoom gevoelig is.

Voorafgaand aan een klinische toepassing werd eerst een studie bij honden verricht.

Achtentwintig honden met een osteosarcoom werden behandeld met een cisplatinum

HILP. Het effect van de behandeling werd geëvalueerd aan de hand van klinische,

radiologische en histologische parameters. Zes weken na behandeling toonde het

looppatroon en de radiologische parameters een significante verbetering. Het

histologisch effect van de behandeling was bescheiden volgens de classificatie van

Huvos. Vier honden overleden binnen 30 dagen na behandeling. Door de temperatuur

van de perfusie met 1 °C te verhogen naar 42 °C werd geen significante verbetering

gezien ten opzichte van de groep honden die met een lagere temperatuur waren

behandeld.

Geconcludeerd kon worden dat HILP bij honden met een osteosarcoom goed

uitvoerbaar was met verbetering van de klinische en radiologische parameters. Het

histologisch effect was echter onvoldoende en het zoeken naar het ideale perfusie

chemotherapeuticum voor behandeling van het osteosarcoom moest daarom worden

voortgezet.

135


Hoofdstuk 3

Met de introductie van ‘tumor necrosis factor’ gebruikt bij HILP, ontstond de vraag

of het histologisch effect van cisplatinum bij honden met een osteosarcoom wellicht

verbeterd kon worden als TNF werd toegevoegd. Alvorens honden met een

osteosarcoom te behandelen werd eerst bij gezonde honden gekeken naar het effect

van een perfusie met TNF alleen en de combinatie met TNF en cisplatinum.

Bij 6 honden werden 7 perfusies verricht met milde hyperthermie. Gedurende 5

perfusies werd alleen TNF gebruikt en in twee gevallen werd hier cisplatinum aan

toegevoegd. Perfusie condities zoals pH, pCO2, pO

2, de stroom van het perfusaat en

de druk bleven binnen fysiologische grenzen. Drie honden overleden binnen 24 uur

na de perfusie ondanks een gemeten subletale systemische dosis van TNF. Drie honden

moesten worden afgemaakt. Eén hond omdat deze nog een tweede perfusie aan een

andere poot onderging; conform de Nederlandse ethische regelgeving mag na een

tweede experiment een dier niet meer uit een narcose ontwaken. Eén hond kreeg na

behandeling een ileus berustend op een invaginatie van de darm. De derde hond

ontwikkelde een totale necrose van de met TNF en cisplatinum geperfundeerde poot.

Gezien de onacceptabele mortaliteit en morbiditeit bij deze experimenten werd het

niet zinvol geacht om een experiment te starten waarbij honden met een osteosarcoom

op deze manier behandeld zouden worden.

Hoofdstuk 4

Om het effect van HILP met cisplatinum bij patiënten met een weke delen sarcoom

of een osteosarcoom te beoordelen werden vier patiënten op deze wijze behandeld

onder milde hyperthermie. De toxiciteit na perfusie was gering, erytheem en oedeem

verdwenen tussen de 7de tot 14de dag postoperatief evenals de lichte motorische en

sensorische neuropathie. Klinisch stond met name een vermindering van de pijn op

de voorgrond. Twee weken na behandeling werden histologische biopten van de tumor

genomen. Bij twee patiënten werden in deze biopten geen vitale tumorcellen

gevonden, bij één patiënt was er sprake van meer dan 90% necrose in de biopten en

bij één patiënt werd geen histologisch effect van de perfusie gezien. De resultaten

van deze studie waren veel belovend, maar verder onderzoek naar de

toepassingsmogelijkheden van cisplatinum als perfusie chemotherapeuticum werd

niet meer uitgevoerd, omdat het gebruik van TNF in combinatie met melphalan toen

in zwang kwam.

Hoofdstuk 5

De eerste patiënt die in Groningen met het perfusie-regime van Lejeune behandeld

werd (TNF in combinatie met IFN en melphalan) was drie jaar eerder behandeld met

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Chapter 9

lokale excisie gevolgd door bestraling met 40 Gy gegeven in 2 Gy per dag gedurende

4 weken. Twee jaar later werd een eerste recidief van de tumor behandeld met een

cisplatinum perfusie. Na een complete regressie recidiveerde de tumor opnieuw en

omdat perfusie met TNF, IFN en melphalan het enige alternatief was voor amputatie

werd de patiënte op deze manier behandeld. Kort na perfusie trad er een blauwe

verkleuring op van de tumor maar ook van het gebied dat 3 jaar eerder bestraald was.

Negen dagen na perfusie was alsnog een amputatie van de voet noodzakelijk wegens

de ernstige necrose die was opgetreden. Histologisch onderzoek toonde necrose van

de tumor aan maar ook trombusvorming in de capillairen in het bestraalde gebied.

TNF bleek niet alleen een effect te hebben op het endotheel van de tumorvaten, maar

ook op het endotheel van bloedvaten ontstaan na bestraling. Een mogelijk

werkingsmechanisme werd beschreven en de casus diende als waarschuwing voor

andere chirurgen die zich met TNF HILP bezig hielden om voorzichtig te zijn bij

patiënten die reeds eerder waren bestraald.

Hoofdstuk 6

Om het glucose metabolisme van weke delen sarcomen te bestuderen voor en na

behandeling met TNF HILP, werd bij twintig patiënten voor perfusie, twee weken na

perfusie en acht weken na perfusie een positron emissie tomografie (PET) studie

verricht met 18Fluorine-gelabeled 2-fluoro-2-deoxy-D-glucose (FDG). Na de laatste

FDG-PET studie werd het restant van de tumor geëxcideerd en pathologisch

onderzocht. Deze pathologische bevindingen werden met de gegevens van de PET-

studie vergeleken. Bij een pathologisch complete response (pCR), werden er geen

vitale tumorcellen in het resectie preparaat gevonden. Indien er nog wel vitale

tumorcellen werden gevonden werd een pathologisch partiele response (pPR)

geclassificeerd. Zeven patiënten hadden een complete respons (35%), 12 patiënten

een partiële respons (60%) en bij één patiënt (5%) werd geen pathologisch onderzoek

verricht. Bij tumoren die een complete respons toonden na de perfusie was het

glucosemetabolisme voor de perfusie significant verhoogd. Visuele analyse van de

PET-studies na perfusie lieten een rand van verhoogd glucosemetabolisme zien om

een kern van verminderd glucosemetabolisme bij 12 patiënten. Bij patiënten met een

complete respons bevatte deze rand een pseudo-kapsel met ontstekingscellen, echter

bij patiënten met een partiele respons werd nog vitaal tumorweefsel in de rand

gevonden. De kern bestond uit necrotisch vervallen tumorweefsel. Het

glucosemetabolisme van de patiënten met een complete respons daalde significant

na de perfusie, terwijl deze daling bij patiënten met een partiele respons minder

uitgesproken was.

Aan de hand van het glucosemetabolisme voor perfusie viel te voorspellen of een

137


patiënt gunstig zou reageren op een behandeling met een perfusie van TNF en

melphalan. Tevens gaf FDG-PET na de behandeling een indicatie over het bereikte

effect, maar het was niet goed mogelijk om te differentiëren tussen een complete of

een partiele respons, gezien de overlap in glucosemetabolisme van tumoren

ontstekingsweefsel.

Hoofdstuk 7

Nu duidelijk was dat zich bij het bestuderen van het glucosemetabolisme met behulp

van PET een differentiatieprobleem van ontstekingsweefsel en tumorweefsel voor

deed, werd een nieuwe weg ingeslagen. Besloten werd om het eiwitmetabolisme van

tumoren voor en na behandeling met een TNF/melphalan perfusie te bestuderen. Als

tracer voor het eiwitmetabolisme werd L-[1-11C]-tyrosine (TYR) gebruikt. Zeventien

patiënten werden in deze studie bestudeerd. Ook nu werd een TYR-PET studie voor,

twee weken na en acht weken na perfusie verricht en werden de resultaten vergeleken

met die van pathologisch onderzoek van het gereseceerde tumorweefsel. Zes patiënten

(35%) hadden bij pathologisch onderzoek geen vitaal tumorweefsel meer en toonden

een complete respons. Elf patiënten (65%) hadden nog vitaal tumorweefsel in het

gereseceerde preparaat en toonden daarmee een partiele respons. Voor behandeling

waren alle tumoren goed zichtbaar met TYR-PET. Patiënten die na perfusie een

complete respons toonden hadden een significante daling van het eiwitmetabolisme,

dit in tegenstelling tot patiënten met een partiële respons. Bij een eiwitmetabolisme-

grenswaarde van 0,91 of hoger was er na perfusie nog altijd sprake van vitaal

tumorweefsel bij pathologisch onderzoek. Indien het eiwitmetabolisme lager was

dan 0,91 bleek er bij twee patiënten bij pathologisch onderzoek nog sprake te zijn

van vitaal tumorweefsel. Dit vitaal ogende tumorweefsel werd echter omgeven door

necrotisch tumorweefsel. Aan de hand van het berekende eiwitmetabolisme na perfusie

was het dus mogelijk om het effect van de behandeling te beoordelen.

Ontstekingsweefsel intervereerde niet met vitaal tumorweefsel voor wat betreft het

eiwitmetabolisme. Deze studie, in combinatie met die beschreven in hoofdstuk 6,

levert op dat met FDG-PET accuraat te voorspellen is of een tumor goed op de

perfusiebehandeling zal reageren; bovendien is het met TYR-PET mogelijk om het

resultaat van die behandeling te evalueren op de aanwezigheid van vitaal

tumorweefsel.

Hoofdstuk 8

Aangezien er tijdens perfusie met hoge dosis chemotherapeutica wordt gewerkt in

het geïsoleerde circuit is het van belang om elke vorm van lekkage naar de rest van

het lichaam te voorkomen. Mocht lekkage toch optreden, dan is het van belang om te

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Chapter 9

meten in welke mate. Met name sinds de introductie van TNF, dat al in een zeer lage

dosering sterke toxische gevolgen kan hebben, is dit aspect belangrijk geworden.

Van oudsher wordt lekkage tijdens perfusie met radioactief Jodium-131 gelabeled

serum albumine (RISA) gemeten. Hierbij wordt een lage dosis RISA aan de

systemische circulatie van de patiënt toegediend en een tien keer hogere dosering

aan het geïsoleerde perfusie circuit. Bij lekkage van het perfusie circuit naar de

systemische circulatie zal er een toename van de radioactiviteit in het lichaam van de

patiënt optreden. Dit wordt met behulp van een teller geplaatst boven het hart van de

patiënt gemeten. In dit hoofdstuk werd gekeken naar de correlatie tussen deze

radioactief gemeten lekkage en de systemisch gemeten waarden van TNF.

Achtenveertig patiënten ondergingen 51 perfusies met TNF en melphalan. In 22

gevallen betrof het een perfusie op iliacaal niveau, in 16 gevallen op popliteaal niveau,

in zeven gevallen op femoraal niveau en zes patiënten ondergingen een perfusie van

de arm. Lekkage waarden, systemische en perfusie circuitspiegels van TNF, IL-6 en

CRP werden bepaald alsmede systemische hematologische en metabolische spiegels.

Tevens werd er gekeken of er een verband was tussen lekkage en tumorresponse.

Gemiddeld was de lekkage 2,9% (spreiding 0-15,5%). Bij 28 perfusies was de lekkage

≤2% en bij 23 perfusies >2%. De correlatiecoëfficiënt tussen de maximaal gemeten

lekkage gedurende de perfusie en de maximale TNF-spiegel in de systemische

circulatie van de patiënt was 0.7114 (P<0.0001). De oppervlakte onder de

concentratiecurve van TNF gemeten in het perfusiecircuit, als indicator voor de

blootstelling van de tumor aan de hoeveelheid TNF, was 18,7% lager in de groep van

patiënten met >2% lekkage (P=0.0457). Er werd geen significant verschil gezien in

tumorrespons tussen beide lekkage groepen ondanks het feit dat de blootstelling van

de tumor aan TNF in de lage lekkage groep hoger was. Vergelijkenderwijs werd de

systemische circulatie van de patiënt in de groep met >2% lekkage aan een 18,1

maal hogere TNF dosering blootgesteld hetgeen resulteerde in een significante daling

van het leukocyten- en trombocytengetal. Ook werd vaker een hyperbilirubinaemie,

een hypocholestoroleamie en een proteïnemie vastgesteld. Een gunstig effect van de

verhoogde concentratie van TNF in de >2% lekkage groep op het in de follow-up

optreden van afstandsmetastasen werd niet gezien. Perfusies op iliacaal en femoraal

niveau hadden een significant hoger lekkage-percentage in vergelijking met perfusies

op popliteaal niveau. Gezien de goede correlatie tussen lekkage en gemeten

systemische TNF waarden kon worden gesteld dat lekkage gemeten door middel van

RISA een goede richtlijn geeft gedurende de perfusie over de lekkage. Indien de

lekkage ≤2% is wordt de tumor in de geperfundeerde extremiteit aan een hogere

dosering TNF blootgesteld en de systemische circulatie van de patiënt aan een

significant lagere dosering.

139


Conclusies

1. HILP met cisplatinum bij honden met een osteosarcoom is goed uitvoerbaar met

verbetering van de klinische en radiologische parameters na behandeling. Het

histologisch effect is echter onvoldoende.

2. Gezien de onacceptabele mortaliteit en morbiditeit bij HILP met TNF en

cisplatinum bij gezonde honden is het niet zinvol een experiment bij honden

met een osteosarcoom uit te voeren.

3. HILP met cisplatinum bij patiënten met een wekedelen sarcoom of een

osteosarcoom resulteert met name in een afname van pijn na behandeling. Ook

hier zijn de histologische resultaten echter bescheiden.

4. HILP met TNF, IFN en melphalan kan naast een effect op de neovascularisatie

van tumoren ook invloed hebben op endotheel ontstaan na radiotherapie.

5. Door het glucose metabolisme van een tumor voor HILP met TNF en melphalan

te bestuderen middels FDG-PET is het mogelijk om te voorspellen of een tumor

goed op de behandeling zal reageren.

6. Om het resultaat van HILP met TNF en melphalan te bestuderen is het bepalen

van het eiwitmetabolisme door middel van TYR-PET na perfusie de aangewezen

methode.

7. Om lekkage van TNF vanuit de geïsoleerde circulatie in de extremiteit naar de

rest van het lichaam te bepalen is continue meeting met radioactief Jodium-131

gelabeled serum albumine waardevol.

140

DANKWOORD

Ook dit proefschrift is kunnen ontstaan door de samenwerking van velen. Het is

moeilijk om volledig te zijn maar al diegene die een bijdrage geleverd hebben wil ik

hartelijk bedanken voor hun inzet en hulp. Hierbij gaan mijn gedachten uit naar de

verpleegkundigen van de verpleegafdeling chirurgische oncologie, de verpleeg-

kundigen van het operatie centrum, de verpleegkundigen van de chirurgische intensive

care, de registratie assistenten van de polikliniek chirurgie, de laboranten van de diverse

laboratoria, en de perfusionisten van het Academisch Ziekenhuis Groningen. De

medewerkers van het PET-centrum en de afdeling nucleaire geneeskunde van het

zelfde ziekenhuis, alsmede de medewerkers van het Centraal Dierenlaboratorium van

de Rijksuniversiteit Groningen.

In het bijzonder dank ik prof. dr. H.J. Hoekstra, prof. dr. Schraffordt Koops en prof.

dr. W. Vaalburg voor het in mij gestelde vertrouwen, hun steun en begeleiding.

Prof. Dr. H.J. Hoekstra, beste Harald, na mijn diensttijd bood jij mij als arts-

onderzoeker de gelegenheid en vrijheid om bij de chirurgische oncologie te komen

werken als arts onderzoeker. Enkele gingen mij vooraf en velen zouden mij volgen

en dat kan ook niet anders met jou enthousiasme en vindingrijkheid op

onderzoeksgebied. Op naar het volgende decennium.

Prof. Dr. H. Schraffordt Koops, als een van de pioniers in Nederland op het gebied

van de TNF perfusies legde u de basis voor een diversiteit aan onderzoeken. Het was

een leerzame ervaring een radar van het geheel te zijn.

Prof. Dr. W. Vaalburg, bedankt voor de prima sfeer op het PET-centrum met een

groep van enthousiaste medewerkers en een diversiteit aan specialismen die op het

PET-centrum onderzoek doen.

De leden van de beoordelingscommissie, bestaande uit prof. dr. B.B.R. Kroon, prof.

dr. M.F. von Meyenfeldt en prof. dr. W.M. Molenaar ben ik dank verschuldigd voor

de beoordeling van dit proefschrift.

Ik wil alle patiënten, honden en hun eigenaren bedanken voor hun onmisbare bijdrage

bij het tot stand komen van dit proefschrift.

141

Alle chirurgen en mijn collega assistenten in het Academisch Ziekenhuis Groningen

en het Medisch Centrum Leeuwarden wil ik bedanken voor de samenwerking en

hetgeen ik heb mogen leren tijdens mijn opleiding tot chirurg.

Drs. D. Klees en drs. R.P. Winkel, beste Dirk en Robert, bij de gedachte aan paranimfen

kwamen jullie als eerste in beeld. Hoe zou dat toch komen ?

Grote dank ben ik verschuldigd aan mijn ouders die mij alle kans hebben geboden

om mijzelf te ontplooien. Pa heeft de afronding van mijn promotie helaas niet meer

mee kunnen maken. In dierbare herinnering draag ik dit proefschrift aan hem op

wetende dat hij het weet.

Lieve Klaar, jij bent onmisbaar. Na tien jaar, drie kinderen, een opleiding en een

eigenzaak verder, blijft thuis het belangrijkste gelukkig ben jij er om alles in goede

banen te leiden.

Joris, Ties en Maxime, jullie gekwek klinkt als muziek in papa’s oren als ik boven

wat probeer te werken en voor een “koffie koppie” drinken blijf ik graag naar beneden

komen.

142

Curriculum Vitae

Robert Johannes van Ginkel was born on May 12th , 1964 in Amsterdam, the

Netherlands. After finishing high school (Rythoviuscollege, Eersel) in 1982, he went

to the Technical University Eindhoven to study Chemistry. After one year he went to

Medical School at the University of Groningen. After graduation in 1991 he served

in the Royal Dutch Army in the Hague, the Netherlands. From February 1993 until

December 1995 he joined a research program at the Department of Surgical Oncology,

University Hospital Groningen (Prof. dr. H. Schraffordt Koops). In January 1995

until September 1996 he worked as a transplant coordinator of the Northern and

Eastern part of the Netherlands. In September 1996 he started his training in Surgery

at the Surgical Department of the University Hospital Groningen (Prof. dr. R. van

Schilfgaarde) followed in September 1999 at the Surgical Department of the Medical

Center Leeuwarden, the Netherlands (Dr. D.C. Busman).

143

Publications

van Ginkel RJ, Kole AC, Nieweg OE, Molenaar WM, Pruim J, Koops HS, Vaalburg W,

Hoekstra HJ. L-[1-11C]-tyrosine PET to evaluate response to hyperthermic isolated limb

perfusion for locally advanced soft-tissue sarcoma and skin cancer. J Nucl Med 1999; 40:

262-267.

Olieman AF, Pras E, van Ginkel RJ, Molenaar WM, Schraffordt Koops H, Hoekstra HJ.

Feasibility and efficacy of external beam radiotherapy after hyperthermic isolated limb

perfusion with TNF-alpha and melphalan for limb-saving treatment in locally advanced

extremity soft- tissue sarcoma. Int J Radiat Oncol Biol Phys 1998; 40: 807-814.

Olieman AF, van Ginkel RJ, Molenaar WM, Schraffordt Koops H, Hoekstra HJ. Hyperthermic

isolated limb perfusion with tumour necrosis factor- alpha and melphalan as palliative limb-

saving treatment in patients with locally advanced soft-tissue sarcomas of the extremities

with regional or distant metastases. Is it worthwhile? Arch Orthop Trauma Surg 1998; 118:

70-74.

Olieman AF, van Ginkel RJ, Hoekstra HJ, Mooyaart EL, Molenaar WM, Koops HS.

Angiographic response of locally advanced soft-tissue sarcoma following hyperthermic

isolated limb perfusion with tumor necrosis factor. Ann Surg Oncol 1997; 4: 64-69.

Sleijfer S, van Ginkel RJ, van der Mark TW, Hoekstra HJ, Zwaveling JH, Schraffordt Koops

H, Mulder NH. Effects of hyperthermic isolated limb perfusion with tumor necrosis factor-

alpha and melphalan on pulmonary function assessments. J Immunother 1997; 20: 202-207.

Zwaveling JH, Hoekstra HJ, Maring JK, van Ginkel RJ, Schraffordt Koops H, Smit AJ,

Girbes AR. Renal function in cancer patients treated with hyperthermic isolated limb perfusion

with recombinant tumor necrosis factor- alpha and melphalan. Nephron 1997; 76: 146-152.

Kole AC, Pruim J, Nieweg OE, van Ginkel RJ, Hoekstra HJ, Schraffordt Koops H, Vaalburg

W. PET with L-[1-carbon-11]-tyrosine to visualize tumors and measure protein synthesis

rates. J Nucl Med 1997; 38: 191-195.

Kole AC, Nieweg OE, van Ginkel RJ, Pruim J, Hoekstra HJ, Paans AM, Vaalburg W, Koops

HS. Detection of local recurrence of soft-tissue sarcoma with positron emission tomography

using [18F]fluorodeoxyglucose. Ann Surg Oncol 1997; 4: 57-63.

van Ginkel RJ, Schraffordt Koops H, de Vries EG, Molenaar WM, Uges DR, Hoekstra HJ.

Hyperthermic isolated limb perfusion with cisplatin in four patients with sarcomas of soft

tissue and bone. Eur J Surg Oncol 1996; 22: 528-531.

van Ginkel RJ, Hoekstra HJ, Pruim J, Nieweg OE, Molenaar WM, Paans AM, Willemsen

AT, Vaalburg W, Koops HS. FDG-PET to evaluate response to hyperthermic isolated limb

perfusion for locally advanced soft-tissue sarcoma. J Nucl Med 1996; 37: 984-990.

144

Zwaveling JH, Maring JK, Clarke FL, van Ginkel RJ, Limburg PC, Hoekstra HJ, Koops HS,

Girbes AR. High plasma tumor necrosis factor (TNF)-alpha concentrations and a sepsis-like

syndrome in patients undergoing hyperthermic isolated limb perfusion with recombinant

TNF-alpha, interferon- gamma, and melphalan. Crit Care Med 1996; 24: 765-770.

Zwaveling JH, Maring JK, Mulder AB, Bom VJ, van Ginkel RJ, Schraffordt Koops H, Girbes

AR, Hoekstra HJ, van der Meer J. Effects of hyperthermic isolated limb perfusion with

recombinant tumor necrosis factor alpha and melphalan on the human fibrinolytic system.

Cancer Res 1996; 56: 3948-3953.

Nieweg OE, Pruim J, van Ginkel RJ, Hoekstra HJ, Paans AM, Molenaar WM, Koops HS,

Vaalburg W. Fluorine-18-fluorodeoxyglucose PET imaging of soft-tissue sarcoma. J Nucl

Med 1996; 37: 257-261.

van Ginkel RJ, Hoekstra HJ, Eggermont AMM, Pras E, Koops HS. Isolated limb perfusion

of an irradiated foot with tumor necrosis factor, interferon, and melphalan. Arch Surg 1996;

131: 672-674.

Zwaveling JH, Maring JK, Moshage H, van Ginkel RJ, Hoekstra HJ, Schraffordt Koops H,

Donse IF, Girbes AR. Role of nitric oxide in recombinant tumor necrosis factor-alpha- induced

circulatory shock: a study in patients treated for cancer with isolated limb perfusion. Crit

Care Med 1996; 24: 1806-1810.

Mulder AB, Zwaveling JH, Smid WM, Maring JK, van Ginkel RJ, Girbes AR, Schraffordt

Koops H, van der Meer J. Augmented procoagulant activity in cancer patients, treated with

recombinant interferon-gamma in addition to recombinant tumor necrosis factor-alpha and

melphalan. Thromb Haemost 1996; 76: 897-901.

van Ginkel RJ, Hoekstra HJ, Meutstege FJ, Oosterhuis JW, Uges DRA, Schraffordt Koops

H. Hyperthermic isolated regional perfusion with cisplatin in the local treatment of

spontaneous canine osteosarcoma: assessment of short-term effects. J Surg Oncol 1995; 59:

169-176.

university of groningen different aspects of hyperthermic

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