Vol. 4, 2357-2362, October 1998 Clinical Cancer Research 2357

Augmented Capillary Leak during Isolated Hepatic Perfusion (IHP)

Occurs via Tumor Necrosis Factor-independent Mechanisms

H. Richard Alexander,’ Charles K. Brown,

David L. Bartlett, Steven K. Libutti,

William D. Figg, Sangeeta Raje, and Ewa TurnerSurgical Metabolism Section, Surgery Branch, and the Clinical

Pharmacokinetics Section, Medicine Branch, Division of Clinical

Sciences, National Cancer Institute, Bethesda, Maryland 20892

ABSTRACTIsolated organ perfusion of the liver or extremity with

tumor necrosis factor (TNF) and melphalan results in re-

gression of bulky tumors in the majority of patients. The

efficacy of TNF in this setting is not known, although data

suggest that it may exert antitumor effects primarily on

tumor-associated neovasculature. We studied the effects of

TNF on capillary leak in liver and tumor tissue during

isolated hepatic perfusion (IHP) with melphalan. Twenty-

seven patients with unresectable cancer confined to the liver

underwent a 60-mm hyperthermic 11W using 1.5 mg/kg

melphalan alone (n = 7) or with 1.0 mg of TNF (n = 20).

Complete vascular isolation was confirmed in all patients

using an intraoperative leak monitoring 1-131 radiolabeled

albumin technique. Samples of tumor and liver were col-

lected just prior to and immediately after IIIP. There was no

difference in 1-131 radiolabeled cpm/g of tissue (cpm) in

liver versus tumor at baseline (P2 0.44). After DIP, 1-131

albumin cpm were higher in tumor versus liver (10,999 ±

1,976 versus 3,821 ± 780, respectively; P2 < 0.005). How-

ever, 1-131 albumin cpm in tumor were not effected by TNF

(11,636 ± 2,518 with TNF versus 9,180 ± 2,674 without

TNF; P2 = 0.59). TNF did not affect melphalan concentra-

tions in tumor (1,883 ± 540 ng/g versus 1,854 ± 861 nglg

without TNF; P2 0.9). Capillary leak, as reflected by

diffusion of 1-131 radiolabeled albumin into the interstitial

space, is comparable in liver and tumor before IHP but is

significantly higher in tumor after IHP. The increased dif-

fusion in the capillary tumor bed must occur through TNF-

independent mechanisms such as intrinsic features of tumor

neovasculature, hyperthermia, or other unrecognized perfu-

sion-related factors. These data indicate that TNF must

continue to be critically evaluated in clinical trials before it

is routinely used with melphalan in isolated organ perfusion.

Received 4/29/98; revised 6/22/98; accepted 7/6/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I To whom requests for reprints should be addressed, at Surgery Branch,

National Cancer Institute, NIH, Building 10, Room 2B07, Bethesda,

Maryland 20892. Phone: (301) 496-2195; Fax: (301) 402-1788; E-mail:

Richard_Alexander@nih.gov.

INTRODUCTIONIsolated organ perfusion with TNF2 and melphalan has

resulted in remarkably high response rates in patients with

intransit melanoma or unresectable high-grade sarcoma of the

extremity or unresectable cancers confined to the liver (1-4). In

1992, Li#{233}nard et a!. ( 1 ) published their initial results of ILP with

TNF, melphalan, and IFN-�y for patients with intransit mela-

noma or extremity sarcoma. They reported an 89% complete

response rate and an overall response rate of 100% ( 1). Theseresults were interpreted to be superior to those obtained in

previous reports of ILP using melphalan alone (2, 5), and based

on these data, a number of centers initiated ILP clinical trials

using TNF and melphalan administered via ILP to critically

evaluate the potential therapeutic efficacy of this regimen. Sub-

sequent series have reported complete response rates between

75 and 90% for patients with intransit melanoma and a limb

salvage rate of over 80% in patients with unresectable high-

grade extremity sarcoma (6-8). A Phase II trial of IHP using

TNF and melphalan resulted in an overall response rate of 74%

in patients with unresectable hepatic malignancies, including

those with numerous metastases, bulky sites of disease, or

patients who had failed extensive prior treatment (9).

When TNF is used with melphalan, there is a characteristic

tumor response distinct to that observed following ILP with

melphalan alone. There is often rapid necrosis of tumors with

eschar formation of bulky cutaneous lesions and abrupt cystic

changes in deeper lesions on computed tomography or contrast-

enhanced magnetic resonance imaging. There are considerable

experimental data ( 10-12) and clinical observations ( 13, 14)

that indicate that the primary antitumor effects of TNF admin-

istered via isolated organ perfusion are on the tumor-associated

neovasculature. In experimental models, TNF administration

systemically results in rapid necrosis of tumors, and on histo-

logical evaluation, there is primarily endothelial cell injury with

intravascular erythrostasis and fibrin deposition (10). Turnors

resistant to direct TNF cytotoxic effects in vitro may be sensi-

tive to TNF effects in vivo, particularly when > 1 cm in diam-

eter, a size at which the tumor neovasculature has been estab-

lished (15). In ILP with TNF-based regimens, there is cornplete

obliteration of the tumor-associated neovasculature after treat-

ment (14).

Despite considerable clinical evaluation, the exact contri-

bution of TNF to the antitumor effects during hyperthermic

isolated organ perfusion with melphalan are not known. Phase

III random assignment trials are under way in Europe and the

United States comparing these two treatment regimens admin-

istered via ILP for extremity melanoma. In a preliminary anal-

2 The abbreviations used are: TNF, tumor necrosis factor; ILP, isolated

limb perfusion; IHP, isolated hepatic perfusion; IVC, inferior vena cava;

GDA, gastroduodenal artery.

2358 IHP and Capillary Leak

ysis of a random assignment trial comparing these regimens in

patients with stage lilA or IIIAB intransit melanoma, the corn-

plete response and overall response rate between groups were

not significantly different, although patients with bulky disease

or large lesions may have had a better response to TNF and

melphalan compared with melphalan alone.3 Although TNF has

potential effects on the tumor-associated neovasculature, when

it is given alone via hyperthermic isolated organ perfusion of the

limb or liver, there are no significant antitumor effects (16). In

addition, leak of perfusate containing TNF can result in poten-

tially significant systemic toxicity (5). Insight into the mecha-

nisms of the effects of TNF in isolated organ perfusion will be

important to use it most efficaciously in this setting. The present

study was undertaken to determine the effects of TNF on endo-

thelial cell permeability as measured by diffusion of radiola-

beled 1-131 albumin in normal and tumor tissue before and after

hyperthermic IHP with rnelphalan.

PATIENTS AND METHODSPatient Population. Between August 1996 and May

1997, 27 patients with primary or metastatic unresectable can-

cers confined to the liver were treated with a 60-mm hyperther-

mic IHP using 1.5 mg/kg of melphalan with (n = 20) or without

(n 7) tumor necrosis factor. The treatment protocols were

approved by Institutional Review Board and the Cancer Ther-

apeutics Evaluation Program of the National Cancer Institute.

Patients were treated on two consecutive Phase II protocols

using identical IHP treatment parameters; the latter without

TNF. All patients had multiple bibobar hepatic tumors. All

patients who had tumors accessible for biopsy were included in

the study. All patients had measurable, unresectable, biopsy-

proven primary metastatic cancers confined to the liver. Patients

underwent standard staging studies including computed tomog-

raphy scan of the chest, abdomen, and pelvis and, as clinically

indicated, brain magnetic resonance imaging or bone scan.

Eligibility criteria included Eastern Cooperative Oncology

Group performance status of 0 or 1 , a serum bilirubin <2.0

mg/dl, a platelet count > l50,000/ml, and a serum creatinine

�l.5 mg/dl.

IHP. The treatment technique of IHP was performed as

described previously (4). Briefly, via a laparotomy the liver is

extensively mobilized by dividing the falciform ligament and

the right and left triangular ligaments. The duodenum is exten-

sively mobilized and reflected medially. The right lobe of the

liver is retracted anteriorly and medially and the IVC from the

level of the renal veins to the diaphragm is completely dissected

from the retroperitoneum. The portahepatis structures are com-

pletely dissected and skeletonized, and a cholecystectomy is

performed. A 2-cm segment of GDA is dissected and serves as

the arterial cannulation site during IHP. The portal vein and

common bile duct are mobilized from the head of the pancreas

3 D. L. Fraker, H. R. Alexander, D. L. Bartlett, and S. A. Rosenberg. Aprospective randomized trial oftherapeutic isolated limb perfusion (ILP)

comparing melphalan (M) versus melphalan, tumor necrosis factor(TNF) and interferon-rny (IFN): an initial report, submitted for publica-

tion.

to the inferior border of the liver. All lymph node-bearing

tissues around the portahepatis structures are resected. A saphe-

nous vein and left axillary cutdown are performed. The patient

is systemically heparimzed with 200 units/kg, and after -5 mm,

a cannula is inserted into the saphenous vein and advanced into

the inferior vena cava just below the renal veins. A second

venous cannula is inserted into the axillary vein, and these are

connected to a veno-veno bypass circuit. The IVC is occluded

above the renal veins, and infrahepatic IVC blood flow is

shunted to the axillary vein using a centrifugal pump. A short

segment of infrahepatic IVC is isolated between vascular oc-

cluding clamps, and a 20-24 French venous cannula is inserted

through a venotomy and positioned behind the retrohepatic IVC

just beneath the hepatic veins. This cannula is connected to the

venous outflow line of the extracorporeal bypass circuit. The

portal vein blood flow is shunted by inserting a cannula distally

and incorporating it into the veno-veno bypass circuit. The GDA

is ligated, the common hepatic artery is occluded, and a

3-4-mm GDA cannula is positioned at the orifice of the corn-

mon hepatic artery. Finally, the suprahepatic IVC is cross-

clamped just below the diaphragm and isolated hepatic perfu-

sion is initiated.

The extracorporeal bypass circuit consists of a roller pump,

membrane oxygenator, and heat exchanger. The perfusate con-

sists of 700 ml of balanced salt solution primed with 300 ml of

packed RBCs and 2000 units of heparin. Arterial and venous

perfusate blood gases are obtained at regular intervals, and

sodium bicarbonate was added to the perfusion circuit to main-

tam an arterial perfusate pH between 7.2 and 7.3. Hepatic

parenchymal temperature probes are placed at various positions,

and perfusate was temperature controlled using a Hemotherm

cooler heated model #4 (Cincinnati SubZero Products, Cincin-

nati, Ohio). Flow rates are adjusted upward while monitoring for

a stable reservoir volume and acceptable line pressures. Usually,

stable perfusion parameters are achieved almost immediately,

and there is rapid and uniform heating of the liver to target

temperatures of 39.5-40#{176}C. Melphalan at a dose of 1.5 mg/kg

(Glaxo-Wellcome, Research Triangle Park, NC) and 1.0 mg

TNF (Knoll Pharmaceuticals, Whippany, NJ) are added sequen-

tially to the arterial inflow line of the perfusion circuit at time 0,

and the perfusion continued for 60 mm. At the conclusion of the

perfusion, the liver is flushed through the arterial inflow cannula

with 1500 ml of crystalloid, followed by 1500 ml of colloid, and

the proximal portal vein is flushed with 1 liter of normal saline.

After decannulation and repair of the P/C and portal venoto-

mies, normal physiological blood flow is reestablished promptly

to the liver.

Continuous Intraoperative Leak Monitoring with 1-131

Radiolabeled Human Serum Albumin. A continuous intra-

operative leak monitoring system as described previously for

isolated limb and liver perfusion with TNF and melphalan was

used in all patients. In addition, because the 1-131 radiolabeled

albumin was being used to assess capillary endothelial leak in

the liver and tumor vascular beds just before and after IHP,

tissue samples were obtained at consistent predetermined points.

Once stable perfusion parameters were established, a gamma

detection camera was positioned over the centrifugal pump

housing, which served as a stable reservoir of systemic blood

flow for the purposes of calculating leak rates. A 20 pCi dose of

Clinical Cancer Research 2359

Table 1 Patient demographics

Total TNF No TNF

n 27 20 7Male:female 18:9 13:7 5:2

AgeMean (range) 50 ± 2.2 49.6 ± 2.7 52.4 ± 3.5Range (yr) 24-71 24-7 1 37-65

Previous treatment (%) 13 (49%) 9 (45%) 4 (56%)Diagnoses

Colon adenocarcinoma 17 13 4Ocular melanoma 5 4 1

Hepatocellular carcinoma 2 2 0Carcinoid 2 0 2Leiomyosarcoma 1 1 0

radiolabeled 1-131 human serum albumin (Merck-Frosst, Que-

bec, Canada) was administered via a central vein, and a baseline

level of radioactive cpm (cpm) was determined on a stripchart

recorder. Subsequently, a 10-fold higher dose of 1-131 radiola-

beled albumin was then injected into the perfusion circuit. If any

increase in cpm were detected on the stripchart recorder, this

would indicate a leak from the perfusion circuit into the sys-

temic circuit circulation.

Tissue Samples. Between 3 and 5 mm after the perfusate

dose of radiolabeled I- 13 1 albumin had been administered, liver

and tumor biopsy samples were obtained, maintained on ice, and

immediately transported to the laboratory. At the completion of

IHP, after the flush of perfusate from the circuit and prior to

reestablishing native liver blood flow, a second biopsy of liver

and tumor tissue was obtained and transported immediately to

the laboratory on ice. If sufficient tissue had been obtained, a

portion of each sample was immediately frozen at - 80#{176}Cfor

subsequent melphalan tissue concentrations. The remaining

samples were immediately weighed and placed in scintillation

vials. 1-131 albumin was quantitated as cpm/g of tissue (cpm)

using a gamma counter.

Melphalan Assay. The melphalan tissue samples were

analyzed using an acid precipitation method, and reverse-phase

HPLC assay was used for quantification of melphalan. The

standard curve preparation procedure involved weighing 0.3 g

of blank liver tissue, adding 300 �l of water, and adding known

amounts of melphalan using a stock that was prepared in 0. 12 N

HC1. The tissue was then homogenized, and 300 p.1 of the

homogenate were transferred to a cold 1 .5-ml centrifugation

tube. The precipitation of proteins was facilitated by the addition

of 75 ml of ice-cold 4.6 N perchloric solution. The solution was

then vortexed and refrigerated at 4#{176}Cfor 20-30 mm to aid in

further protein precipitation. The homogenate was then centri-

fuged for 4-5 rain at 13,500 rpm, and 200 �i.l of the supernatant

were transferred into injector vials. A total of 170 pA was

injected into the reverse phase-HPLC system composed of a � 8

column and a isocratic mobile phase consisting of 0.24 N HC1:methanol (53:47% v/v) with a flow rate of 1 mI/mm. Fluores-

cence detection was used to detect melphalan, which had an

excitation wavelength of 260 nm and an emission wavelength of

365 nm. The patient samples were weighed, and 300 pA of water

added and the samples were processed with the same method as

the standard curve. The equation produced using the standard

TabI e 2 IHP parameters

Total TNF No TNF

n

Melphalan dose

Mean (mg)

Range (mg)

Duration (mm)

Flow rateMean (mL/min)

Range (mL/min)

Mean central hepatic

temperature (#{176}C)

Mean perfusion pressure

(mm Hg)

Perfusion pressure range(mm Hg)

Mean change reservoir

volume (ml)

%leak

27

117 ± 4.1

75-160

60

853 ± 25600-1350

40.0 ± 0.06

176 ± 5.7

105-240

98 ± 14

0

20

115 ± 4.375-144

60

840 ± 21600-1050

40. 1 ± 0.07

177 ± 6.3

1 10-240

1 10 ± 17

0

7

124 ± 9.4

95-160

60

889 ± 75720-1350

39.9 ± 0.07

171 ± 12.9

105-223

64 ± 23

0

curve was used to calculate the mass of melphalan present in

each sample. The mass of melphalan was then divided by the

weight of the tissue to give the final concentration in ng/g tissue.

Statistics. Data are presented as mean ± SE. Data were

analyzed using Student’s t test, and significance of differences

between data was determined by using the Bonferoni correction

for multiple comparisons.

RESULTSPatient demographics are shown in Table 1 . There was a

similar mean age, a 2: 1 male:female ratio, and a predominance

of colon adenocarcinoma diagnoses in both groups. Twenty-

seven patients were treated with a 60-mm hyperthermic IHP

using 1.5 mg/kg melphalan; 20 were treated with TNF, and 7

without. The perfusion parameters are shown in Table 2. The

mean flow rate, central hepatic temperature, line pressure, and

other parameters were comparable between groups. Using the

radiolabeled 1-131 albumin continuous leak monitoring system,

there was no identifiable leak of perfusate into the systemic

circulation in any patient. Conversely, the mean change in

reservoir volume in each group was < 100 ml, indicating no leak

of systemic blood into the perfusion circuit. Small changes in

reservoir volume are typically observed during the 60-mm IHP

and usually reflect small changes in passive filling or emptying

of the hepatic vascular bed.

Fig. 1 illustrates the protocol for administration of radio-

labeled 1-131 albumin and biopsies of liver and tumor just

before and immediately after Il-IF. After establishing a stable

hepatic perfusion circuit, the systemic dose of I- 13 1 albumin

was administered through a central vein. Biopsies of liver and

tumor were obtained 3-5 mm after administration of the circuit

dose of 1-131 radiolabeled albumin. On the basis of mean flow

rates of almost 850 mi/mn and a circuit volume of 1 liter, this

allowed the perfusion circuit containing 1-131 albumin to recir-

culate through the hepatic vascular bed -4 times. Melphalan,

with or without TNF, was then added via the arterial line of the

perfusion circuit. After 60 mm, the circuit was flushed to re-

move melphalan and TNF from the intravascular space and also

served to remove any intravascular I- 1 3 1 radiolabeled albumin.

Establish IHP circuit

15000 -

0�.2� �oooo

E

�

Fig. 3 1-131 albumin cpm in tumor samples post-IHP with or without

TNF (P1 = 0.59). Bars, SE.

1 .) Biopsy liver and tumor

2.) Add melphalan± TNF�

C)

a)� 2000 -

-�

0.. �

4)

0�

= Liver

- TumorFig. 4 Melphalan concentrations (ng/gm) in tumor samples after IHPwith n = 14 and without TNF (n 5, P2 0.96). Bars, SE.

___ 15000a)

�C

E

g�Pre-IHP Post-IHP

Fig. 2 Mean 1-131 albumin cpm in liver and tumor samples obtainedimmediately before and after IHP. Post-IHP 1-131 albumin was signif-icantly greater than post-IHP liver levels (*, P2 0.005) and pretreat-

ment tumor levels (*, P2 = 0.006). There was no difference in 1-131

albumin cpm in liver compared with tumor at baseline and a marginal

increase in 1-131 albumin cpm in post- compared with pre-IHP liver(P2 = 0.06). Bars. SE.

Therefore, all 1-131 albumin detected in these samples is in the

interstitial space.

Overall, there was no difference in 1-131 albumin cprn in

liver compared with tumor at baseline (P2 = 0.44; Fig. 2). There

was a marginal increase in I- 1 3 1 albumin cpm in post- corn-

pared to pre-IHP liver tissue (P2 = 0.06). I- 13 1 albumin cpm

were significantly higher in tumor after IHP compared with

pre-IHP tumor (P2 = 0.006) and post-IHP liver (P2 = 0.005;

Fig. 2). Interestingly, the 1-13 1 albumin in tumor was not

2360 IHP and Capillary Leak

1T3 to 5 mm

20 uCi 1-131 albumin�

� systemically

3to5min

200 uCi 1-131 albumin intoIHP circuit

5mm

60 mm 3000 ----�-�--

I .) Flush HP circuit

2.) Biopsy liver and tumorj

Fig. 1 Protocol for obtaining liver and tumor samples before and

immediately after isolated hepatic perfusion.

TNF NoTNF

TNF NoTNF

statistically different between patients treated with or without

TNF (P, = 0.59; Fig. 3). Furthermore, TNF did not affect

melphalan tumor concentrations after IHP when compared with

melphalan alone (P2 = 0.97; Fig. 4). These last data are based

on sample analyses of 14 patients treated with TNF and 5

without. One other patient treated without TNF had a melphalan

concentration in tumor that was >2-fold higher than any other

value, almost 10-fold higher than the mean of either group and,

therefore, was excluded from the statistical analysis. However,

the outcome of the statistical analysis between the groups re-

mained nonsignificant when the value was included in the data.

DISCUSSION

Because of the significant effects on tumor neovasculature

that have been observed after hyperthermic isolated limb per-

fusion with TNF and melphalan (13, 14) and the experimental

data suggesting that the primary antitumor effects of TNF may

be exerted on the tumor neovasculature ( 1 1 , 14), this study was

performed to determine whether hyperthermic isolated hepatic

perfusion may cause augmentation in tumor-associated micro-

vascular permeability, thereby facilitating the selective delivery

of chemotherapeutics to the tumor.

The data presented here show that comparable amounts of

Clinical Cancer Research 2361

4 H. R. Alexander, unpublished observations.

radiolabeled 1-131 albumin are present in liver and tumor tissue

just prior to treatment with isolated hepatic perfusion. However,

after a 60-mm hyperthermic hepatic perfusion using rnelphalan

with or without TNF, there is a significant and selective increase

in interstitial 1-13 1 albumin in tumor compared with both pre-

treatment tumor levels and posttreatrnent liver tissue. The pre-

treatment 1-131 albumin cprn in liver and tumor also reflect

some I- 13 1 albumin contained in the intravascular space in the

tissue biopsies obtained. However, posttreatrnent values reflect

true interstitial amounts of 1-131 albumin because the circuit is

flushed with 3 liters of crystalloid and colloid solution, which

effectively removes the 1-131-containing perfusate from the

intravascular space. The 27 patients analyzed in this report

underwent an identical hepatic perfusion treatment protocol.

This cohort represents consecutively treated patients who had

tumor that could be easily biopsied before and after treatment.

All but two patients had metastatic cancers to the liver, and

two-thirds of those were colorectal tumors. All identifiable

perfusion-related parameters that may have influenced micro-

vascular permeability were comparable between the 20 patients

treated with TNF and the 7 who received no TNF. Remarkably,

using the maximum safe tolerated dose of TNF when combined

with 1.5 mg/kg rnelphalan, there was no significant difference in

interstitial 1-131 albumin in patients treated with TNF compared

with those who received no TNF. This suggests that other

unrecognized perfusion-related factors must be accounting for

the augmented capillary leak in the tumor bed during isolated

perfusion. The number of possibilities are considerable and

would include the artificial flow dynamics of the extracorporeal

bypass circuit, local pH changes potentially related to nonphysi-

ological flow rates, or local production of cytokines or other

factors that may render the tumor-associated neovasculature

selectively sensitive to hyperthermia or melphalan. Interest-

ingly, the initial overall response rates between the groups are

similar.4

The comparable levels of rnelphalan in liver tissue after

IHP in patients treated with or without TNF may be related to

the increased diffusability of 1-131 albumin. The larger macro-

rnolecule, I- 13 1 albumin, has a molecular weight of 66,000,

whereas melphalan is a considerably smaller molecule with a

molecular weight of 305. Therefore, its delivery into the inter-

stitial space may not be as dependent on changes in endothelial

permeability as 1-131 albumin. Although mean melphalan con-

centrations were similar in patients treated with TNF compared

with those who were not, because there was only a single time

point available for analysis, there are limited conclusions that

can be drawn from these data. The tissue concentrations of

melphalan over time or the possible cellular or molecular effects

of rnelphalan with or without TNF have not been addressed in

this study.

A large number of institutions are evaluating the use of

TNF in isolated limb perfusion for intransit melanoma or unre-

sectable high grade extremity sarcoma, and a smaller number of

centers are evaluating its use in IHP for unresectable cancers

confined to the liver. In both limb and liver perfusion TNF is

associated with considerable regional and systemic toxicities

(9, 17, 1 8) and has not been conclusively demonstrated to

improve response rates.3 On the basis of presently available

data, it appears that TNF causes a very characteristic pattern of

tumor regression manifested by rapid tumor necrosis with es-

char formation of cutaneous lesions, significant angiographic

and radiographic changes of large extremity sarcomas, and a

more rapid time course of response compared with patients

treated with melphalan alone (2, 14, 19). Taken together, these

points highlight the fact that the use of TNF in isolated perfusion

must be critically evaluated in appropriately designed clinical

trials to determine efficacy and mechanisms of its antitumor

effects, optimal dosing schedules, and appropriate patient selec-

tion.

We have recently reported results of a Phase II trial of IHP

with melphalan and TNF for patients with unresectable cancers

confined to the liver (9). The 60-mm perfusion interval has been

used based upon previously conducted studies, and there are no

data indicating that a longer or shorter interval would be supe-

nor. The overall response rate in 33 evaluable patients was 74%

including patients with large or numerous lesions as well as

those who had undergone extensive previous treatment. Seventy-

five percent of all patients experienced transient grade 3 or 4

(National Cancer Institute common toxicity criteria) hepatic

toxicity, and one patient developed fatal hepatic veno-occlusive

disease. Complete vascular isolation was achieved during IHP

on the basis of the results of continuous intraoperative leak

monitoring and the fact that systemic levels of TNF could not be

detected, except for low transient levels in two patients who had

a correctable small (<4%) leak of perfusate during treatment.

However, there are significant hernodynamic changes associated

with this procedure, and circulating levels of interleukin 6 and

interleukin 8 between 2 and 12 h after the procedure are as high

as those reported in patients with life-threatening septic shock

(20-22). These secondary cytokines are presumably released by

the liver in response to the perfusion. In patients undergoing

isolated limb perfusion with TNF and melphalan, systemic

toxicity is associated with the presence of a perfusate leak

during treatment, and leak rates of 5% or greater occur in -15%

of patients (23, 24). The question as to whether the routine use

of TNF is justified in isolated organ perfusion has not yet been

determined.

In summary, the data presented in this report show that

hyperthermic IHP results in a selective and significant increase

in capillary endothelial permeability in tumor-associated neo-

vasculature. This has important implications for the use of

isolated organ perfusion with various therapeutic agents. Al-

though the exact mechanisms for this increase in microvascular

permeability are not known, it does not appear that TNF con-

tributes significantly to this effect.

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