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Annals of Surgical Oncology 9:355-363 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Value of Continuous Leakage Monitoring With Radioactive Iodine-131–Labeled Human Serum Albumin During Hyperthermic Isolated Limb Perfusion With Tumor Necrosis Factor-{alpha} and Melphalan

Robert J. van Ginkel, MD, Pieter C. Limburg, PhD, D. Albertus Piers, MD, PhD, Heimen Schraffordt Koops, MD, PhD and Harald J. Hoekstra, MD, PhD

From the Departments of Surgical Oncology (RJVG, HSK, HJH), Internal Medicine (PCL), and Nuclear Medicine (DAP), Groningen University Hospital, Groningen, the Netherlands.

Correspondence: Address correspondence and reprint requests to: Harald J. Hoekstra, MD, PhD, Department of Surgery, Division of Surgical Oncology, Groningen University Hospital, PO Box 30.001, 9700 RB Groningen, the Netherlands; Fax: 0031-503614873; E-mail: h.j.hoekstra@ chir.azg.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: 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 with tumor necrosis factor-{alpha} (TNF{alpha}) and melphalan.

Methods: 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 at the axillary level in 6 cases. Leakage rates and perfusion circuit and systemic levels of TNF{alpha}, interleukin-6, and C-reactive protein were determined, as were systemic hematological and metabolic profiles and tumor response.

Results: The mean isotopically measured leakage was 2.9%. Systemic leakage was <=2% in 28 perfusions and >2% in 23 perfusions. The correlation between the maximal monitored leakage and maximal systemic TNF{alpha} levels was .7114. The area under the curve for TNF{alpha} in the perfusion circuit, indicating the exposure of the perfused limb to TNF{alpha}, was 18.7% lower in the >2% leakage group. No significant differences in tumor response were found between groups. The area under the curve for systemic TNF{alpha}, indicating the exposure of the patient to TNF{alpha}, was 18.1 times higher in the >2% leakage group, 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.

Conclusions: A good correlation between RISA leakage measurement and TNF{alpha} exposure during and after hyperthermic isolated limb perfusion with TNF{alpha} 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{alpha}, increased exposure of the patient systemic circulation to TNF{alpha}, and more systemic side effects can be expected.

Key Words: Hyperthermic isolated limb perfusion • Leakage • Iodine-131–labeled human serum albumin • Sarcoma • Melanoma • Tumor necrosis factor-{alpha}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Creech et al.1 developed isolated limb perfusion with chemotherapy for the treatment of extremity melanoma in humans in 1958. Stehlin2 modified the technique in 1969 to include hyperthermia. 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 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{alpha}) to melphalan seems promising.4,5

With the introduction of TNF{alpha}, monitoring of leakage of the isolated circuit into the systemic circulation has been mandatory because TNF{alpha} levels in the perfusion circuit are approximately 10 times the maximally tolerated systemic levels.6 If significant leakage occurs during HILP, the resultant TNF{alpha}-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.8 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). Although determination of blood samples takes time and is discontinuous, it is frequently used by other groups.9,10 To overcome these disadvantages, Stehlin et al.11 were the first to describe a method of continuous external leakage monitoring with RISA. Because of safety regulations, nuclear medicine techniques are not always allowed in the operating room. 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{alpha} and melphalan, with or without interferon {gamma} (IFN{gamma}), 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{alpha} perfusion with respect to systemic levels of TNF{alpha}, interleukin (IL)-6, and C-reactive protein (CRP), as well as hematological and metabolic profiles and tumor response.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Forty-eight patients with melanoma (n = 14) or soft tissue sarcoma (n = 34) of the extremity underwent 51 perfusions with a combination of TNF{alpha} and melphalan, with or without IFN{gamma}. Twenty-one men and 27 women, 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 (14%), and at the axillary level in 6 cases (12%). All patients were treated after informed consent was obtained according to institutional guidelines.

Perfusion Technique
The perfusion technique used 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 of heparin per kilogram body weight (Thromboliquine; Organon BV, Oss, the Netherlands), the axillary, iliac (the internal iliac artery was temporarily closed), 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. 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 minutes under mild hyperthermia (39°C–40°C) and physiologically optimal conditions.16 At the start of perfusion, 3 mg (upper extremity) or 4 mg (lower extremity) of TNF{alpha} (Boehringer, Ingelheim, Germany) was injected as a bolus into the arterial line. Eighteen patients also received a dose of .2 mg of IFN{gamma} (Boehringer) subcutaneously 1 and 2 days before perfusion, followed by .2 mg of IFN{gamma} injected into the arterial line at the start of perfusion. Melphalan (L-phenylalanine mustard; Glaxo-Wellcome, London, England) was administered 30 minutes later as 10 mg/L of extremity volume (leg) or 13 mg/L of 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 oxygen and CO2 and consisted of 350 mL of 5% dextran 40 in glucose 5% (Isodex; Pharmacia AB, Uppsala, Sweden), 500 mL of blood (250 mL of red blood cells and 250 mL of plasma), 30 mL of 8.4% NaHCO3, and .5 mL of 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 minutes of perfusion, the limb was flushed with 2 L of dextran 40 in glucose 5% (Isodex) and 500 mL of blood (250 mL of red blood cells and 250 mL of plasma), catheters were removed, the circulation was restored, and the heparin was 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

Leakage Measurement
Any leakage into the systemic circulation was continuously monitored with radioactive tracers. A small calibration dose of RISA (.5 MBq) and a dose of radioactive 99mTc-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 were measured with an 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 was 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 on-line data processing by a personal computer. The count rate of the .5-MBq RISA determined a baseline count level, corrected for room background. The 10-MBq RTcSA served to check the volume dilution caused by 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 et al.11 were the first to describe the LF on the basis of the following equation:equation


(1)

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 plus the patient’s systemic circulation), and Vsystemic is the blood volume of the patient’s systemic circulation.

Blood Sampling Procedure and Assays
A baseline blood sample from the patient’s systemic circulation was taken from an indwelling radial artery cannula before the start of the operation and at 5, 30, 60, and 89 minutes 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 minutes 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, {gamma}-glutamyl transpeptidase, protein, cholesterol, lactic dehydrogenase (LDH) with its isoenzymes, 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 1 month after perfusion. Blood samples (3 mL) were collected in ethylenediaminetetraacetic acid Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) and kept on melting ice during transport to a centrifuge. Samples were centrifuged for 10 minutes at 3000 rpm at 0°C, and the separated plasma was kept at -80°C until analysis.

TNF{alpha} 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 enzyme-linked immunosorbent assays, as described previously,19 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 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 were performed.

Statistical Analysis
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. GraphPad Prism version 2.0 for Windows (GraphPad, San Diego, CA) statistical software was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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. Because this was the cause of the high leakage and perfusion was not completed, the data from this patient were 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 < .0001 and .0159, respectively). There was no leakage encountered in patients with axillary perfusions.



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  		FIG. 1. Scatter diagram of leakage at different perfusion levels; the uninterrupted line represents mean values. A significant difference was observed between perfusions performed at the iliac and femoral levels compared with leakage values at the popliteal level (P < .0001 and .0159, respectively).

 
Perfusion Circuit Levels
At 5 minutes, mean TNF{alpha} levels in the perfusion circuit were 6798 ± 528 ng/mL (Fig. 2). During perfusion, a significant decrease in TNF{alpha} levels in the perfusion circuit occurred, with a significantly lower concentration of TNF{alpha} in the perfused limb in patients with >2% leakage at 30 minutes (P = .0201), 60 minutes (P = .0337), and 89 minutes (P = .002). The calculated mean AUC, indicating the exposure of the perfused limb to TNF{alpha}, was 18.7% less in the >2% leakage group (P = .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 group and 11.7 ± 3.5 ng/mL in the >2% leakage group (P = .0455). CRP levels in the perfusion circuit remained at the detection level, and no significant differences were observed between the leakage groups.



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  		FIG. 2. Tumor necrosis factor-{alpha} (TNF{alpha}) levels in the perfusion circuit (mean ± SEM). A significant decrease in TNF{alpha} levels occurred with significantly lower concentrations of TNF{alpha} in the perfused limb in patients with >2% leakage at 30 (P = .0201), 60 (P = .0337), and 89 minutes (P = .002). Mean area under the curve, indicating the exposure of the perfused limb to TNF{alpha}, was 18.7% less in the >2% leakage group.

 
Systemic Levels
Systemic TNF{alpha} levels in patients with >2% leakage were already significantly higher at 5 minutes after TNF{alpha} injection compared with the group of patients with <=2% leakage. Peak systemic TNF{alpha} 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 < .0001; Fig. 3). The calculated mean systemic AUC, indicating the exposure of the patient to TNF{alpha}, was 18.1 times higher in the >2% leakage group (P < .0001). Ten minutes after release of the tourniquet, we observed a significant systemic peak level of TNF{alpha} in the <=2% group, possibly caused by the TNF{alpha} still present in the perfused limb after the washout procedure (P = .026). To calculate the correlation between maximal systemic TNF{alpha} levels and the maximal monitored leakage by using the RISA measured during perfusion, Pearson’s correlation (two tailed) was used. Figure 4 illustrates the observed correlation with r = .7114 and P < .0001. A strong correlation was also found between the maximal observed leakage and the maximal IL-6 concentration measured in the postoperative period (r = .7737, P < .0001).



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  		FIG. 3. Tumor necrosis factor-{alpha} (TNF{alpha}) levels in the systemic circulation of the patients (mean ± SEM). A significant difference was found between the >2% leakage group and the <=2% leakage group starting 5 minutes after TNF{alpha} injection and lasting until the second postoperative day (P < .05). The mean systemic area under the curve, indicating the exposure of the patient to TNF{alpha}, was 18 times higher in the >2% leakage group.

 


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  		FIG. 4. Pearson’s correlation (two tailed) between maximal systemic tumor necrosis factor-{alpha} (TNF{alpha}) levels measured during perfusion and maximal monitored leakage using radioactive iodine-131–labeled human serum albumin (r = .7114, P < .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 with <=2% leakage vs. 77.7 ± 20.8 ng/mL with >2% leakage; P = .0089). The AUC of IL-6 was 4.7 times higher in the >2% leakage group compared with the <=2% leakage group (P < .0243).

CRP started to increase 6 hours after HILP and reached its maximal value 2 days after perfusion (185.8 ± 25.5 mg/L with <=2% leakage vs. 226.7 ± 32.7 mg/L with >2% leakage; not significant). The AUC of CRP between the groups, however, was not significantly different.

Hematological and Metabolic Parameters
Leukocyte counts increased from 7.7 ± .3 x 109/L to 13.0 ± .6 x 109/L 1 day after perfusion; 5, 6, and 7 days after perfusion, a significant difference between the two leakage groups was observed (Fig. 5). Platelet counts decreased from 303.6 ± 13.4 x 109/L before perfusion to 124.3 ± 10.7 x 109/L 4 days after perfusion. 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 ± .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 the 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 {gamma}-glutamyltransferase 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 isoenzymes 1 and 2 showed a decrease, whereas LDH isoenzymes 4 and 5 increased 1 day after perfusion. LDH isoenzyme 3 remained at the same level. Creatine phosphokinase levels increased from 28.3 ± 2.4 U/L to a maximum of 496.4 ± 197.6 U/L on 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 1 day after perfusion. None of these variables showed a significant difference between the leakage groups.



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  		FIG. 5. White blood count (WBC) and platelet (PLT) levels from before perfusion to 30 days after perfusion (mean ± SEM). *Significant difference between the leakage groups (P < .05).

 


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  		FIG. 6. Bilirubin, protein, and cholesterol levels from before perfusion to 30 days after perfusion (mean ± SEM). *Significant difference between the leakage groups (P < .05).

 
Tumor Response
In the <=2% leakage group, 14 patients showed a CR, and the same number of patients showed a PR. In the >2% leakage group, 11 patients showed a CR, 10 patients showed a PR, and 2 patients had no change. No significant differences in response to TNF{alpha} 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).



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  		FIG. 7. Absence of distant metastasis in patients with grade II or III soft tissue sarcoma. No significant difference was found between the >2% leakage group and the <=2% leakage group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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{alpha} levels after HILP with TNF{alpha}, from Gerain et al.6 in 1992, demonstrated no significant correlation between leakage and cytokine levels at any time, raising questions about the value of the leakage measurement procedure.

The aim of this study was to investigate whether or not the RISA leakage measurements during HILP with TNF{alpha} used in the Groningen University Hospital are accurate in predicting systemic TNF{alpha} levels. We observed a good correlation between maximal systemic TNF{alpha} levels and the maximal monitored leakage (r = .7114, P < .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{alpha} levels (r = .7737 vs. r = .7114). IL-6 levels occurred in response to TNF{alpha}, with a high correlation between maximal levels of both cytokines (r = 8097). Stam et al.22 also found a strict correlation between the degree of leakage estimated by isotope monitoring and the measured maximal systemic TNF{alpha} levels in the same treatment setting (r = .7886, P = .0067; calculation based on their data). They also found a sharper relation between systemic IL-6 curves and duration of exposure to high TNF{alpha} 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.,23 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. 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 an 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{alpha} levels in the perfusion circuit were approximately 7000 ng/mL, approximately 50 times higher than peak systemic levels. A significantly lower concentration of TNF{alpha} 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{alpha} in the > 2% leakage group. This decrease in TNF{alpha} exposure, however, did not result in a significant reduction of tumor response between the groups. This result supports the initiation of TNF{alpha} dose-reduction studies. Thom et al.24 observed the same decreased TNF{alpha} perfusion circuit levels in patients with >=1% leakage. The Rotterdam perfusion group did not demonstrate a significant difference in perfusion circuit TNF{alpha} levels between a high- and low-leakage group, possibly because of a limited number of samples available.22

TNF{alpha} levels in the systemic circulation of the patients were approximately 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{alpha} exposure was 18.1 times less as calculated by the AUC. On the basis of the hypothesis that micrometastatic disease is attacked by the leaked TNF and melphalan, a higher systemic exposure of TNF could have its effect on the occurrence of distant metastasis during follow-up. However, subanalysis of the occurrence 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 maximal levels reached 2 hours after HILP. CRP levels started to increase 6 hours after HILP and reached their maximum 2 days after perfusion. A three-wave pattern was seen; the first wave was caused by the systemically leaked TNF{alpha} that generated a second wave of IL-6 some hours after perfusion, followed by a third wave of CRP that lasted for several days.

TNF{alpha} leakage was associated with a decrease in leukocyte and platelet count, with significantly lower values in the >2% leakage group. Representing cytolytic liver toxicity, significant hyperbilirubinemia, hypocholesterolemia, and proteinemia were observed in the >2% leakage group. An increase in the activity of the fraction of LDH isoenzymes 4 and 5 after perfusion was partly related to hepatotoxicity and partly to muscle damage. No significant difference between the leakage groups was found for creatine phosphokinase levels or myoglobin levels, although both parameters showed a significant increase after HILP. The same results were obtained by Sorkin et al.,25 who diminished TNF{alpha} leakage after flow rate reduction during TNF{alpha} HILP. Analysis of our own flow data in relation to systemic leakage revealed a weak negative correlation of r = -.2910 and P = .0448 with a mean flow of 455 ± 172 mL/min in our perfusions.

Like others, we also found a significant systemic TNF{alpha} 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 normal saline, TNF{alpha} in the limb reaches the systemic circulation. A corresponding increase 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{alpha} perfusions to reduce TNF{alpha} release.

In a previous study we described the clinical features of HILP with TNF{alpha} characterized by a short-lived sepsis-like syndrome.28 This, best called SIRS, was seen in all patients and was accompanied by fever, an 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 maximal TNF{alpha} concentrations and systemic vascular resistance and cardiac index. The National Cancer Institute perfusion group demonstrated the relation between the vascular response and the need for vasopressor support and systemic TNF{alpha} levels in patients with TNF{alpha} leakage as well.24 Lienard et al.4 and Gerain et al.6 also demonstrated severe toxicity in patients with leaks of >5%. Vrouenraets et al.26 reported an absence of severe systemic toxicity of TNF{alpha} in patients without systemic leakage. Stam et al.22 observed only a mild postoperative toxicity in the event of significant leakage during perfusion. This was easily managed in 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{alpha} systemically. Currently, SIRS is only seldom seen, because the majority of the institutions performing HILP with TNF and melphalan are experienced and are using a more extensive washout procedure. One could ask if leakage measurements during HILP are still worthwhile when the side effects of TNF{alpha} leakage are so easily dealt with. In this study we demonstrated a good correlation between RISA leakage measurement and TNF{alpha} exposure during and after HILP with TNF{alpha} 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{alpha}, increased exposure of the patient’s systemic circulation to TNF{alpha}, and more systemic side effects can be expected. Because leakage of >2% did not influence the tumor response, further dose-reduction studies of TNF in the HILP setting are warranted.

Received for publication September 21, 2001. Accepted for publication February 9, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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