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Clinical Cancer Research Vol. 5, 1650-1657, July 1999
© 1999 American Association for Cancer Research

Clinical Trials

Hyperthermic Isolated Limb Perfusion with Tumor Necrosis Factor-{alpha} and Melphalan in Patients with Locally Advanced Soft Tissue Sarcomas: Treatment Response and Clinical Outcome Related to Changes in Proliferation and Apoptosis1

Boudewijn E. C. Plaat2, Willemina M. Molenaar, Mirjam F. Mastik, Jan Koudstaal, Eva van den Berg, Heimen Schraffordt Koops and Harald J. Hoekstra

Departments of Pathology [B. E. C. P., W. M. M., M. F. M., J. K.] and Surgical Oncology [H. S. K., H. J. H.], University Hospital Groningen, 9700 RB Groningen, the Netherlands, and Department of Medical Genetics, University of Groningen, 9713 AW Groningen, the Netherlands [E. v. d. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperthermic isolated limb perfusion with tumor necrosis factor-{alpha} and melphalan (HILP-TM) with or without IFN-{gamma} is a promising local treatment in patients with locally advanced extremity soft tissue sarcomas (STSs), with response rates of up to 84%. The mechanisms of the treatment response are poorly understood. Here, we determined the HILP-TM-induced changes in mitotic activity, proliferation, and apoptosis in 37 STSs; the additional effect of IFN-{gamma}; and the association of HILP-TM with treatment response and clinical outcome.

On archival material, obtained before and 6–8 weeks after HILP-TM with (n = 15) or without (n = 22) IFN-{gamma}, the number of mitoses was counted, and the proliferation fraction was determined by immunohistological staining for the proliferation associated Ki-67 antigen (MIB1). Apoptosis was visualized by enzymatic detection of DNA fragmentation (terminal deoxynucleotidyl transferase-mediated nick end labeling method). Clinical and histological response, follow-up status, and survival were recorded.

The number of mitoses dropped 57% and proliferation rate decreased with 40% after HILP-TM, whereas the amount of apoptosis after HILP-TM more than doubled as before HILP-TM. The addition of IFN-{gamma} to HILP-TM did not influence the changes in tumor parameters and did not affect treatment response. A better clinical response to HILP-TM was correlated with high mitotic activity and low amount of apoptosis in tumor samples before HILP-TM. Patients with highly proliferative STS before and after HILP-TM had a relatively poor prognosis. Furthermore, patients who developed distant metastases after HILP-TM had a relatively high number of dividing cells in the tumor remnants after treatment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
STSs3 constitute a heterogeneous group of malignant tumors that arise from tissue of mesenchymal origin and account for ~1% of all malignancies. STSs predominantly affect the limbs, and surgical resection in combination with high-dose adjuvant external beam radiotherapy, after marginal resection, is the treatment of choice (1, 2, 3) . Because of the size and extent of the tumor or the proximity of vital structures, limb-saving surgery is not always possible, and mutilating surgical procedures or amputation are occasionally necessary (2 , 4, 5, 6, 7, 8) . Currently, one of the most promising limb-saving procedures is the combination of HILP with delayed surgical tumor excision (9 , 10) . The major advantages of HILP are that regional cytostatic concentrations can be 15–20 times as high as after systemic administration and that hyperthermia may enhance the cytotoxic effect (11 , 12) . In 1992, Lienard et al. (13) used HILP to administer high-dose recombinant TNF-{alpha} in combination with melphalan, an alkylating chemotherapeutic agent, resulting in encouraging outcomes. In HILP-TM with or without IFN-{gamma} response rates of up to 84% have been reported in the treatment of STS (9 , 10 , 13, 14, 15) . Because the mechanism of the antitumor effects of HILP-TM is still poorly understood and studies suggest that chemotherapeutic drugs in general inhibit proliferation by generating cell cycle arrest and subsequent apoptosis, these tumor parameters were investigated in HILP-TM (16, 17, 18, 19, 20, 21, 22, 23) .

The objective of this study was to investigate whether the treatment response and clinical outcome of patients treated with HILP-TM is associated with tumor grade, mitotic activity, proliferation, and apoptosis in the tumor specimens of STS obtained before and after HILP-TM. Furthermore, the influence of HILP-TM on these tumor markers and additional effect of IFN-{gamma} was evaluated.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
The inclusion criteria for this study were: (a) histological diagnosis of a malignant mesenchymal tumor, located in the soft tissues; (b) primary nonresectable tumor of lower or upper limb, which was treated with HILP-TM with or without IFN-{gamma}; and (c) the availability of paraffin-embedded tumor material obtained within 3 months before HILP-TM, with a maximum period between pretreatment and posttreatment tumor samples of 5 months.

Between 1991 and 1997, 37 patients with primary nonresectable STS of the lower or upper limb underwent HILP-TM. HILP-TM was combined with IFN-{gamma} in 15 of the 37 analyzed patients. The mean age (± SD) of the 18 men and 19 women, at the time of diagnosis, was 46 ± 16.7 years (range, 18–80 years; median, 48 years). Four tumors were located in the upper limb (11%), and 33 were located in the lower limb (89%). At the time of HILP-TM, six patients (16%) had distant metastases, and three patients (8%) had regional lymph node metastases. The mean time (± SD) between incisional biopsy and perfusion was 22 ± 17 days (range, 0–83 days; median, 17 days). In one patient, an incisional biopsy of multiple superficial sarcomas was performed at the day of HILP-TM to obtain fresh tumor material. In 35 of the 37 patients, HILP-TM was followed by surgical resection 6–8 weeks later, according to the study protocol. In two patients (5%), histological examination after HILP-TM was not possible: one patient had progressive lung metastases requiring chemotherapy, and in the other patient, the limb had to be amputated because of vascular occlusion 2 days after HILP-TM. Local progressive disease or treatment-related morbidity were reasons for performing the delayed local surgical resection earlier or later than planned in the protocol. Specimens for the assessment of histological reaction were obtained after a mean period of 61 ± 16 days following HILP-TM (range, 12–103 days; median, 60 days). In three patients, in which HILP-TM was performed twice, histological tumor reaction was assessed after the first HILP-TM in two patients and after the second HILP-TM in one patient, of whom no tumor material was available after the first HILP-TM. The mean time between the pretreatment sample and the posttreatment sample was 83 ± 22 days (range, 33–138 days; median, 80 days). No additional treatment was given between HILP-TM and the delayed resection. Patient characteristics, histology, and clinical data are presented in Table 1Citation .


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  		Table 1 Patient characteristics, histology, HILP-TM modality, treatment (clinical, histological, and overall) response, and clinical outcomea

 
Histology.
In all cases, the histological diagnosis was made on H&E-stained paraffin sections of incisional biopsies with or without additional immunohistological stains. All cases were classified according to Enzinger and Weiss (1) , who revealed 16 different histological types (Table 1)Citation . The STSs were graded according to the grading system of Coindre et al. (24) , in which points are assigned to differentiation level, mitotic index, and necrosis. This resulted in 7 grade I (19%), 19 grade II (51%), and 11 grade III (30%) STSs (Table 1)Citation . In the resection specimens, the amount of necrosis was estimated on gross examination. For histology, at least one section per centimeter of the largest tumor diameter was taken. Care was taken to histologically document the presence of necrosis, viable tumor, or fibrosis.

Tumor Parameters.
The following tumor parameters were studied on adjacent slides of the macroscopically and microscopically most viable parts of the tumor before and after HILP-TM: mitotic index, proliferation, and apoptosis.

The number of mitoses per 2 mm2 (mitotic index) in adjacent fields was counted on H&E-stained paraffin sections.

For proliferation, the monoclonal antibody MIB1 (Immunotech S.A., Marseille, France) was used. This antibody recognizes an epitope of the Ki-67 antigen, which is present in the nucleus of cells in all phases of the cell cycle except G0 and early G1 (25) . Immunohistochemistry was performed on paraffin sections (4 µm) according to a method modified from Shi et al. (26 , 27) . Briefly, after heating on a hot plate, slides were dewaxed in xylene and rehydrated in serial ethanol washes (100, 96, and 70%). After two incubations in an autoclave for 10 min at 110°C in a 20 mM blocking reagents (Boehringer Mannheim, Mannheim, Germany; pH 6.0), slides were incubated with a 1:400 dilution of the antibody in BSA buffer (pH 7.4). The primary antibody was detected with a biotinylated secondary antibody (multilink), followed by a streptavidin-alkaline phosphatase conjugate (Ready-to-Use Link and Label; Biogenex, San Ramon, CA). Final color was developed by the bromochloroindolyl-phosphate 4-nitroblue tetrazolium chloride method (Boehringer Mannheim).

Apoptosis was studied in 4-µm sections of formaldehyde-fixed and paraffin-embedded tissue using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. After deparaffination, sections were subjected to RNase (1 mg/ml in 50 mM Tris-HCl and 150 mM NaCl, pH 7.5; Aldrich, Milwaukee, WI) treatment for 30 min at room temperature to prevent nonspecific binding to DIG-11-dUTP. Slides were subsequently incubated with 15 µl of a 100-µl solution containing terminal deoxynucleotidyl transferase buffer (Pharmacia, Uppsala, Sweden) with 5 mM cobalt chloride and 1% BSA (Aldrich) together with 5 nM DIG-11-dUTP (Boehringer Mannheim) for 60 min at 37°C. After this reaction, slides were rinsed twice in a 0.1x SSC solution for 15 min at room temperature. Nonspecific background staining was prevented by preincubating slides with 5% blocking reagents (Boehringer Mannheim). Sections were incubated overnight at 6°C with alkaline phosphatase-labeled sheep anti-DIG Fab fragments diluted 1:300 in 2% blocking reagents (Boehringer Mannheim). After extensive washing, alkaline-phosphatase was visualized with nitroblue tetrazolium 5-bromo-4-chromo-3-indolyl-phosphatase (Boehringer Mannheim) for 50 min at room temperature. Sections were counterstained with hematoxylin and coverslipped with a mounting medium soluble in xylene.

Quantification of Ki-67 and Apoptosis.
All sections stained for proliferation and apoptosis were quantified without knowing whether this section was before or after HILP-TM. For measuring the Ki-67 LI and the apoptosis LI, we used ocular micrometry on a Leitz microscope by using an eyepiece grid at x400 magnification. Fifteen adjacent fields in histologically viable areas were quantified. The positive and negative nuclei were counted. Endothelial cells, inflammatory cells, and necrosis were excluded. The number of positive nuclei was then divided by the total number of nuclei in each of the 15 randomly selected fields to calculate an index per field. The Ki-67 LI (representing proliferative activity) and the apoptosis LI (representing apoptotic activity) were defined as the mean of the indices of the 15 randomly selected fields.

Drugs and Treatment Schedule.
The technique of HILP-TM with or without IFN-{gamma} for upper and lower limb was recently described extensively (9 , 10) . Briefly, recombinant human TNF-{alpha} (0.2 mg per ampule, 4.9 x 107–5.8 x 107 units/mg per ampule; Boehringer Ingelheim, GmbH, Ingelheim/Rhein, Germany), and in 16 patients, recombinant human IFN-{gamma} (0.2 mg or 1.5 x 106 units per ampule; Boehringer Ingelheim) was used. The cytostatic drug melphalan (Alkeran) was obtained as a sterile powder (100 mg) that was dissolved aseptically using solvent and diluent, as provided by Glaxo Burroughs Wellcome (London, United Kingdom). After isolation of the systemic blood circulation under general anesthesia and with heparinization (3 mg/kg), the limb was perfused with 3 mg (upper limb) TNF-{alpha} to 4 mg of TNF-{alpha} (lower limb) administered as a bolus and, after 30 min, with 13 mg/liter (upper limb) or 10 mg/liter (lower limb) melphalan at 39–40°C with or without addition of 0.2 mg of IFN-{gamma}. The HILP treatment with TNF-{alpha} and melphalan with or without IFN-{gamma} was approved by the local medical ethical committee of the Groningen University Hospital.

Assessment of Tumor Response.
Tumor response was assessed as described previously by the European TNF perfusion group (9 , 10) . To estimate the tumor response, we validated both clinical and histological reaction on HILP-TM. Briefly, the clinical response was regularly determined by standard physical and radiodiagnostic examination, i.e., magnetic resonance imaging or computed tomography during the 6–8-week period between HILP-TM and protocol planned delayed resection. The largest diameter of the tumor in centimeters before and after HILP-TM was used as the clinical parameter. Clinical CR was defined as the disappearance of all measurable disease in the limb for >4 weeks; clinical PR was defined as a regression of the tumor size of >50% for >4 weeks; a regression of the tumor size <50% or a progression of the tumor size <25% for >4 weeks was interpreted as clinical NC; and clinical progressive disease was defined as a progression of the tumor size >25% for >4 weeks. Histological CR was defined as 100% necrosis after HILP-TM, histological PR was defined as >=50% and <100% necrosis, and histological NC was defined as >=0% and <50% necrosis. Overall response was determined as the combination of both clinical and histological tumor response. A clinical CR was downgraded to overall PR when there was a histological PR or NC. Otherwise, a clinical PR could be upgraded to overall CR when a histological CR was observed. In addition, a clinical tumor regression of <50% was upgraded to overall PR when a histological PR or CR was present. When a clinical PR was observed but the histological examination revealed <50% necrosis (NC), the overall response was considered a PR.

Statistics.
A Mann-Whitney U test was carried out to analyze the differences in tumor parameters before and after HILP-TM of all 37 studied patients. A Wilcoxon matched-pairs signed rank sum test was used to analyze the differences in tumor parameters before and after HILP-TM in the 29 patients with viable tumor both before and after HILP-TM. To quantitate the degree of correlation between parameters, we used the Spearman’s rank test. A two-tailed P of <0.05 was considered to be significant. Statistical analysis was performed using Graphpad Prism software (Version 2.0; Graphpad Software Inc., San Diego, CA). Actuarial survival curves were constructed by the Kaplan-Meier method to compare OS and DFS for the different patient groups in relation to HILP-TM modality as well as high (i.e., above the median value) or low (i.e., smaller or equal to the median value) tumor parameters. Survival curves in the different groups were compared by the log-rank test using SPSS Version 8.0 for Windows.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HILP-TM with or without IFN-{gamma}.
As shown in Table 2Citation , tumor grade, mitotic activity, amount of proliferation, and apoptosis before and after HILP-TM of the 15 STSs treated with HILP-TM in combination with IFN-{gamma} did not differ from the values obtained from the 22 STSs treated with HILP-TM alone. Furthermore, the changes in these tumor parameters after HILP-TM as compared to pretreatment values did not differ significantly between the two groups. Treatment responses, clinical outcome, and survival did not differ between the IFN-{gamma}-treated patient group and the patients who received HILP-TM without IFN-{gamma}. Therefore, the statistical analyses were performed on the whole group.


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  		Table 2 Tumor parameters of tumors treated with HILP-TM with (n = 15) or without (n = 22) IFN-{gamma}a

 
Treatment Response Related to Tumor Parameters before HILP-TM.
A clinically assessed CR was observed in 5 patients (14%), a PR was noticed in 27 patients (73%), and in 5 patients (14%), no response could be detected clinically (Table 1)Citation . After histological evaluation, the overall tumor response after HILP-TM was considered to be complete in 6 patients (16%) and partial in 23 patients (62%). In six patients (16%) the tumor did not respond, and in two patients, the histological response could not be evaluated. Both mitotic rate and the amount of apoptosis before HILP-TM did correlate (P < 0.05) with the clinical response (r = 0.41 and r = -0.33, respectively); i.e., high mitotic index and low amount of apoptosis before HILP-TM were associated with a relatively good clinical response. However, these parameters did not correlate with the histological or overall response. Tumor grade and amount of proliferation before HILP-TM did not significantly correlate with clinical, histological, or overall response, although a nearly significant correlation (r = 0.31; P = 0.06) between tumor grade and clinical response was found; i.e., clinical response was relatively good in high-grade tumors. The clinical, histological, and overall responses were not associated with the period between HILP-TM and the delayed local excision.

HILP-TM-induced Changes in Tumor Parameters.
The changes in tumor parameters in tumors of individual patients could be assessed before and after HILP-TM in the 29 patients with viable tumor both before and after HILP-TM (Fig. 1)Citation . Changes in tumor markers or posttreatment values did not correlate with the period between HILP-TM and the collection of the posttreatment samples, nor did they correlate with the time between both samples. The mean number of mitoses in these patients declined from 14 before HILP-TM to 6 after HILP-TM (P < 0.01; Table 3Citation ). The changes in mitotic index were correlated (r = 0.47; P < 0.05) with the clinical response but not with the histological or overall response. Furthermore, no correlation with other clinical or tumor markers was found. In 4 of 29 patients, the number of mitoses after HILP-TM was elevated as compared to before HILP-TM. The two patients with the highest increase developed distant metastases after HILP-TM.



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  		Fig. 1. Paired analysis of the studied tumor parameters (A, mitotic count; B, proliferation; C, apoptosis) of the 29 patients with viable tumor both before and after HILP-TM.

 

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  		Table 3 Tumor parameters before and after HILP-TMa

 
Mean proliferative activity dropped significantly (P < 0.0001), from 18.2% before HILP-TM to 11.0% after HILP-TM. As shown in Fig. 1BCitation , 23 patients had a decrease in proliferation, whereas 6 patients had an increase. The two patients with the largest increase in proliferation developed distant metastases and died of disease. However, there was no significant correlation between the change in proliferation and other tumor or clinical parameters. The mean percentage of apoptotic cells increased from 0.87% before HILP-TM to 2.09% after HILP-TM (P < 0.01). In seven patients (Fig. 1C)Citation , a decrease in apoptosis was observed, including two patients who had an increase in proliferation after HILP-TM. The increase in apoptosis was not correlated with other tumor or clinical parameters.

Follow-up, Metastasis, and Survival in Relation to the Studied Tumor Parameters.
After a mean follow-up period of 29 ± 17.7 months (range, 3–64 months; median, 27 months) after HILP-TM, 19 patients were alive without evidence of disease, 3 patients were alive with disease, and 15 patients died of disease (Table 1)Citation . In the tumors of the patients who died of disease, a higher number of mitoses and higher proliferation rates were observed (P < 0.05) both before and after HILP-TM, as compared to the patients with NED (Table 4)Citation . Amount of apoptosis (before or after) did not differ between those groups. The change in mitotic, proliferative, and apoptotic activity after HILP-TM was not related to follow-up status.


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  		Table 4 Tumor parameters before and after HILP related to follow-up status NED and DODa

 
Six patients (16%) developed a local recurrence or regional lymph node metastasis after a mean DFS of 4.3 ± 2.8 months (range, 1–9 months; median, 3.5 months). Twelve of the 31 patients (39%) without distant metastases at the time of HILP-TM developed distant metastases after a mean DFS of 13.7 ± 8.9 months (range, 5–35 months; median, 11 months). In one patient with a sarcoma not otherwise specified, who developed a malignant tumor 11 months after HILP, it was uncertain whether this was a metastatic lesion or a second malignant primary tumor. Tumor grade, mitotic activity, proliferation, and apoptosis before HILP-TM were not related to the development of metastases. However, in the patients who developed new distant metastases after HILP-TM, the number of mitoses in the post-HILP-TM specimens was higher (P = 0.05) than that in the patients who did not (Table 5)Citation . In contrast, proliferation and apoptosis after HILP-TM were not significantly associated with development of distant metastases. Development of metastatic disease or clinical outcome were not associated with the time between HILP-TM and the delayed local resection or with the period between the pretreatment and posttreatment samples.


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  		Table 5 Tumor parameters before and after HILP-TM related to newly developed distant metastases after HILP-TMa

 
The 2-year OS for the 37 HILP-TM-treated patients was 62%, and the 5-year OS was 51%. Mean OS of the patients with a clinical CR was 51 months (PR, 42 months; NC, 42 months). Patients with a complete overall response had a mean OS of 51 months, which differed (P = 0.12) from that of the patients who had no response (mean OS, 29 months). Mean OS of patients with an overall PR was 47 months. As shown in Table 6Citation , large (i.e., above the median) or small (i.e., below or equal to the median) changes in mitotic rate, proliferation, or apoptosis after HILP-TM did not influence OS. However, the patients with a high amount of proliferation after HILP-TM had a shorter (P < 0.05) OS than the patients with a low amount of proliferation after HILP-TM. The amount of mitotic rate or amount of apoptosis did not influence OS.


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  		Table 6 Mean OS in relation to tumor parameters before and after HILP-TM

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since it was first described by Creech et al. (28) in 1958, HILP has predominantly been used in the treatment of melanomas and sarcomas (13 , 15 , 29, 30, 31, 32, 33, 34, 35) . Traditional cytostatic agents like melphalan and doxorubicin yielded unsatisfactory response rates after HILP for STS (36, 37, 38, 39, 40) . HILP with TNF-|ga and melphalan with or without IFN-{gamma} has dramatically improved the local tumor control of primary nonresectable STS, and response rates of up to 84%, resulting in preservation of the perfused limb without major dysfunction in >80% of the patients, have been reported (9 , 10 , 13, 14, 15) . This study did correlate tumor markers with treatment response and clinical outcome. It was found that a good clinical response was associated with high mitotic index, high tumor grade, and low amount of apoptotic cells before treatment. A relation between tumor grade and treatment response was also suggested by Eggermont et al. (10) , who noted that high-grade tumors grade tend to respond better to HILP-TM. Tumor grade is determined by differentiation level, necrosis, and number of mitoses. The latter seems to be more associated with clinical response than amount of necrosis or degree of differentiation because the proportion of the shift in mitotic index was also correlated with clinical outcome. The pretreatment apoptosis values were positively correlated with clinical treatment response and negatively correlated with pretreatment mitotic rates. This might indicate that the response to HILP-TM is enhanced in STS with an apparently disturbed balance between cell growth and programmed cell death. The absence of a correlation between the studied tumor markers before HILP-TM and the histological or overall tumor response can be explained by the differences between the clinical and histological assessments. The clinical evaluation of tumor regression depends on the estimated size of the perfused tumor, which is measured several times during the period between HILP-TM and the delayed resection by both radiodiagnostic and physical examination. The relatively late definitive histological examination 6–8 weeks after HILP-TM is carried out on the samples after HILP-TM representing the nonresponding, viable parts of the tumor. Furthermore, the way of assessing histological treatment response could also explain the lack of correlation because it is based on the macroscopically and microscopically estimated percentage of necrosis after HILP-TM without taking into account the amount of necrosis before HILP-TM. Moreover, tumor parameters could not be studied in the post-HILP-TM samples of eight patients.

The additive effect of IFN-{gamma} on HILP-TM is subject of debate (10) . In this in vivo study, the changes in proliferation or cell death after HILP-TM did not differ between the patients treated with or without IFN-{gamma}. Furthermore, IFN-{gamma} did not influence the changes in tumor parameters, treatment response, or clinical outcome. It can be concluded from this study that the addition of IFN-{gamma} in HILP-TM for STS does not substantially affect treatment response or clinical outcome.

The biological mechanisms that induce changes in tumor parameters during the 6–8 weeks between HILP-TM and resection remain unclear. Several authors postulated vascular occlusion as a result of intravascular coagulation after endothelial damage by TNF-{alpha} followed by hemorrhagic necrosis (41, 42, 43, 44) . Because animal models and in vitro studies have reported the synergistic effect of TNF-{alpha} and melphalan resulting in cell death and tumor regression, some authors have postulated a more complex process in which vascular endothelium of the tumor, adhesion molecules, and direct cytotoxic effects of melphalan and/or TNF-{alpha} may play a role (45, 46, 47, 48, 49, 50, 51) . This clinical study shows a significant decrease in number of both dividing and proliferating cells as well as an increase in apoptosis after HILP-TM. It remains unclear, however, whether the observed changes in proliferation and apoptosis are a secondary phenomenon occurring 6–8 weeks after HILP-TM induced necrosis, or the remnants of a massive chemotherapeutically generated programmed cell death induced by cell cycle blockage with or without DNA damage (50) . However, the significant increase in number of apoptotic cells after HILP-TM indicates a more subtle process of cell death induced by TNF-{alpha} and/or melphalan, than hemorrhagic necrosis after vascular occlusion alone.

It is shown that the tumors of the patients who died of disease showed higher amounts of proliferative and mitotic activity before and after HILP as compared to those patients who were alive without disease. These findings suggest that patients with STS treated with HILP with a high proliferation index and high number of mitoses still have a worse prognosis, due to a known more aggressive behavior of high-grade STS (1) . The HILP-TM-generated changes in these tumor markers did not predict clinical outcome. With regard to the development of metastatic disease after HILP-TM, it should be noted that, in the patients with newly developed distant metastases, the number of mitotic cells was higher after HILP-TM as compared to the patients who did not develop distant metastases. Interestingly, this was not the case before HILP-TM, which implies that a high amount of dividing cells detected after HILP-TM could indicate patients who are at risk for the development of distant metastases. Although not significant, posttreatment proliferation was higher, and posttreatment apoptosis was lower in the patients who developed new distant metastases after HILP-TM as compared to patients who did not. The importance of posttreatment proliferation index is also observed in the survival analysis, in which a low proliferative activity after HILP-TM is associated with a significantly better OS. Mitotic and apoptotic activity after HILP-TM is not clearly associated with survival.

In conclusion, the results of this study indicate that a better clinical response in HILP-TM is observed in patients with STS displaying high mitotic activity and low amounts of apoptosis. The antitumor effects of TNF-|ga and melphalan seem to result in a decrease in both mitotic activity and proliferation as well as an increase in apoptosis. Furthermore, patients with a favorable clinical outcome and survival have low proliferative STS both before and after HILP-TM, whereas patients who develop distant metastases after HILP-TM have relatively high numbers of dividing cells in the tumor remnants after treatment.


   FOOTNOTES
 
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.

1 Supported by the Dutch Cancer Society (Koningin Wilhelmina Fonds) Grant 95-1085. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, University Hospital Groningen, P. O. Box 30.001, 9700 RB Groningen, the Netherlands. Phone: (31) 50 3619530; Fax: (31) 50 3632510; E-mail: b.e.c.plaat{at}path.azg.nl Back

3 The abbreviations used are: STS, soft tissue sarcoma; HILP, hyperthermic isolated limb perfusion; TNF, tumor necrosis factor; HILP-TM, HILP with TNF-{alpha} and melphalan; DIG, digoxigenin; LI, labeling index; CR, complete response; PR, partial response; NC, no change; OS, overall survival; DFS, disease-free survival. Back

Received 6/26/98; revised 3/ 9/99; accepted 3/25/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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