P.T.G.A. Nooijen

Synergistic effects of TNF-alpha and melphalan in an isolated limb perfusion model of rat sarcoma. A histopathological, immunohistochemical and electron microscopical study

Isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan has shown impressive results in patients with irresectable soft tissue sarcomas and stage III melanoma of the extremities. The mechanisms of the reported in vivo synergistic anti-tumour effects of TNF-alpha and melphalan are not precisely understood. We have developed an ILP model in the rat using a non-immunogenic sarcoma in which similar in vivo synergy is observed. The aim of this present study was to analyse the morphological substrate for this synergistic response of TNF-alpha in combination with melphalan to shed more light on the pathomechanisms involved. Histology of the tumours from saline- (n = 14) and melphalan-treated (n = 11) rats revealed apparently vital tumour cells in over 80% of the cross-sections. Interstitial oedema and coagulation necrosis were observed in the remaining part of the tumour. Haemorrhage was virtually absent. TNF-alpha (n = 22) induced marked oedema, hyperaemia, vascular congestion, extravasation of erythrocytes and haemorrhagic necrosis (20-60% of the cross-sections). Oedema and haemorrhage suggested drastic alterations of permeability and integrity of the microvasculature. Using light and electron-microscopy, we observed that haemorrhage preceded generalised platelet aggregation. Therefore, we suggest that the observed platelet aggregation was the result of the microvascular damage rather than its initiator. Remarkably, these events hardly influenced tumour growth. However, perfusion with the combination of TNF-alpha and melphalan (n = 24) showed more extensive haemorrhagic necrosis (80-90% of the cross-sections) and revealed a prolonged remission (mean 11 days) in comparison with the other groups of rats. Electron microscopical analysis revealed similar findings as described after TNF-alpha alone, although the effects were more prominent at all time points after perfusion. In conclusion, our findings suggest that the enhanced anti-tumour effect after the combination of TNF-alpha with melphalan results from potentiation of the TNF-alpha-induced vascular changes accompanied by increased vascular permeability and platelet aggregation. This may result in additive cytotoxicity or inhibition of growth of residual tumour cells.

Low-dose tumor necrosis factor-α augments antitumor activity of stealth liposomal doxorubicin (DOXIL®) in soft tissue sarcoma-bearing rats

It has previously been demonstrated in the setting of an isolated limb perfusion that application of high‐dose TNF‐α in combination with chemotherapy (melphalan, doxorubicin) results in strong synergistic antitumor effects in both the clinical and preclinical settings. In this study, we demonstrate that systemic administration of low‐dose TNF‐α augments the antitumor activity of a liposomal formulation of doxorubicin (DOXIL®). Addition of TNF‐α to a DOXIL® regimen, which by itself induced some tumor growth delay, resulted in massive necrosis and regression of tumors. Furthermore, we could demonstrate a significant increase of liposomal drug in the tumor tissue when TNF‐α had been co‐administered. Administration of TNF‐α augmented DOXIL® accumulation only after repeated injections, whereas accumulation of free doxorubicin was not affected by TNF‐α. Drug levels in the tumor interstitium appeared crucial as intracellular levels of free or liposome‐associated doxorubicin were not increased by TNF‐α. Therefore, we hypothesize that low‐dose TNF‐α augments leakage of liposomal drug into the tumor interstitium, explaining the observed improved antitumor effects. Regarding the effects of systemic administration of low doses of TNF‐α, these findings may be important for enhanced tumor targeting of various liposomal drug formulations. Int. J. Cancer 87:829–837, 2000.

Tumor necrosis factor‐alpha in isolated perfusion systems in the treatment of cancer: The Rotterdam Preclinical‐Clinical Program

The clinical success of the application of tumor necrosis factor-alpha (TNFalpha) in isolated limb perfusions in patients with advanced sarcomas, melanomas and other tumors has renewed the interest in this agent as an anticancer drug. At the Rotterdam Cancer Center, we have developed an interactive preclinical-clinical TNFalpha program that explores new methods to use TNFalpha in various settings. Regional organ perfusion models were developed and the effectivity of targeting of TNFalpha to the tumor by means of systemic administration of liposomes are tested. Furthermore various drugs and mechanisms that may enhance the activity of TNFalpha are under investigation. A summary of this comprehensive program is presented here.

Transient induction of E-selectin expression following TNF alpha-based isolated limb perfusion in melanoma and sarcoma patients is not tumor specific.

Endothelial injury of the tumor microvasculature after isolated limb perfusion (ILP) with TNF-α and melphalan is considered to play an important role in the pathogenesis of tumor necrosis. It is thought to follow endothelial cell activation and subsequent attraction of polymorphonuclear cells (PMNs). The observed selectivity for the tumor could be due to preferential overex-pression of cell-adhesion molecules by the tumor vasculature. We tested this proposition by analyzing sequential biopsies from both tumor and normal distant skin, taken from melanoma and sarcoma patients before ILP and at 30 min and 24 h after ILP. Histopathologically confirmed complete response was observed in six of seven melanoma patients, 1–8 months after ILP. By using immunohistochemistry on the light- and electron-microscopic level, the expression patterns of intercellular adhesion molecules-1 (ICAM-1), E-selectin (ELAM-1), VCAM-1, and PECAM-1 were examined. In addition, the results were compared with the effects on HUVECs (human umbilical vein endothelial cells) in vitro of transient exposure of the agents used during ILP. ICAM-1 and PECAM-1 were constitutively expressed on vascular endothelial cells, both in normal tissues and in the tumor lesions. In biopsies taken 30 min after termination of the perfusion, a moderate induction of E-selectin expression on the vascular endothelium in the tumors and a marked expression on the vasculature in the perfused normal skin were observed. It decreased within 24 h after perfusion in both normal skin and in the tumor. The upregulation of E-selectin was accompanied neither by an influx of neutrophils nor by hemorrhagic necrosis. There were no drastic changes in the expression of VCAM-1, ICAM-1, or PECAM-1. These findings imply that the upregulation of E-selectin after ILP is not restricted to the tumor microvasculature and that, therefore, these microvascular events seem not to be the decisive pathomechanism responsible for tumor regression.

In vivo isolated kidney perfusion with tumour necrosis factor α (TNF-α) in tumour-bearing rats

SummaryIsolated perfusion of the extremities with high-dose tumour necrosis factor α (TNF-α) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-α and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 μg TNF-α. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-α and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-α alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-α, the minimal threshold concentration of TNF-α to exert its anti-tumour effects was not reached. The applicability of TNF-α in isolated kidney perfusion for human tumours seems, therefore, questionable.

Erratum: Immunohistochemical analysis of integrin αvβ3 expression on tumor‐associated vessels of human carcinomas. Int. J. Cancer, 71, 320–324 (1997)

Max, Regina, Gerritsen, Roland R.C.M., Nooijen, Peet T.G.A., Goodman, Simon L., Sutter, Arne, Keilholz, U., Ruiter, Dirk J. and De Waal, Robert M.W., Immunohistochemical analysis of integrin αvβ3 expression on tumor‐associated vessels of human carcinomas. Int. J. Cancer, 71, 320–324 (1997).