TNF-alpha and melphalan-based isolated limb perfusion: no evidence supporting the early destruction of tumour vasculature

Background:

Isolated limb perfusion with TNF-alpha and melphalan (TM-ILP) is a highly effective treatment for locally advanced tumours of the extremities. Previous research suggests an almost immediate disintegration of the blood supply of the tumour. The aim of the present study was to verify this hypothesis using non-invasive measurements of microvascular perfusion and tissue oxygenation.

Methods:

A total of 11 patients were included in the study. TM-ILP was performed under mildly hyperthermic conditions (39 °C) in the extremities via proximal vascular access. Capillary-venous microvascular blood flow, haemoglobin level (Hb) and oxygen saturation (SO₂) were determined using laser Doppler and white-light spectroscopy, respectively, before TM-ILP and at 30 min, 4 h, 1 day, 4 days, 1 week, 2 weeks and 6 weeks after TM-ILP from tumour and healthy muscle tissues.

Results:

Blood flow and Hb were mostly higher, whereas SO₂ was lower, in tumour tissue compared with muscle tissue. In both tumour and muscle tissues, blood flow significantly increased immediately after TM-ILP and remained elevated for at least 2 weeks, followed by a return to the initial values 6 weeks after the procedure.

Conclusion:

No signs were found of early destruction of the tumour vasculature. The observations suggest that an inflammatory reaction is one of the key elements of TM-ILP.     

Lack of efficacy of Doxil in TNF-alpha-based isolated limb perfusion in sarcoma-bearing rats

Here we show that Doxil has minimal antitumour activity in the isolated limb perfusion (ILP) setting and its activity was not enhanced by the addition of tumour necrosis factor (TNF). Doxil accumulation in tumour tissue was low and also not augmented by TNF. In contrast, activity of free conventional doxorubicin was enhanced by TNF. We conclude that application of Doxil in a TNF-based ILP is not a useful alternative to free conventional doxorubicin or melphalan.     

p53 status correlates with histopathological response in patients with soft tissue sarcomas treated using isolated limb perfusion with TNF-alpha and melphalan.

BACKGROUND: Recombinant tumor necrosis factor-alpha (TNF-alpha) combined to melphalan is clinically administered through isolated limb perfusion (ILP) for regionally advanced soft tissue sarcomas of the limbs. In preclinical studies, wild-type p53 gene is involved in the regulation of cytotoxic action of TNF-alpha and loss of p53 function contributes to the resistance of tumour cells to TNF-alpha. The relationship between p53 status and response to TNF-alpha and melphalan in patients undergoing ILP is unknown. PATIENTS AND METHODS: We studied 110 cases of unresectable limbs sarcomas treated by ILP. Immunohistochemistry was carried out using DO7mAb, which reacts with an antigenic determinant from the N-terminal region of both the wild-type and mutant forms of the p53 protein, and PAb1620mAb, which reacts with the 1620 epitope characteristic of the wild-type native conformation of the p53 protein. The immunohistochemistry data were then correlated with various clinical parameters. RESULTS: P53DO7 was found expressed at high levels in 28 patients, whereas PAb1620 was negative in 20. The tumours with poor histological response to ILP with TNF-alpha and melphalan showed significantly higher levels of p53-mutated protein. CONCLUSIONS: Our results might be a clue to a role of p53 protein status in TNF-alpha and melphalan response in clinical use.     

TNF-alpha augments intratumoural concentrations of doxorubicin in TNF-alpha-based isolated limb perfusion in rat sarcoma models and enhances anti-tumour effects

We have shown previously that isolated limb perfusion (ILP) in sarcoma-bearing rats results in high response rates when melphalan is used in combination with tumour necrosis factor alpha (TNF-alpha). This is in line with observations in patients. Here we show that ILP with doxorubicin in combination with TNF-alpha has comparable effects in two different rat sarcoma tumour models. The addition of TNF-alpha exhibits a synergistic anti-tumour effect, resulting in regression of the tumour in 54% and 100% of the cases for the BN175-fibrosarcoma and the ROS-1 osteosarcoma respectively. The combination is shown to be mandatory for optimal tumour response. The effect of high dose TNF-alpha on the activity of cytotoxic agents in ILP is still unclear. We investigated possible modes by which TNF-alpha could modulate the activity of doxorubicin. In both tumour models increased accumulation of doxorubicin in tumour tissue was found: 3.1-fold in the BN175 and 1.8-fold in the ROS-1 sarcoma after ILP with doxorubicin combined with TNF-alpha in comparison with an ILP with doxorubicin alone. This increase in local drug concentration may explain the synergistic anti-tumour responses after ILP with the combination. In vitro TNF-alpha fails to augment drug uptake in tumour cells or to increase cytotoxicity of the drug. These findings make it unlikely that TNF-alpha directly modulates the activity of doxorubicin in vivo. As TNF-alpha by itself has no or only minimal effect on tumour growth, an increase in local concentrations of chemotherapeutic drugs might well be the main mechanism for the synergistic anti-tumour effects.     

Nifedipine improves blood flow and oxygen supply,but not steady-state oxygenation of tumours in perfusion pressure-controlled isolated limb perfusion

Isolated limb perfusion allows the direct application of therapeutic agents to a tumour-bearing extremity. The present study investigated whether the dihydropyridine-type Ca(2+)-channel blocker nifedipine could improve blood flow and oxygenation status of experimental tumours during isolated limb perfusion. Perfusion was performed by cannulation of the femoral artery and vein in rats bearing DS-sarcoma on the hind foot dorsum. Perfusion rate was adjusted to maintain a perfusion pressure of 100-140 mmHg throughout the experiment. Following equilibration, nifedipine was continuously infused for 30 min (8.3 microg min(-1) kg(-1) BW). During constant-pressure isolated limb perfusion, nifedipine can significantly increase perfusion rate (+100%) and RBC flux (+60%) through experimental leg tumours. "Steal phenomena" in favour of the surrounding normal tissue and oedema formation were not observed. Despite the increased oxygen availability (+63%) seen upon application of this calcium channel blocker, nifedipine does not result in a substantial reduction of tumour hypoxia, most probably due to an increase in O(2) uptake with rising O(2) supply to the tumour-bearing hind limb. Nifedipine application during isolated limb perfusion can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled isolated limb perfusion.     

Isolated limb perfusion with melphalan,tumour necrosis factor‐alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in‐transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer‐selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour‐targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor‐alpha (TNF‐α) and Lister strain vaccinia virus (GLV‐1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF‐α, melphalan and GLV‐1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV‐1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF‐α/GLV‐1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF‐α/melphalan‐based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.     

Isolated limb perfusion with biochemotherapy and oncolytic virotherapy combines with radiotherapy and surgery to overcome treatment resistance in an animal model of extremity soft tissue sarcoma

The management of locally advanced or recurrent extremity sarcoma often necessitates multimodal therapy to preserve a limb, of which isolated limb perfusion (ILP) is a key component. However, with standard chemotherapeutic agents used in ILP, the duration of response is limited. Novel agents or treatment combinations are urgently needed to improve outcomes. Previous work in an animal model has demonstrated the efficacy of oncolytic virotherapy when delivered by ILP and, in this study, we report further improvements from combining ILP‐delivered oncolytic virotherapy with radiation and surgical resection. In vitro, the combination of radiation with an oncolytic vaccinia virus (GLV‐1h68) and melphalan demonstrated increased cytotoxicity in a panel of sarcoma cell lines. The effects were mediated through activation of the intrinsic apoptotic pathway. In vivo, combinations of radiation, oncolytic virotherapy and standard ILP resulted in delayed tumour growth and prolonged survival when compared with standard ILP alone. However, local disease control could only be secured when such treatment was combined with surgical resection, the timing of which was crucial in determining outcome. Combinations of oncolytic virotherapy with surgical resection and radiation have direct clinical relevance in extremity sarcoma and represent an exciting prospect for improving outcomes in this pathology.     

Role of isolated limb perfusion with recombinant human tumor necrosis factor α and melphalan in locally advanced extremity soft tissue sarcoma

The management of locally advanced extremity soft tissue sarcoma of the limbs is challenging, particularly for recurrent tumors and those adjacent to neurovascular bundles and joints. Typically, the tumors are large, below the fascia, and high‐grade (T2b or stage III according to the American Joint Committee on Cancer) and thus require multimodal therapy. Treatment options must be tailored to patient and tumor characteristics. Isolated limb perfusion with recombinant human tumor necrosis factor α and melphalan (TNF‐ILP) adds a therapeutic option to radiation therapy (RT) and systemic chemotherapy. Although the procedure is somewhat sophisticated to learn, it is a safe method and has been used now for almost 2 decades at more than 50 centers worldwide. TNF‐ILP yields a high rate of complete or nearly complete pathologic tumor remission. In combination with surgical resection of the tumor remnant after isolated limb perfusion, the limb salvage rate is close to 90%. Often, patients can be spared adjuvant RT without long‐term local tumor control rates being compromised. Nevertheless, TNF‐ILP has never been compared with another treatment regimen in a randomized trial. This review summarizes the mode of action and standard application of TNF‐ILP and focuses on a critical discussion of the role of TNF‐ILP in the multimodal treatment of locally advanced primary and recurrent extremity sarcoma. Cancer 2016 . © 2016 American Cancer Society. Cancer 2016;122:2624–2632. © 2016 American Cancer Society.