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.     

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.     

Synergistic antitumor response of interleukin 2 with melphalan in isolated limb perfusion in soft tissue sarcoma-bearing rats

The cytokine interleukin 2 (IL-2) is a mediator of immune cell activation with some antitumor activity, mainly in renal cell cancer and melanoma. We have previously shown that tumor necrosis factor (TNF)-alpha has strong synergistic antitumor activity in combination with chemotherapeutics in the isolated limb perfusion (ILP) setting based on a TNF-mediated enhanced tumor-selective uptake of the chemotherapeutic drug followed by a selective destruction of the tumor vasculature. IL-2 can cause vascular leakage and edema and for this reason we examined the antitumor activity of a combined treatment with IL-2 and melphalan in our well-established ILP in soft tissue sarcoma-bearing rats (BN175). ILP with either IL-2 or melphalan alone has no antitumor effect, but the combination of IL-2 and melphalan resulted in a strong synergistic tumor response, without any local or systemic toxicity. IL-2 enhanced significantly melphalan uptake in tumor tissue. No signs of significant vascular damage were detected to account for this observation, although the tumor sections of the IL-2- and IL-2 plus melphalan-treated animals revealed scattered extravasation of erythrocytes compared with the untreated animals. Clear differences were seen in the localization of ED-1 cells, with an even distribution in the sham, IL-2 and melphalan treatments, whereas in the IL-2 plus melphalan-treated tumors clustered ED-1 cells were found. Additionally, increased levels of TNF mRNA were found in tumors treated with IL-2 and IL-2 plus melphalan. These observations indicate a potentially important role for macrophages in the IL-2-based perfusion. The results in our study indicate that the novel combination of IL-2 and melphalan in ILP has synergistic antitumor activity and may be an alternative for ILP with TNF and melphalan.     

Outcome and prognostic factor analysis of 217 consecutive isolated limb perfusions with tumor necrosis factor-alpha and melphalan for limb-threatening soft tissue sarcoma

BACKGROUND: Extensive and mutilating surgery is often required for locally advanced soft tissue sarcoma (STS) of the limb. As it has become apparent that amputation for STS does not improve survival rates, the interest in limb-preserving approaches has increased. Isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNF) and melphalan is successful in providing local tumor control and enables limb-preserving surgery in a majority of cases. A mature, large, single-institution experience with 217 consecutive ILPs for STS of the extremity is reported. METHODS: At a prospectively maintained database at a tertiary referral center, 217 ILPs were performed from July 1991 to July 2003 in 197 patients with locally advanced STS of the extremity. ILPs were performed at mild hyperthermic conditions with 1-4 mg of TNF and 10-13 mg/L limb-volume melphalan (M) for leg and arm perfusions, respectively. RESULTS: The overall response rate was 75%. Limb salvage was achieved in 87% of the perfused limbs. Median survival post-ILP was 57 months and prognostic factors for survival were Trojani grade of the tumor and ILP for single versus multiple STS. The procedure could be performed safely, with a perioperative mortality of 0.5% in all patients with no age limit (median age, 54 yrs; range, 12-91). Systemic and locoregional toxicity were modest and easily manageable. CONCLUSION: TNF+M-based ILP can provide limb salvage in a significant percentage of patients with locally advanced STS and has therefore gained a permanent place in the multimodality treatment of STS.     

The palliative value of tumor necrosis factor alpha-based isolated limb perfusion in patients with metastatic sarcoma and melanoma

BACKGROUND: Both patients with soft tissue sarcoma (STS) and patients with melanoma have limited treatment possibilities once the tumor has metastasized systemically. In patients with extremity STS or bulky melanoma in-transit metastases, the local tumor burden may be so problematic that, even in patients with systemically metastasized disease, an amputation may be inevitable. Isolated limb perfusion (ILP) has proven to be an excellent, local, limb-saving treatment option in patients with locally advanced extremity tumors. In this study, the authors investigated the palliative value of the ILP procedure to avoid amputation in patients who had Stage IV STS and melanoma. METHODS: From 1991 to 2003, of 339 tumor necrosis factor alpha (TNF)-based ILPs, 51 procedures were performed for either Stage IV STS (n = 37 patients) or Stage IV melanoma (n = 14 patients). All patients underwent an ILP with TNF and melphalan of the upper limb (n = 4 patients) or the lower limb (n = 47 patients) with 26-140 mg melphalan and 2-4 mg TNF. RESULTS: The overall response in patients with Stage IV STS was 84%, and their median survival was 12 months after ILP. Limb salvage was achieved in 36 of 37 patients, with 1 patient undergoing amputation due to treatment toxicity. In the patients with Stage IV melanoma, the complete response rate was 43%. All patients with melanoma preserved their limb during a median survival of 7 months. CONCLUSIONS: TNF-based ILP is an excellent procedure that provided tumor control and limb salvage for the short survival of patients with metastasized, very bulky, limb-threatening tumors of the extremity.     

Isolated limb perfusion for unresectable extremity sarcoma: results of 2 single-institution phase 2 trials

BACKGROUND:

Controversy has surrounded the role of isolated limb perfusion (ILP) for unresectable extremity sarcomas. However, there remains a group of sarcoma patients for whom amputation is the only potential treatment. Because systemic therapies are limited, the authors evaluated ILP in an effort to provide a limb‐salvage option.

METHODS:

Since 1995, patients with unresectable extremity sarcomas were entered in 2 prospective trials using ILP. Study 1 used tumor necrosis factor (TNF) and melphalan in the perfusion circuit at hyperthermic temperatures (39‐41°C). Study 2 used doxorubicin at normothermic temperatures. All ILPs were performed using the standard, previously described technique.

RESULTS:

Seventeen patients were entered into study 1; there were 10 (58%) partial responses, 1 (6%) near complete response (CR), 1 (6%) CR, and 5 (30%) no response/minor response. Fourteen patients died of their disease, with a median follow‐up of 17 months. Seven (41%) patients maintained their limb intact until the time of death. Twelve patients were entered into study 2; there were no partial or CRs and 2 (20%) minor responses. With a median follow‐up of 35 months, there are 3 patients alive (2 with their extremity intact and 1 with an amputation). Six patients developed myonecrosis with creatine phosphokinase levels up to 54,000 U/dL.

CONCLUSIONS:

Although doxorubicin is active systemically, TNF and melphalan appear to have greater activity and less toxicity during ILP. Future clinical trials are needed to clearly identify the role for ILP in patients with unresectable extremity sarcomas. Cancer 2011. © 2011 American Cancer Society.     

Rapid increase in plasma tenascin-C concentration after isolated limb perfusion with high-dose tumor necrosis factor (TNF), interferon gamma (IFNγ) and melphalan for regionally advanced tumors

The matrix protein tenascin-C (TN-C) is present in the blood of healthy individuals at concentrations around 1 mg/1. Elevated serum levels have been reported in cancer patients. In this study we have measured the concentration of circulating TN-C in 40 patients with melanoma, soft-tissue sarcoma (STS) or squamous-cell carcinoma (SCC) of the limbs, and have found a minor increase in the mean concentration compared with healthy subjects. Only 10 patients had TN-C levels above the normal range. No correlation was observed between TN-C levels and tumor burden. Nineteen patients were treated by isolation limb perfusion (ILP) with TNF, IFNγ, melphalan (11 melanoma, 2 SCC and 1 STS), melphalan alone (3 melanoma) or hyperthermia at 41.5°C (2 melanoma). ILP with TNF, IFNγ and melphalan induced a rapid increase in plasma TN-C levels, peaking in most patients between 24 to 48 hr after ILP. Two patients treated with hyperthermia only had a slow increase in TN-C concentration peaking at day 4, while the patients treated with melphalan alone had no significant change. In some cases elevated TN-C levels persisted for over 8 weeks after ILP. The early rise in TN-C concentration correlates with the increase in circulating C-reactive protein. Our findings suggest that circulating TN-C behaves, at least in part, as an acute-phase protein and that it may play a role in the inflammatory response. © 1995 Wiley-Liss, Inc.     

Regional toxicity after isolated limb perfusion with melphalan and tumour necrosis factor- alpha versus toxicity after melphalan alone.

AIMS: To determine whether the addition of high-dose tumour necrosis factor-alpha (TNF alpha) to isolated limb perfusion (ILP) with melphalan increases acute regional tissue toxicity compared to ILP with melphalan alone. METHODS: A retrospective, multivariate analysis of toxicity after normothermic (37--38 degrees C) and 'mild' hyperthermic (38--40 degrees C) ILPs for melanoma was undertaken. Normothermic ILP with melphalan was performed in 294 patients (70.8%), 'mild' hyperthermic ILP with melphalan in 71 patients (17.1%) and 'mild' hyperthermic ILP with melphalan combined with TNF alpha in 50 patients (12.0%). Toxicity was nil or mild (grades I--II according to Wieberdink et al.) in 339 patients (81.7%), and more severe acute regional toxicity (grades III--V) developed in 76 patients (18.3%). A stepwise logistic regression procedure was performed for the multivariate analysis of prognostic factors for more severe toxicity. RESULTS: On univariate analysis, 'mild' hyperthermic ILP with melphalan plus TNF alpha significantly increased the incidence of more severe acute regional toxicity compared to normothermic and 'mild' hyperthermic ILP with melphalan alone (36% vs 16% and 17%; P=0.0038). However, after ILP using TNF alpha no grade IV (compartment compression syndrome) or grade V (toxicity necessitating amputation) reactions were seen. Significantly more severe toxicity was seen after ILPs performed between 1991 and 1994 compared with earlier ILPs (33%vs 14%P=0.0001). Also, women had a higher risk of more severe toxicity than men (22% vs 7%; P=0.0007). After multivariate analysis, prognostic factors which remained significant were: sex (P=0.0013) and either ILP schedule (P=0.013) or treatment period (P=0.0003). CONCLUSIONS: Regional toxicity after 'mild' hyperthermic ILP with melphalan and TNF alpha was significantly increased compared to ILP with melphalan alone. This may be caused by increased thermal enhancement of melphalan due to the higher tissue temperatures (39--40 degrees C) at which the melphalan in the TNF alpha-ILPs was administered or by an interaction between high-dose TNF alpha and melphalan. Copyright Harcourt Publishers Limited.     

Multifocal soft tissue sarcoma: limb salvage following hyperthermic isolated limb perfusion with high-dose tumor necrosis factor and melphalan

BACKGROUND AND OBJECTIVES: The prognosis for recurrent multifocal limb soft tissue sarcoma (STS) is dismal due to systemic spread. However, many of these patients undergo amputation due to ineffective local control. The purpose of the present study was to determine whether isolated limb perfusion (ILP) with tumor necrosis factor (TNF) and melphalan permits limb salvage and palliation for such patients. METHODS: Of 53 STS patients treated with hyperthermic ILP with TNF (3-4 mg) and melphalan (1-1.5 mg/kg), 13 (25%) had multifocal STS and were candidates for amputation. RESULTS: The overall response rate was 92% (12/13) with 38% complete response and 54% partial response. Two patients died during the early postoperative period. Limb salvage was achieved in 85% of patients. One patient (8%) had only stable disease and underwent amputation. Local recurrence occurred in 38% but did not result in amputation. CONCLUSIONS: Although the number of patients in this study is too small to allow definitive conclusions, it seems that ILP/TNF offer limb salvage and palliation for recurrent multifocal STS patients.     

Isolated Limb Perfusion for Malignant Melanoma: Systematic Review on Effectiveness and Safety

Background.

Isolated limb perfusion (ILP) involves the administration of chemotherapy drugs directly into a limb involved by locoregional metastases. Unresectable locally advanced melanoma of the limbs represents one of the clinical settings in which ILP has demonstrated benefits.

Methods.

A systematic review of the literature on ILP for patients with unresectable locally advanced melanoma of the limbs was conducted. MEDLINE, EMBASE, and Cochrane database searches were conducted to identify studies fulfilling the following inclusion criteria: hyper- or normothermic ILP with melphalan with or without tumor necrosis factor (TNF) or other drugs providing valid data on clinical response, survival, or toxicity. To allocate levels of evidence and grades of recommendation the Scottish Intercollegiate Guidelines Network system was used.

Results.

Twenty-two studies including 2,018 ILPs were selected with a clear predominance of observational studies (90.90%) against experimental studies (9.10%). The median complete response rate to ILP was of 58.20%, with a median overall response rate of 90.35%. ILP with melphalan yielded a median complete response rate of 46.50%, against a 68.90% median complete response rate for melphalan plus TNF ILP. The median 5-year overall-survival rate was 36.50%, with a median overall survival interval of 36.70 months. The Wieberdink IV and V regional toxicity rates were 2.00% and 0.65%, respectively.

Conclusions.

ILP is effective in achieving clinical responses in patients with unresectable locally advanced melanoma of the limbs. The disease-free and overall survival rates provided by ILP are acceptable. ILP is safe, with a low incidence of severe regional and systemic toxicity.     

Isolated limb perfusion for extremity soft-tissue sarcomas, in-transit metastases, and other unresectable tumors: Credits, debits, and future perspectives

Isolated limb perfusion (ILP) with melphalan is effective against melanoma in-transit metastases but has failed in the treatment of limb-threatening extremity sarcomas. Tumor necrosis factor-α (TNF) has changed this situation completely. Now, ILP with TNF + melphalan is a very successful treatment to prevent amputation. In a multicenter European trial, ILP with TNF + melphalan resulted in a 76% response rate and a 71% limb salvage rate in patients with limb-threatening soft-tissue sarcomas, deemed unresectable by independent review committees, leading to approval of TNF in Europe. We have also reported on the success of this regimen against bulky melanomas, multifocal skin cancers, and drug-resistant bony sarcomas. High-dose TNF destructs tumor vasculature, and, most importantly, it enhances tumor-selective drug uptake (ie, melphalan and doxorubicin) by threefold to sixfold. Similar synergy is observed in well-vascularized liver metastases after isolated hepatic perfusion with TNF and melphalan. New (vasoactive) drugs and mechanisms of action and interaction with chemotherapy are in development. ILP is also a promising treatment modality for adenoviral vector-mediated gene therapy. Many clinical phase I/II evaluations in ILP are now underway.     

Prerequisites for effective isolated limb perfusion using tumour necrosis factor alpha and melphalan in rats.

An isolated limb perfusion (ILP) model using soft tissue sarcoma-bearing rats was used to study prerequisites for an effective ILP, such as oxygenation of the perfusate, temperature of the limb, duration of the perfusion and concentration of tumour necrosis factor (TNF). Combination of 50 microg TNF and 40 microg melphalan demonstrated synergistic activity leading to a partial and complete response rate of 71%. In comparison to oxygenated ILP, hypoxia was shown to enhance anti-tumour activity of melphalan alone and TNF alone but not of their combined use. Shorter perfusion times decreased anti-tumour responses. At a temperature of 24-26 degrees C, anti-tumour effects were lost, whereas temperatures of 38-39 degrees C or 42-43 degrees C resulted in higher response rates. However, at 42-43 degrees C, local toxicity impaired limb function dramatically. Synergy between TNF and melphalan was lost at a dose of TNF below 10 microg in 5 ml perfusate. We conclude that the combination of TNF and melphalan has strong synergistic anti-tumour effects in our model, just as in the clinical setting. Hypoxia enhanced activity of melphalan and TNF alone but not the efficacy of their combined use. For an optimal ILP, minimal perfusion time of 30 min and minimal temperature of 38 degrees C was mandatory. Moreover, the dose of TNF could be lowered to 10 microg per 5 ml perfusate, which might allow the use of TNF in less leakage-free or less inert perfusion settings.     

Tumor vasculature‐targeted delivery of tumor necrosis factor‐α*

BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor‐α (TNF‐α) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF‐α, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF‐α targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno‐associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF‐α (AAVP‐TNF‐α). The activity of AAVP‐TNF‐α was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP‐TNF‐α infection of human melanoma cells resulted in high levels of TNF‐α expression. Systemic administration of targeted AAVP‐TNF‐α to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF‐α, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF‐α specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF‐α is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy. Cancer 2009. Published 2008 by the American Cancer Society.     

Isolated limb perfusion with tumour necrosis factor-alpha and melphalan for unresectable bone sarcomas of the lower extremity.

AIMS: Isolated limb perfusion (ILP) with recombinant tumour necrosis factor-alpha (rTNF-alpha) and melphalan has recently been reported to induce major tumour responses and permit limb salvage in over 80% of patients with unresectable soft-tissue sarcomas of the extremities. We investigated whether TNF-based ILP could allow limb-sparing surgery in patients with primary, recurrent or metastatic bone sarcoma to the lower extremity who met the criteria for an amputation and had failed or refused chemotherapy. METHODS: From August 1992 to December 1997, we employed ILP with rTNF-alpha and melphalan in 13 patients with unresectable bone sarcoma of the lower extremity, all of whom were candidates for amputation. The aim was to reduce tumour size and allow the performance of a limb-sparing surgery (LSS). RESULTS: Following ILP, none of the patients had severe local toxicity and only one patient experienced significant systemic side-effects. LSS was subsequently performed in nine of the 13 patients. LSS was feasible in an additional three patients but was not performed because of the emergence of diffused metastatic disease. CONCLUSIONS: ILP with rTNF-alpha and melphalan can allow limb salvage in patients wih locally advanced bone sarcomas who had failed standard treatment options. Its potential role in the treatment of unresectable bone sarcomas of the extremities merits further evaluation.     

Harly endothelium activation and polymorphonuclear cell invasion precede specific necrosis of human melanoma and sarcoma treated by intravascular high-dose tumour necrosis factor alpha (rTNFα)

Twenty-seven patients treated with high-dose rTNFα, IFNγ and melphalan by isolated limb perfusion were histologically documented. There were 20 cases of melanoma-in-transit metastases and 7 cases of high-grade soft-tissue sarcoma. Biopsies were taken before IFN7, after IFNγ, before TNFα and between 2 hr and 60 days after the TNFα perfusion. Immunohistochemistry was performed for adhesion molecules ICAM-I, ELAM-I (E selectin), VCAM-I and PECAM. During the first hours after beginning perfusion, the endothelial cells of the tumour capillaries appeared swollen. Significant tumour necrosis was already observed 3 hours after the perfusion in melanoma cases. The overall predominant feature was coagulative necrosis associated or not with haemorrhagic necrosis. TNFα induced increased expression of ELAM-I and VCAM-I adhesion molecules on intratumoral endothelial cells. The activated tumour vessels were progressively destroyed. Significant intra-vascular recruitment of polymorphonuclear cells (PMNs) was observed 3 hr after starting TNFα; it was followed by diapedesis and tumour colonization 3 days later. T lymphocytes and macrophages were detected during the first 7 days and B lymphocytes during the second week. Melanoma in transit metastases treated with alkylating agent alone did not show significant necrosis and did not express high levels of adhesion molecules (ELAM-1, VCAM-I) nor infiltration by PMN.     

31Phosphorus-magnetic resonance spectroscopy to assess histologic tumor response noninvasively after isolated limb perfusion for soft tissue tumors

BACKGROUND: In patients with unresectable soft tissue sarcoma of the extremities, isolated limb perfusion (ILP) has been reported to result in significant tumor regression enabling limb-sparing resection in the majority of patients. However, clinical tumor response as evaluated by imaging and histopathology (extent of tumor necrosis) often differ significantly. The current study was initiated to evaluate prospectively the role of 31phosphorus-magnetic resonance spectroscopy (31P-MRS) in the noninvasive assessment of histologic response in patients treated with ILP. METHODS: Thirty-two patients with locally advanced and unresectable soft tissue tumors (sarcoma in 28 patients and bulky melanoma in 4 patients) were treated by ILP with recombinant human tumor necrosis factor-alpha and melphalan or with cytostatics. 31P-MRS was performed prior to treatment and at regular intervals after ILP until definite tumor resection. Clinical response parameters according to the World Health Organization as well as the histopathologic necrosis rate of the resection specimen were correlated with changes in the energy-rich phosphorous metabolites phosphocreatine (PCR); alpha-, beta-, gamma-adenosine triphosphate (ATP); phosphomonoesters (PME); and inorganic phosphate (Pi). RESULTS: Clinically, 15 of 32 patients (response rate [RR] of 47%) demonstrated a partial response (PR). The ratios of PME/PCR and PME/beta-ATP decreased significantly after ILP in comparison with preoperative values (P < 0.001). The changes in the PME/beta-ATP ratio were significantly different between clinical responders and nonresponders (P < 0.02) in contrast with the PME/PCR ratios (P < 0.09). Histologic necrosis of > 90% (pathologic (p) PR) was present in 17 resection specimens, 7 of which demonstrated no clinical response. Seven tumors demonstrated a pathologic complete response (pCR). When combining PR, pPR, and pCR (RR of 68%), 31P-MRS was able to predict response with a specificity of 94% and a sensitivity of 68% (P < 0.006, by the chi-square test). CONCLUSIONS: The considerable difference between clinical and pathologic RR after ILP underlines the shortcomings of established response criteria. Utilizing changes in PME/beta-ATP ratios, 31P-MRS is a highly specific tool with which to predict histologic response in this setting. This finding may be of major value in those patients in whom the decision to perform a major resection or amputation must be made for local tumor control.     

Isolated Limb Perfusion with Melphalan and TNF-α in the Treatment of Extremity Sarcoma

Opinion statement Isolated limb perfusion (ILP) with chemotherapy alone has uniformly failed in the treatment of irresectable extremity soft tissue sarcomas. The addition of tumor necrosis factor-alpha (TNF-α) to this treatment approach contributed to a major step forward in the treatment of locally advanced extremity soft tissue sarcoma (STS). High response rates and limb salvage rates have been reported in multicenter trials, which combined ILP with TNF-α plus melphalan, which resulted in the approval of TNF-α for this indication in Europe in 1998. Subsequently a series of confirmatory single institution reports on the efficacy of the procedure have now been published. TNF-α has an early and a late effect; it enhances tumor-selective drug uptake during the perfusion and plays an essential role in the subsequent selective destruction of the tumor vasculature. These effects result in a high response rate in high-grade soft tissue sarcomas. This induction therapy thus allows for resection of tumor remnants some 3 months after ILP and thus avoidance of limb amputation. TNF-α-based ILP is a well-established treatment to avoid amputations. It represents an important example of tumor vasculatory-modulating combination therapy and should be offered in large volume tertiary referral centers.     

Modulation of RIP1 ubiquitylation and distribution by MeBS to sensitize cancer cells to tumor necrosis factor α‐induced apoptosis

Overexpression of anti‐apoptosis protein cIAP1 due to its genetic amplification is found in certain cancers such as esophageal squamous cell carcinoma, hepatocellular carcinoma, cervical cancer and lung cancer, and plays a significant role in resistance to cancer therapy. We previously reported that a class of small molecules represented by (?)‐N‐[(2S, 3R)‐3‐amino‐2‐hydroxy‐4‐phenyl‐butyryl]‐L‐leucine methyl ester (MeBS) activates auto‐ubiquitylation of cIAP1 for proteasomal degradation, and enhances apoptosis of various cancer cells. However, the molecular mechanism of how MeBS sensitizes cancer cells to apoptosis via downregulation of cIAP1 is not well understood. Here, we show that ubiquitylation and distribution of RIP1, a protein ubiquitylated by cIAP1, is modulated by MeBS. Upon tumor necrosis factor (TNF)α stimulation, ubiquitylated RIP1 associates with the TNF‐receptor (TNFR) complex, whereas non‐ubiquitylated RIP1 associates with caspase8. MeBS reduces the ubiquitylated RIP1 in the TNFR complex and increases non‐ubiquitylated RIP1 bound to caspase8. Downregulation of RIP1 by siRNA reduces apoptosis induced by TNFα plus MeBS treatment. These results indicate an important role of RIP1 in apoptosis induced by combined treatment with TNFα and MeBS, suggesting that MeBS sensitizes cancer cells to apoptosis by modulating RIP1 ubiquitylation and distribution. (Cancer Sci 2010; 101: 2425–2429)     

TNF-based isolated limb perfusion in unresectable extremity desmoid tumours.

BACKGROUND: Desmoid tumours are soft tissue sarcomas with local aggressive behaviour and a high rate of local recurrence after treatment. Although they do not tend to metastasise systemically, the local aggressiveness can lead to situations in which limb-preserving surgery cannot be performed without severe disability. As isolated limb perfusion (ILP) with TNF and melphalan has proven to be extremely effective in the treatment of soft tissue sarcoma, we studied its potential in locally advanced extremity desmoid tumours. METHODS: Prospectively maintained database in a tertiary referral centre. Between 1991 and 2003, 12 ILP procedures were performed in 11 patients for locally advanced desmoid tumours. Local surgical therapy with preservation of limb function was impossible in all patients due to large or multifocal tumours, multiple recurrences or extensive previous treatment. Perfusions were performed with 4-3mg TNF and 10-13 mg/l limb volume melphalan form leg and arm perfusions, respectively. RESULTS: Overall response rate was 75%: Two complete responses were recorded (17%) and seven patients had a partial response (58%). Amputation could be avoided in all cases. Local control was obtained after 10/12 ILPs and in the other two patients through repeat ILP and systemic chemotherapy, thus leading to an overall local control rate of 100%. Local toxicity was mild and systemic toxicity was absent in all patients. CONCLUSION: ILP is a very effective treatment option in the multimodality treatment of limb desmoid tumours. It should be considered in patients with aggressive and disabling disease where resection without important functional sacrifice is impossible.     

Repeat isolated limb perfusion with TNFalpha and melphalan for recurrent limb melanoma after failure of previous perfusion.

AIM: To assess the effectiveness of isolated limb perfusion (ILP) with tumour necrosis factor-alpha (TNFalpha) and melphalan for recurrent or persistent melanoma lesions after previous ILP. METHODS: Between 1978 and 2001, 21 patients (mean age 65, range 29-83 years) underwent repeat ILP for recurrent or persistent melanoma after a previous ILP. First ILPs had been performed with melphalan alone in 13 patients and with addition of TNFalpha in eight, for a median of nine lesions (interquartile (IQ) range 2-23 lesions). Repeat ILP was performed with TNFalpha and melphalan in all 21 patients for a median of nine lesions (IQ range 5-25 lesions). Median follow-up after repeat ILP was 18 months (IQ range 6-36 months). RESULTS: Thirteen patients attained a complete response (CR) after repeat ILP compared to 11 of 17 with measurable lesions after the first ILP. Nine patients relapsed after CR. Median limb recurrence-free survival was 13 months. Fourteen patients had mild acute regional toxicity after repeat ILP compared to 18 after the first ILP (n.s.). One patient underwent amputation for critical limb ischemia 10 months following repeat ILP. The limb salvage rate was 95%. Overall median survival was 62 months after CR compared to 13 months for those without CR (P=0.05). CONCLUSION: Repeat ILP with TNFalpha and melphalan is feasible after previous ILP with mild regional toxicity. The CR rate is relatively high and comparable to the first procedure with good limb recurrence-free survival and high limb salvage rate.