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.

Prognostic value of sentinel node biopsy in 327 prospective melanoma patients from a single institution.

AIM: To confirm the accuracy of sentinel node biopsy (SNB) procedure and its morbidity, and to investigate predictive factors for SN status and prognostic factors for disease-free survival (DFS) and disease-specific survival (DSS). MATERIALS AND METHODS: Between October 1997 and December 2004, 327 consecutive patients in one centre with clinically node-negative primary skin melanoma underwent an SNB by the triple technique, i.e. lymphoscintigraphy, blue-dye and gamma-probe. Multivariate logistic regression analyses as well as the Kaplan-Meier were performed. RESULTS: Twenty-three percent of the patients had at least one metastatic SN, which was significantly associated with Breslow thickness (p<0.001). The success rate of SNB was 99.1% and its morbidity was 7.6%. With a median follow-up of 33 months, the 5-year DFS/DSS were 43%/49% for patients with positive SN and 83.5%/87.4% for patients with negative SN, respectively. The false-negative rate of SNB was 8.6% and sensitivity 91.4%. On multivariate analysis, DFS was significantly worsened by Breslow thickness (RR=5.6, p<0.001), positive SN (RR=5.0, p<0.001) and male sex (RR=2.9, p=0.001). The presence of a metastatic SN (RR=8.4, p<0.001), male sex (RR=6.1, p<0.001), Breslow thickness (RR=3.2, p=0.013) and ulceration (RR=2.6, p=0.015) were significantly associated with a poorer DSS. CONCLUSION: SNB is a reliable procedure with high sensitivity (91.4%) and low morbidity. Breslow thickness was the only statistically significant parameter predictive of SN status. DFS was worsened in decreasing order by Breslow thickness, metastatic SN and male gender. Similarly DSS was significantly worsened by a metastatic SN, male gender, Breslow thickness and ulceration. These data reinforce the SN status as a powerful staging procedure.

Sentinel lymph node involvement and a high Breslow index are independent factors of risk for early relapse of melanoma.

AIM: The clinical relevance of sentinel lymph node (SLN) analysis was evaluated prospectively and compared with other known risk factors of relapse in early stage melanoma. METHODS: Surgery was guided by lymphoscintigraphy, blue dye and gamma probe detection. SLN were analysed by haematoxylin eosin (HE) histochemistry and multimarker immunohistochemistry (IHC). Disease free survival (DFS) was evaluated with Kaplan-Meier plots according to different parameters and Cox analyses of variance. RESULTS: From 210 patients a total of 381 SLN were excised. Lymphoscintigraphy identified all excised SLN with only 2 false positive lymphatic lakes. Fifty patients (24%) had tumour positive SLN. With a mean follow-up of 31.3 months, 29 tumour recurrences were observed, 19 (38%) in 50 SLN positive and 10 (6%) in 160 SLN negative patients. Strong predictive factors for early relapse (p < 0.0005) were SLN positivity and a high Breslow index. CONCLUSION: SLN tumour positivity is an independent factor of high risk for early relapse with a higher power of discrimination than the Breslow index.

Recombinant human tumor necrosis factor: an efficient agent for cancer treatment.

Recombinant human TNF (rhTNF) has a selective effect on endothelial cells in tumour angiogenic vessels. Its clinical use has been limited because of its property to induce vascular collapsus. TNF administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs was shown to be safe and efficient. When combined to the alkylating agent melphalan, a single ILP produces a very high objective response rate. ILP with TNF and melphalan provided the proof of concept that a vasculotoxic strategy combined to chemotherapy may produce a strong anti-tumour effect. The registered indication of TNF-based ILP is a regional therapy for regionally spread tumours. In soft tissue sarcomas, it is a limb sparing neoadjuvant treatment and, in melanoma in-transit metastases, a curative treatment. Despite its demonstrated regional efficiency TNF-based ILP is unlikely to have any impact on survival. High TNF dosages induce endothelial cells apoptosis, leading to vascular destruction. However, lower TNF dosage produces a very strong effect that is to increase the drug penetration into the tumour, presumably by decreasing the intratumoural hypertension resulting in better tumour uptake. TNF-ILP allowed the identification of the role of alphaVbeta3 integrin deactivation as an important mechanism of antiangiogenesis. Several recent studies have shown that TNF targeting is possible, paving the way to a new opportunity to administer TNF systemically for improving cancer drug penetration. TNF was the first agent registered for the treatment of cancer that improves drug penetration in tumours and selectively destroys angiogenic vessels.

Phase I-II study on isolation antiblastic fotemustine perfusion after dacarbazine chemosensitization for advanced melanoma of the extremities.

Isolation limb perfusion (ILP) is the treatment of choice for locally advanced limb melanoma. With melphalan, the referral drug, complete response (CR) is achieved in about 50% of patients, but significant local toxicity occurs in up to 30%. The aim of the present phase I-II study was to challenge fotemustine (F) in ILP after systemic chemosensitization with dacarbazine (DTIC), given its lower toxicity and greater efficacy, as reported in a previous pilot study. Eleven patients with locally advanced limb melanoma were subdivided into triplets, and given F ILP at escalating doses (starting from 25 mg/l) after intravenous administration of 500 mg/m2 DTIC. Acute and chronic locoregional and systemic toxicity, tumour response and clinical outcome were evaluated. Two patients in the first triplet had G3-G4 local toxicity, so that the scheduled F dosage was halved. At drug levels of 12.5, 15.6 and 18.2 mg/l, local toxicity decreased, but only one of eight patients showed CR. The trial was then interrupted due to the low tolerability and poor efficacy of this perfusion regimen. At present, F ILP after DTIC chemosensitization should not be recommended for the treatment of locally advanced limb melanoma.

Isolated liver perfusion for non-resectable liver tumours: a review.

Many treatments have been proposed for non-resectable primary or secondary hepatic cancer but the results have generally been disappointing. Isolated Hepatic Perfusion (IHP) was first attempted four decades ago but it gained acceptance only recently, after spectacular tumour responses were obtained by isolated limb perfusion with melphalan and tumour necrosis factor (TNF) for melanomas and sarcomas. Surgical isolation of the liver is a technically demanding operation that allows the safe administration of high doses of chemotherapeutics and TNF. Percutaneous techniques using balloon occlusion catheters are simpler but result in higher leakage rates from the perfusion circuit into the systemic circulation. Several phase I-II trials indicate that IHP can yield high tumour response rates, even when there is resistance to systemic chemotherapy. However, no significant advantage in overall survival has been demonstrated so far. IHP offers unique pharmacokinetic advantages for locoregional chemotherapy and biotherapy. It might also allow gene therapy with limited systemic exposure and toxicity. At present, IHP nevertheless remains an experimental treatment modality which should therefore be used in controlled trials only.

Isolated limb perfusion: the European experience.

Isolated limb perfusion (ILP) is a method of cancer treatment allowing the administration of high doses of anticancer agents in a limb surgically isolated from systemic circulation. By using continuous leakage monitoring and using the drug melphalan, a high complete remission rate is obtained in patients with melanoma. In patients with sarcomas, ILP with tumor necrosis factor and melphalan represents a neoadjuvant treatment for limb-sparing surgery. This treatment is the first demonstration of an active anti-angiogenic regimen in the clinic.

Limb salvage by neoadjuvant isolated perfusion with TNFalpha and melphalan for non-resectable soft tissue sarcoma of the extremities.

AIMS: Patients with non-resectable soft tissue sarcomas of the extremities do not live longer if they are treated by amputation or disarticulation. In order to avoid major amputations, we tested isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF)+melphalan+/-interferon-gamma (IFN) as a pre-operative, neoadjuvant limb salvage treatment. METHODS: Twenty-two patients were included (six men and 16 women; three upper limb and 19 lower limb tumours). The AJCC stage was IIA in four patients, III in seven and IV in 11. Thirteen cases were recurrent or progressive after previous therapy; five tumours had a diameter >/=20 cm, and four were multiple or regionally metastatic. There were six malignant fibrous histiocytomas, five liposarcomas, four malignant peripheral nerve sheath tumours, three rhabdomyosarcomas, two leiomyosarcomas, one recurrent extraskeletal osteosarcoma and one angiosarcoma. RESULTS: Twenty-four ILPs were performed in the 22 patients, and 18 (82%) experienced an objective response: this was complete in four (18%) and partial in 14 (64%). Three patients had a minimal or no response and the tumour progressed in one case. All patients had fever for 24 hours but only one developed a reversible grade 3 distributive shock syndrome with no sequelae. There was no grade 4 toxicity. Seventeen patients (77%) underwent limb-sparing resection of the tumour remnants after a median time of 3.4 months: 10 resections were intracompartmental and seven extracompartmental. Surgery included flaps or skin grafts in five patients, arterial replacement in two and knee arthrodesis in one. Adjuvant chemotherapy was given to eight patients and radiotherapy to six. In one patient amputation was necessary after a second ILP. Secondary amputations were performed for recurrence in two patients, resulting in an overall limb salvage rate of 19/22 (86%). After a median follow-up of 18.7 months, 10 recurrences were recorded: seven were both local and systemic and three were only local. The median disease free and overall survival times have been >12.5 and 18.7 months respectively: this is similar to the outcome after primary amputations for similar cases. CONCLUSION: ILP with TNF and chemotherapy is an efficient limb sparing neoadjuvant therapy for a priori non-resectable limb soft tissue sarcomas.

High tPA-expression in primary melanoma of the limb correlates with good prognosis.

To investigate whether the course of primary melanoma disease correlates with expression of the various components of the proteolytic plasminogen activation (PA) system, immunohistochemical stainings for activators of plasminogen (tissue type (tPA) and urokinase type (uPA)), inhibitors of plasminogen activation (type 1 (PAI-1) and type 2 (PAI-2)) and the receptor for uPA (uPAR) were performed on 214 routinely processed melanoma lesions. All lesions were primary cutaneous melanomas, minimally 1.5 mm thick, and derived from patients with only local disease at the moment of diagnosis (clinically stage II (T(3-4)N(0)M(0)), American Joint Committee on Cancer). Median patient follow-up was 6.1 years. Single variables as immunohistochemical staining results (extent of tumour cell staining, pattern of tumour cell staining and for some components also staining of stromal cells), histopathological and clinical parameters as well as treatment variables were analysed in order to assess their prognostic importance, in terms of time to recurrence, time to distant metastasis and duration of survival. The extent of tPA tumour cell positivity, categorized as 0-5%, 6-50% and 51-100%, appeared to be of importance for these end-points. Lesions with 51-100% tPA-positive tumour cells were found to have the best prognosis, whereas lesions with 6-50% tPA-positive tumour cells had the worst. Moreover, the prognostic significance of Breslow thickness, microscopic ulceration and sex was confirmed in this study. Multivariate analyses, incorporating these relevant factors, showed that the extent of tPA tumour cell positivity was an independent prognostic factor for distant metastasis-free interval (P = 0.012) and for the duration of survival (P = 0.043).

Isolated limb perfusion with tumour necrosis factor-alpha and melphalan with or without interferon-gamma for the treatment of in-transit melanoma metastases: a multicentre randomized phase II study.

This open, multicentre, randomized phase II trial was conducted to determine the effect of isolated limb perfusion (ILP) with tumour necrosis factor-alpha (TNFalpha) in combination with melphalan with or without interferon-gamma (IFNgamma) in patients with in-transit metastases of melanoma of the limbs (MD Anderson stage IIIA or IIIAB, AJCC stage III). The 64 patients included were randomized to receive either a two- drug regimen consisting of TNFalpha and melphalan (TM-ILP) or a three-drug regimen consisting of TNFalpha, melphalan and INFgamma (TIM-ILP). Patients randomized to receive IFNgamma were pretreated for 2 days before the ILP with once daily 0.2 mg IFNgamma subcutaneously and also received the same amount of IFNgamma during ILP. A total of 47 complete responses (73%) were reported, 22 (69%) of which occurred in the TM-ILP group and 25 (78%) in the TIM-ILP group; the difference was not significant. The 14 partial responses (22%) were split evenly between the treatment groups. In the TM-ILP group, two cases of stable disease and one case of progressive disease were reported. The overall response rate (complete plus partial responses) was 100% in the TIM-ILP group and 91% in the TM-ILP group, yielding an overall response of 95% for this study. In the historical control data, where 103 patients had received melphalan alone (M-ILP), there were 54 records of complete responses (52%) and 80 of complete or partial responses (78%). The median survival time estimated by the Kaplan-Meier method was 819 days for the TM-ILP group, > 705 days for the TIM-ILP group and 873 days for the combined study population; estimates for time to local progression or recurrence were 327 days, in excess of 498 days and 405 days, respectively. The corresponding figure for the historical controls was 338 days. These data suggest that TNFalpha associated with melphalan may be superior to melphalan alone for ILP.

Sunscreen use and duration of sun exposure: a double-blind, randomized trial.

BACKGROUND: In epidemiologic studies, sunscreen use is associated with increased risk of cutaneous melanoma, basal cell skin cancer, and higher numbers of nevi. It has been proposed that sunscreens may encourage prolonged sun exposure because they delay sunburn occurrence. We examined whether, under habitual conditions of sunscreen use, the sun-protection factor (SPF) had an influence on sun-exposure duration. METHODS: Before the 1997 summer holidays, we randomly assigned 87 French and Swiss participants who were 18-24 years of age to receive an SPF 10 or an SPF 30 sunscreen. Neither medical personnel nor study participants were aware of their sunscreen assignment. Participants were asked to complete daily records of their sun exposure. To avoid influencing the recreational sun-exposure habits of the study participants, no recommendation was made about sun exposure or sun protection. Furthermore, participants were told that the trial end point was the number of pigmented skin lesions before and after the holidays. One subject was lost to follow-up. All statistical tests were two-sided. RESULTS: The SPF 10 (n = 44) and SPF 30 (n = 42) groups had equivalent mean holiday durations (19.4 days versus 20.2 days) and mean quantities of sunscreen used (72.3 g versus 71.6 g). The mean cumulative sun exposures for the two groups were 58.2 hours and 72.6 hours, respectively (P =.011). The mean daily durations of sunbathing were 2.6 and 3.1 hours, respectively (P =.0013), and, for outdoor activities, they were 3.6 and 3.8 hours, respectively (P =.62). There was no difference in sunburn experience between the two groups. CONCLUSIONS: Use of higher SPF sunscreen seems to increase the duration of recreational sun exposure of young white Europeans.

Hyperthermic isolated limb perfusion with tumor necrosis factor alpha, interferon gamma, and melphalan for locally advanced nonmelanoma skin tumors of the extremities: a multicenter study.

BACKGROUND: Hyperthermic isolated limb perfusion (HILP) with tumor necrosis factor alpha (TNF-alpha), interferon gamma, and melphalan has proved to be useful in the treatment of recurrent malignant melanoma and of locally advanced soft tissue sarcomas of the extremities. OBJECTIVE: To determine whether this modality is also effective in the treatment of locally advanced nonmelanoma skin tumors of the extremities. PATIENTS AND METHODS: Fifteen patients with locally advanced primary, recurrent, or metastatic skin tumors of the extremities (12 with squamous cell carcinoma and 3 with Merkel cell carcinoma) underwent HILP with TNF-alpha, interferon gamma, and melphalan. Six tumors were localized in the upper extremity (40%), and 9 in the lower extremity (60%). Treatment-related complications, limb salvage rate, local recurrence, and regional and distant metastases were scored during a median follow-up of 20 months. RESULTS: After HILP, 9 patients (60%) showed a complete response (with histopathological confirmation). Four patients (27%) showed a partial response (with histopathological confirmation in 1 patient), and 2 patients (13%) showed no change (with histopathological confirmation in 1 patient and with clinical evidence in 1 patient). Two patients (13%) showed treatment-related complications. The limb salvage was achieved in 12 patients (80%), and the local recurrences developed in 4 patients (27%). During follow-up, regional lymph node metastases were observed in 2 patients (13%) and distant metastases in 2 patients (13%). CONCLUSION: Based on our results, HILP with TNF-alpha, interferon gamma, and melphalan should be considered as a limb-saving treatment modality in patients with locally advanced nonmelanoma skin tumors of the extremities who would otherwise be candidates for ablative surgery.

Clinical applications of TNF-alpha in cancer.

High-dose TNF-alpha plus chemotherapy, with or without IFN-gamma, can be safely administered regionally through isolated limb perfusion. This procedure produced between 70% and 80% complete remission in cases of in transit melanoma metastases and between 25% and 36% complete remission in cases of inextirpable soft-tissue sarcomas. Dual targeting is involved; TNF-alpha and IFN-gamma induce apoptosis of angiogenic endothelium, while melphalan induces apoptosis of tumour cells.

Hyperthermic isolated limb perfusion with tumour necrosis factor and melphalan as treatment of locally advanced or recurrent soft tissue sarcomas of the extremities.

Hyperthermic isolated limb perfusion (HILP) with various chemotherapeutic agents has been used for the local treatment of high-grade soft tissue sarcomas (STS) of the extremities, but in most cases with a disappointing result. Most regimens should certainly not be considered superior to surgery plus radiotherapy. Although the majority of extremity STS can be resected locally, some have a very large size and are in close proximity to bones, nerves or blood vessels. In these cases, amputation is the only means of resecting the tumour. A new combination of drugs used in the set-up of HILP with tumour necrosis factor-alpha and melphalan has emerged as a very promising option for the limb-saving management of locally advanced STS. In recent studies, complete response rates of approximately 30% and partial remission rates of 50% have been achieved, while the overall limb-salvage rate is more than 80%.

Prophylactic isolated limb perfusion for localized, high-risk limb melanoma: results of a multicenter randomized phase III trial. European Organization for Research and Treatment of Cancer Malignant Melanoma Cooperative Group Protocol 18832, the World Health Organization Melanoma Program Trial 15, and the North American Perfusion Group Southwest Oncology Group-8593.

PURPOSE: Patients with primary cutaneous melanoma > or = 1.5 mm in thickness are at high risk of having regional micrometastases at the time of initial surgical treatment. A phase III international study was designed to evaluate whether prophylactic isolated limb perfusion (ILP) could prevent regional recurrence and influence survival. PATIENTS AND METHODS: A total of 832 assessable patients from 16 centers entered the study; 412 were randomized to wide excision (WE) only and 420 to WE plus ILP with melphalan and mild hyperthermia. Median age was 50 years, 68% of patients were female, 79% of melanomas were located on a lower limb, and 47% had a thickness > or = 3 mm. RESULTS: Median follow-up duration is 6.4 years. There was a trend for a longer disease-free interval (DFI) after ILP. The difference was significant for patients who did not undergo elective lymph node dissection (ELND). The impact of ILP was clearly on the occurrence-as first site of progression - of in-transit metastases (ITM), which were reduced from 6.6% to 3.3%, and of regional lymph node (RLN) metastases, with a reduction from 16.7% to 12.6%. There was no benefit from ILP in terms of time to distant metastasis or survival. Side effects were higher after ILP, but transient in most patients. There were two amputations for limb toxicity after ILP. CONCLUSION: Prophylactic ILP with melphalan cannot be recommended as an adjunct to standard surgery in high-risk primary limb melanoma.

Pulse treatment of human vascular endothelial cells with high doses of tumor necrosis factor and interferon-gamma results in simultaneous synergistic and reversible effects on proliferation and morphology.

Regional administration of high doses of tumor necrosis factor (TNF), interferon gamma (IFNgamma) and melphalan to patients with advanced cancers of the limbs, results in rapid and specific tumor necrosis, while the normal adjacent tissues remain unaffected. The tumor vasculature is selectively destroyed by this treatment, and neovascular endothelial cells appear to be an early and specific target of TNF and IFNgamma. To further understand some of the cellular events underlying these in vivo effects, we have investigated the response of human macro- and microvascular endothelial cells in vitro, after exposure to high doses of TNF and IFNgamma (up to 40 x 10(3) U/ml each). TNF and IFNgamma synergistically inhibited endothelial-cell proliferation by up to 80% after 72 hr of treatment. Achievement of synergy required the simultaneous presence of both cytokines. A cytokine pulse as short as 30 min was sufficient to induce maximal growth inhibition measured after 48 hr. Both cytokines also induced progressive and dose-dependent elongation of the endothelial-cell morphology. The effects on endothelial-cell proliferation and morphology were reversible upon removal of the cytokines. Moreover, replating of treated cells onto a fresh substrate immediately resulted in re-acquisition of their normal shape. In contrast to the effect on cell proliferation, there was little or no effect on the rate of endothelial-cell apoptosis. The presented data extend reports on the effects of TNF and IFNgamma on human endothelial cells in vitro, and suggest that the in vivo disruption of the tumor vasculature caused by high doses of TNF and IFNgamma is not due to a direct cytotoxic effect on endothelial cells but occurs through an indirect mechanism.