Isolated Limb Perfusion for Stage III Melanoma: Does It Still Have a Role in the Present Era of Effective Systemic Therapy?

The management of advanced-stage melanoma has changed dramatically with the introduction of systemic targeted therapy and immunotherapy. Patients with stage IV melanoma currently benefit from agents that are extremely effective, especially when compared with classic chemotherapeutic agents. The field is still evolving, and these newer agents are now used in patients with stage III disease, in the setting of adjuvant trials after resection of the disease bulk. Patients with bulky and numerous in-transit metastases form a very distinct subset of melanoma patients. The disease is classically staged as stage III, but because it is usually unresectable, a regional or systemic rather than a local approach is warranted. Isolated limb perfusion (ILP) is a regional technique that has been shown to provide high response rates and tumor control. The impressive results of ILP were obtained in the era of ineffective systemic agents. Now that this situation has profoundly changed, questions arise as to what the role of ILP is in the treatment of patients with melanoma in-transit metastases, which patients are ideal candidates for ILP, and whether ILP is here to stay-or will become obsolete in the near future.

Introduction

During recent decades, the incidence of melanoma in the United States has increased to an estimated > 76,000 new cases in 2016.[1] The vast majority (84%) of these patients present with disease confined to the local site. Surgery was, is, and will be the mainstay of treatment for these patients, resulting in excellent overall survival rates of > 90% (relative 5-year survival rate of 98.4%).[1] Innovation in these patients with early-stage disease is not to be expected, nor is there a desperate need.

At the other end of the spectrum are the patients who present with metastatic disease. Patients with stage IV melanoma generally do not benefit from surgery; traditionally, systemic chemotherapy was offered, albeit with very limited success. This situation has changed fundamentally with the introduction of molecular targeted therapy and immune checkpoint inhibition. These therapies are currently recommended for first-line treatment of metastatic melanoma.[2] Innovations over the past decade have been practice-changing for patients with stage IV disease.

About 9% of all patients with melanoma present with stage III disease.[1] Patients in this category are the most challenging to treat. Regional metastases can present as satellite lesions close to the primary tumor location, as in-transit metastases between the site of the primary tumor and the draining lymph node basin, as micrometastases in the regional lymph nodes that can be detected only with a sentinel node biopsy, as clinically apparent lymph node metastases, or even as a combination of these features. This wide range of presentations corresponds with a wide range in expected 5- or 10-year survival. Patients with sentinel node micrometastases have 10-year life expectancies of about 70% (> 80% for patients with submicrometastases, according to the Rotterdam/Dewar criteria[3]), whereas patients who present with clinically apparent nodes (eg, in the ilio-inguinal region) have overall survival rates of 10% to 20%. Consequently, the management of stage III melanoma should be tailored to the patient’s specific situation: a one-size-fits-all approach does not exist. It is for this patient subset that innovative approaches to treatment are most eagerly awaited.

In what can be called “early stage III” melanoma, life expectancy in the presence of satellite metastases or lymph node micrometastases is relatively high, and the goal of therapy can therefore be minimizing toxicity and morbidity while obtaining local control. This trend is apparent especially in the approach to the sentinel lymph node. Traditionally, patients with metastatic deposits in the sentinel node were offered completion lymph node dissection (CLND) to achieve maximal local control. It has become apparent that patients with micrometastases in their sentinel node may not benefit from further lymph node dissection and can thus be spared the additional morbidity.[4] This concept is currently being prospectively evaluated in the European Organisation for Research and Treatment of Cancer (EORTC) 1208 (MiniTub) trial.

Furthermore, the results of a German prospective randomized trial of the value of CLND in patients with a positive sentinel lymph node biopsy strongly suggest that this procedure has no benefit, specifically for those patients with sentinel node metastases of < 1 mm.[5] These results reinforce the matched cohort data from multiple centers that did not demonstrate a survival benefit for CLND after a positive sentinel node biopsy.[6] The worldwide Multicenter Selective Lymphadenectomy Trial II (MSLT-II) will provide further insight into this intriguing question: given the morbidity of CLND, will less be more?

Patients with “advanced stage III” melanoma, such as those with multiple in-transit metastases or palpable nodes, require a different approach. Both in-transit metastases and multiple palpable nodes reflect extensive disease, and survival rates are correspondingly low. The prognosis of patients with > 4 palpable lymph nodes is no better than that of patients with stage IV disease.[7] Recently, it has been suggested that more extensive surgery to the deep pelvic and obturator nodes does not improve outcomes for these patients.[8-10] In other words, the prognosis is dictated by the biology of the disease rather than by the extent of surgery.

The goal of therapy in these patients should therefore be to maximize overall survival and to achieve adequate local control. Local and systemic toxicity can be accepted but should obviously be minimized. In this patient category, the development of effective systemic chemotherapy may be resulting in serious paradigm shifts. A surgical approach is no longer the sole treatment option. Adjuvant immunotherapy (high-dose interferon alfa, pegylated interferon alfa-2b, and anti–cytotoxic T-lymphocyte–associated antigen 4 [anti–CTLA-4]) after CLND has been tested in phase III trials and has improved recurrence-free survival in the Eastern Cooperative Oncology Group (ECOG) 1684/Intergroup E1690,[11,12] EORTC 18991,[13] and EORTC 18071 trials.[14] Multiple trials are ongoing in this setting: eg, EORTC 1325/KEYNOTE 054 (anti–programmed death 1 [anti–PD-1]), Southwest Oncology Group (SWOG) S1404 (anti–PD-1 vs high-dose interferon alfa), ECOG 1609 (high-dose anti–CTLA-4 vs low-dose anti–CTLA-4 vs high-dose interferon alfa), BRIM8 (BRAF inhibitor), and COMBI-AD (BRAF inhibitor + MEK inhibitor). For patients with stage IIIC disease, systemic therapy is already registered as a potential first-line treatment, especially when the disease is deemed unresectable. Thus, effective systemic therapy for the treatment of stage III melanoma has emerged. Whether this influences treatment choice in stage III melanoma patients with in-transit metastases is the subject of this review.

In-Transit Metastases

In about 8% of melanoma patients with primary tumors of > 1 mm, in-transit metastases will develop during the course of the disease.[15] These metastases result from tumor emboli trapped within the dermal and subdermal lymphatics and can occur anywhere between the site of the primary tumor and the draining regional lymph node basin. The median time between the diagnosis of the primary tumor and the development of in-transit metastases is about 15 months. The development of in-transit metastases is often a prelude to the appearance of systemic disease.

Various treatment options exist for melanoma in-transit metastases, as the presentation can range from a very few tiny lesions easily amenable to local excision, to > 100 extremely bulky lesions in previously extensively treated extremities. This wide range of clinical presentation requires a tailored approach for each patient. Whereas in some patients, resection of limited disease is part of a curative strategy, other patients may need treatment of in-transit metastases even in the presence of stage IV disease for purposes of palliation.

Finding the best treatment option for in-transit metastases can therefore be challenging. When the interval between the appearance of new lesions is short, when numerous and bulky metastases are present, or when multiple therapeutic modalities have failed, few options are available. The optimal treatment in these settings should be technically feasible, should have the potential for repetitive use if needed, and should limit both local and systemic toxicity.[16]

Creech et al faced these challenges when they developed the concept of isolated limb perfusion (ILP) in 1958.[17] At that time, melanoma was infamously refractory to any kind of systemic treatment. This led to the search for techniques that could deliver high concentrations of chemotherapy or other agents to the affected limb, without the risk of systemic toxicity. In this way, drug concentrations could potentially be made high enough to achieve an antitumor effect. As in-transit metastases of extremity melanomas are, by definition, confined to a limb, isolation of the affected limb from the systemic circulation would offer such an opportunity.

ILP Technique

Isolation of the limb is achieved by surgical access to the artery and vein on the iliac, femoral, popliteal, axillary, or brachial level. The artery and vein are clamped and cannulated, after which the catheters can be connected to a heart-lung machine to get an oxygenated circuit. To further isolate the limb, a tourniquet is placed proximal to the site of the perfusion. The major concern with ILP is potential leakage of the effective agents into the systemic circulation. Therefore, leakage monitoring is mandatory, and a precordial scintillation probe is placed to detect any radioactively labeled albumin administered to the isolated circuit that has potentially leaked to the systemic circulation.

Once an isolated and leakage-free circuit is established, the perfusate is warmed in order to increase limb temperatures to between 38.5°C (101.3°F) and 39.5°C (103.1°F). This mild hyperthermia causes vasodilation in the dermal and subdermal tissue, which improves local drug delivery (twofold at 39.5°C [103.1°F] compared with 37.0°C [98.6°F]).[18] A second effect is the idiosyncratic sensitivity of tumor cells to heat.[19] Higher temperatures lead to increased drug uptake and cell death, but at the cost of severe local toxicity. True hyperthermia (> 40.0°C [104°F]) should therefore be avoided.[20] When adequate tissue temperatures are reached, drugs can be added to the perfusate.

Melphalan (L-phenylalanine mustard) has been the standard drug for ILP because of its efficacy and toxicity profile.[21] With the use of an isolated circuit, drug concentrations in the limb are 20 times higher than can be achieved systemically. Melphalan concentrations of 10 mg/L (leg) or 13 mg/L (arm) are considered standard doses. Melphalan-based ILP (M-ILP) was used for decades during the previous century, and complete response (CR) rates of 40% to 50% and overall response (OR) rates of 75% to 80% were achieved. These rates were unequaled by any other treatment modality.[22]

Several attempts have been made to improve the response to ILP by using cytostatic drugs other than melphalan. Drugs commonly used in the treatment of systemically metastasized melanoma include dacarbazine and cisplatin, either alone or in a combination schedule. These drugs-among others-were tested in the ILP setting, but no drug or drug combination for patients with melanoma has achieved results superior to those of melphalan. Probably the only alternative schedule still in use is the combination of melphalan and actinomycin-D.[23]

Probably the most influential adjustment of ILP was the introduction of tumor necrosis factor (TNF) by Lejeune and Liénard in 1988.[24] TNF was isolated as an endogenous factor, especially active in inflammation, and with a necrotizing effect on tumor cells. TNF has a dual mechanism of action: the direct cytotoxic effect of high-dose TNF on tumor cells certainly plays a role in antitumor activity, but more importantly, the TNF effect on the so-called tumor-associated vasculature induces a rapid change in tumor morphology characterized by hemorrhagic necrosis. However, systemic use in patients with melanoma has been very disappointing. TNF turned out to be a potent mediator of septic shock; therefore, the systemic adverse effects (eg, fever, acute drop in vascular resistance leading to low blood pressure) are the major factors that obviate systemic application of this cytokine. Because the maximum tolerated dose of TNF in humans is 10 to 50 times lower than the dose required for antitumor effect, systemic as well as intralesional administration of TNF is not clinically feasible.

ILP combines the advantages of TNF antitumor activity with the avoidance of systemic effects. Moreover, the cytotoxic effects of TNF are enhanced in hyperthermic conditions and with the addition of alkylating chemotherapeutics,[25] both of which are present during ILP.

TNF-Based ILP in Stage III Melanoma

Response to perfusion

Although the response rates with M-ILP were very satisfactory compared with those of systemic options at that time, the rates were relatively disappointing in patients with bulky melanoma. The tumor bulk of melanoma in-transit metastases can indeed be crucial, since these are large, sarcoma-like lesions. The inhomogeneous drug uptake of soft-tissue sarcomas was once the reason for abandoning M-ILP as a treatment option for these unresectable tumors. The application of TNF to the M-ILP protocol (TM-ILP) has been a fundamental change in the sarcoma setting, and TM-ILP is now used widely for this indication. Tumor bulk can be vital in appraising the TNF effect. Like sarcomas, bulky melanoma in-transit metastases benefit most from the destruction of tumor-associated vasculature by TNF.

Initial studies of TM-ILP for melanoma metastases reported response rates that were far superior to the historical melphalan-only data.[26] These results have led to comparative studies of M-ILP and TM-ILP. A randomized trial published in 2006 that compared M-ILP and TM-ILP showed no beneficial effect of adding TNF.[27] This trial had extremely low CR rates compared with rates in the available case series worldwide, and the true indication for a TM-ILP protocol (bulky disease) could not be analyzed separately. Furthermore, the response rate in this trial was assessed at 3 months rather than at the point when maximum response was reached, which is usually after 3 to 6 months. This led to criticism, and further series have demonstrated improved results for TM-ILP, albeit in a nonrandomized setting.

A retrospective mixed series of M-ILP and TM-ILP by Rossi et al showed a significantly improved CR rate when TM-ILP was used.[28] The results of TM-ILP in a large series published after 2000 showed that CR rates are consistently slightly greater than 60% and OR rates are nearly 90% (Table).[29-32] Of note, the response to ILP is dependent on the extent of disease. Best responses are observed in patients with in-transit metastases with no nodal disease (CR, 77%), followed by in-transit metastases with nodal disease (CR, 49%) and stage IV disease (CR, 38%).[33] These differences in response rate are observed in many series[34,35] and reflect differences in the aggressiveness of melanoma biology.

Infusion vs perfusion

When bulkiness of the tumors is not an issue and the relative complexity of ILP is regarded as a drawback, isolated limb infusion (ILI) may be an excellent alternative to ILP. The principle of ILI is similar to that of ILP, but some technical differences make ILI less complex. During ILI, the catheters are placed percutaneously, which results in a lower blood flow during the procedure (50 to 100 mL/min for ILI vs 150 to 1,000 mL/min for ILP). Furthermore, ILI is a hypoxic procedure, which leads to marked acidosis of the isolated circuit, in contrast to ILP, in which the pump oxygenator maintains oxygenation in the limb. Blood transfusion, or more recently the use of autologous blood, which is required to prime the perfusion circuit in ILP, is unnecessary in ILI.[36] The response to ILI is very good,[34,37] but the high CR rates that are standard with TM-ILP cannot be reached with ILI (see Table).[38]

Local control after perfusion

Although TM-ILP can achieve excellent response rates in patients with melanoma in-transit metastases, the unfavorable nature of the disease dictates that patients often experience locally recurrent disease in the limb. Reported recurrence rates after perfusion are approximately 50%. The duration of response is very acceptable; median disease-free survival is well over 1 year.[39] The management of limb recurrences after ILP is essentially the same as for in-transit metastases in general: local excision if technically feasible, but repeated perfusion for extensive disease.[40]

Survival after perfusion

Melanoma in-transit metastases have a relatively poor prognosis because of the extent of the disease. A local treatment option such as ILP cannot be expected to alter this prognosis because survival is dictated by the presence or appearance of systemic disease. Median survival of patients after a perfusion is approximately 2 years, but interestingly, this is highly correlated with the response to perfusion.[33] This implies that those patients with high response rates to perfusion apparently have more favorable tumor biology.

Toxicity of perfusion

An area of concern is the toxicity that is associated with the perfusion procedure. This can be divided into systemic toxicity resulting from leakage of the perfusate to the systemic circulation, and local toxicity in the treated extremity. When ILP is performed by specialized and experienced teams, clinically relevant leakage percentages (> 10%) are extremely rare and median leakage should be 0%.

Local toxicity of perfusions is scored according to the Wieberdink classification.[41] A few modifications of the ILP protocol have reduced local toxicity significantly.[33] Currently, the TNF dose is reduced from 3–4 mg to 2 mg for leg perfusions, and from 2 mg to 1 mg for the arm. This modification has been widely accepted after a randomized trial in patients with sarcoma showed equal effectiveness of the low-dose perfusions, with decreased local toxicity.[42] The effect of low-dose ILP on local toxicity was confirmed in further case series of patients with sarcoma, but there was no effect in patients with melanoma.[33] Reasons for this difference are unknown.

Another adjustment in ILP that results in less local toxicity is the performance of more distal perfusions in order to reduce the perfused limb volume. This has a profound effect on local toxicity.[43] Major local toxicity (Wieberdink score of greater than 3) is now seldom observed in ILP; it is 3% in recent reports.[33]

Selection of Patients for ILP

Careful patient selection is crucial for ILP, and to a lesser extent for the use of TNF. In particular, proximal extension of in-transit metastases should be carefully examined to ensure that all disease is distal to the planned tourniquet position. Patients with a history of peripheral vascular disease or deep vein thrombosis should have a vascular workup to ascertain adequate flow and vascular diameters. Those patients who have severe cardiac dysfunction are at risk in the event of systemic leakage of TNF because of the drop in vascular resistance. However, this clinically significant leakage is very unlikely when continuous monitoring is performed, since adequate measures (premature ending of perfusion, vasopressor use) can be taken as soon as leakage occurs. Although cardiovascular disease is more common in older adults, it has been shown that TM-ILP is well tolerated by elderly patients.[44] Therefore, age should not be an exclusion criterion when selecting patients for ILP.

Overall, the view of most melanoma centers that perform TM-ILP is that the true indication for TM-ILP is bulky disease or failure after previous M-ILP. For every patient with in-transit metastases, ILP can achieve high response rates and excellent tumor control. It is in these specific situations that the addition of TNF is most effective.[31]

The success of ILP in the treatment of melanoma in-transit metastases has prompted trials to test the efficacy of ILP in the adjuvant setting, after excision of the primary melanoma. After the first disappointing results, this use was largely abandoned by most melanoma centers.[45] Recently, the long-term results of a Swedish trial confirmed that adjuvant ILP after the excision of high-risk primary melanomas does not improve survival.[46]

The classic indications for TM-ILP are well established. Patients who qualify for TM-ILP form a small and highly selected subset that should be taken into account when ILP studies are analyzed. We know which patients are ideal candidates for ILP, but is ILP still the best treatment option for such patients in this era of systemic chemotherapy?

Systemic Therapy for Stage III Melanoma

Until recently, the results of systemic therapy in metastasized melanoma were so disappointing that the use of systemic agents in patients with stage III disease was considered only as a last resort. The introduction of BRAF/MEK/KIT inhibitors, anti–CTLA-4 antibodies, and PD-1 pathway inhibitors may change this situation drastically. Patients with stage IV melanoma now experience response rates ranging from 10% to 75% and, in a subset of patients, long-term benefit can be obtained.[47]

Although the majority of patients who entered the trials of these new drugs had stage IV disease, most protocols allowed patients with unresectable stage III disease to enroll as well. Patients with in-transit metastases who are eligible for perfusion have by definition unresectable stage III disease. To extrapolate trial results to this subset of patients is probably unfair, since only a small minority of patients in these trials had M0 disease. The trials of adjuvant systemic therapy for stage III disease are ongoing. In the meantime, systemic therapy can be considered an alternative to ILP as primary treatment, even though specific data on the effect of systemic therapy in patients with melanoma in-transit metastases only are lacking. To determine the best option-apart from practical aspects, such as availability and costs-response rate, duration of response, and toxicity should be carefully weighed.

The response rates of the new agents are impressive compared with those of standard chemotherapy, but they are far below those that can be achieved with perfusion. Of note, response data on systemic agents are merely from patients with M1b/c disease, whereas data on ILP are predominantly from patients with stage III disease. BRAF inhibitors have impressive and rapid response rates of approximately 60% (OR) in patients with the BRAF mutation.[48,49] Besides the drawback of being effective only in patients with the mutation, the median duration of response is only about 7 months, after which resistance seems almost inevitable. Nor should the toxicity of this agent be overlooked. The combination of BRAF and MEK inhibition has theoretical advantages that result in improved response rates and duration of response.[50-52] However, the toxicity of this combination therapy is severe (grade 3/4 systemic toxicity of > 50%). Therefore, systemic kinase inhibition therapy for patients with advanced melanoma, but confined to a limb, does not seem warranted.

A more durable response can be achieved with immunotherapy, but only a minority of patients will respond. Anti–CTLA-4 was introduced first and is especially recognized for achieving durable responses in some patients.[53,54] The CR and OR rates are such that in patients with locally advanced disease, single-agent anti–CTLA-4 therapy is of limited value. Anti–PD-1 therapy is generally considered standard first-line treatment in patients with BRAF wild-type melanoma,[2] either as a single agent[55] or in combination with anti–CTLA-4. The combination therapy in particular yields high response rates that are durable in more than 20% of patients.[56] Again, it is the toxicity of this schedule that makes combination immunotherapy unattractive for treatment of regional disease.

There are regional therapies that can be considered as alternatives to ILP. We mentioned ILI as a “simplified” alternative to TM-ILP. The obvious drawback of ILI as compared with ILP is the lower rate of CR and OR (33% and 75%, respectively).[34] More recently, oncolytic immunotherapy by intralesional injection of talimogene laherparepvec (T-VEC) has shown interesting response rates (CR, 11%) and is well tolerated.[57] This novel approach is promising as a regional treatment, but the response rates are such that it is unlikely to replace TM-ILP for extremity in-transit metastases. Further exploration of T-VEC is most likely in areas where TM-ILP is not feasible, such as the trunk and head and neck.

For patients with multiple unresectable in-transit metastases, the aim of therapy should ideally be a CR. Partial responses rarely lead to resectable disease in such patients; thus, the benefit is limited to the time until disease progression. The CR rates of ILP of about 60% are still unmatched by any systemic agent. The superior local control rate of a perfusion, coupled with the mild toxicity, means that perfusion remains the optimal treatment option in this highly selected subset of patients.

Future of ILP

In the era of continually improving systemic therapies, it is unlikely that a local therapy such as ILP would still be used on a stand-alone basis in patients with extensive disease. The development of potent agents for the systemic treatment of patients with melanoma offers new possibilities not only for combining drugs, but also for combining two different methods of drug delivery (systemic and in the isolated circuit). It is a logical next step in patients with extensive in-transit metastases to first ensure a rapid response, and then to use systemic therapy for survival benefit. Although BRAF inhibition followed by anti–CTLA-4 therapy is appealing, this regimen has produced disappointing results in retrospective series. The combination of ILP followed by an immunotherapy regimen is theoretically very attractive, but a phase II trial protocol that combined ILP and systemic ipilimumab (ClinicalTrials.gov identifier: NCT02094391) recently closed due to lack of accrual. The combination of T-VEC locally with anti–CTLA-4 systemically has just been tested in a phase Ib trial, with promising results.[58]

In conclusion, ILP provides rapid responses that are still superior to those achieved by systemic therapy, even in the latest trials. The responses are achieved with only very mild local toxicity and virtually no systemic toxicity. Patients with extensive in-transit metastases do need and benefit from local treatment for disease control, but because unresectable stage III disease reflects poor tumor biology, survival remains relatively poor. The rapidly evolving landscape of melanoma treatment options provides tremendous opportunities for tailor-made treatment, and it is for the benefit of patients that all available options-alone or in combination-are evaluated in specialized melanoma centers.

Financial Disclosure:The authors have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.

References:

1. SEER stat fact sheets: melanoma of the skin. hhttp://seer.cancer.gov/statfacts/html/melan.html. Accessed November 7, 2016.

2. Garbe C, Peris K, Hauschild A, et al. Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline-update 2016. Eur J Cancer. 2016;63:201-17.

3. van der Ploeg AP, van Akkooi AC, Rutkowski P, et al. Prognosis in patients with sentinel node-positive melanoma is accurately defined by the combined Rotterdam tumor load and Dewar topography criteria. J Clin Oncol. 2011;29:2206-14.

4. van der Ploeg AP, van Akkooi AC, Verhoef C, Eggermont AM. Completion lymph node dissection after a positive sentinel node: no longer a must? Curr Opin Oncol. 2013;25:152-9.

5. Leiter U, Stadler R, Mauch C, et al. Complete lymph node dissection versus no dissection in patients with sentinel lymph node biopsy positive melanoma (DeCOG-SLT): a multicentre, randomised, phase 3 trial. Lancet Oncol. 2016;17:757-67.

6. van der Ploeg AP, van Akkooi AC, Rutkowski P, et al. Prognosis in patients with sentinel node-positive melanoma without immediate completion lymph node dissection. Br J Surg. 2012;99:1396-405.

7. Young SE, Martinez SR, Faries MB, et al. Can surgical therapy alone achieve long-term cure of melanoma metastatic to regional nodes? Cancer J. 2006;12:207-11.

8. Egger ME, Brown RE, Roach BA, et al. Addition of an iliac/obturator lymph node dissection does not improve nodal recurrence or survival in melanoma. J Am Coll Surg. 2014;219:101-8.

9. Mann GB, Coit DG. Does the extent of operation influence the prognosis in patients with melanoma metastatic to inguinal nodes? Ann Surg Oncol. 1999;6:263-71.

10. van der Ploeg AP, van Akkooi AC, Schmitz PI, et al. Therapeutic surgical management of palpable melanoma groin metastases: superficial or combined superficial and deep groin lymph node dissection. Ann Surg Oncol. 2011;18:3300-8.

11. Kirkwood JM, Strawderman MH, Ernstoff MS, et al. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group Trial EST 1684. J Clin Oncol. 1996;14:7-17.

12. Kirkwood JM, Ibrahim JG, Sondak VK, et al. High- and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. J Clin Oncol. 2000;18:2444-58.

13. Eggermont AM, Suciu S, Testori A, et al. Long-term results of the randomized phase III trial EORTC 18991 of adjuvant therapy with pegylated interferon alfa-2b versus observation in resected stage III melanoma. J Clin Oncol. 2012;30:3810-8.

14. Eggermont AM, Chiarion-Sileni V, Grob JJ, et al. Adjuvant ipilimumab versus placebo after complete resection of high-risk stage III melanoma (EORTC 18071): a randomised, double-blind, phase 3 trial. Lancet Oncol. 2015;16:522-30.

15. Read RL, Haydu L, Saw RP, et al. In-transit melanoma metastases: incidence, prognosis, and the role of lymphadenectomy. Ann Surg Oncol. 2015;22:475-81.

16. Gabriel E, Skitzki J. The role of regional therapies for in-transit melanoma in the era of improved systemic options. Cancers (Basel). 2015;7:1154-77.

17. Creech O Jr, Krementz ET, Ryan RF, Winblad JN. Chemotherapy of cancer: regional perfusion utilizing an extracorporeal circuit. Ann Surg. 1958;148:616-32.

18. Omlor G, Gross G, Ecker KW, et al. Optimization of isolated hyperthermic limb perfusion. World J Surg. 1992;16:1117-9.

19. Cavaliere R, Ciocatto EC, Giovanella BC, et al. Selective heat sensitivity of cancer cells. Biochemical and clinical studies. Cancer. 1967;20:1351-81.

20. Kroon BB, Klaase JM, van Geel AN, Eggermont AM. Application of hyperthermia in regional isolated perfusion for melanoma of the limbs. Reg Cancer Treat. 1992;4:223-6.

21. Thompson JF, Gianoutsos MP. Isolated limb perfusion for melanoma: effectiveness and toxicity of cisplatin compared with that of melphalan and other drugs. World J Surg. 1992;16:227-33.

22. Eggermont AM. Treatment of melanoma in-transit metastases confined to the limb. Cancer Surv. 1996;26:335-49.

23. Sanki A, Kam PC, Thompson JF. Long-term results of hyperthermic, isolated limb perfusion for melanoma: a reflection of tumor biology. Ann Surg. 2007;245:591-6.

24. Liénard D, Ewalenko P, Delmotte JJ, et al. High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol. 1992;10:52-60.

25. Watanabe N, Niitsu Y, Umeno H, et al. Synergistic cytotoxic and antitumor effects of recombinant human tumor necrosis factor and hyperthermia. Cancer Res. 1988;48:650-3.

26. Liénard D, Eggermont AM, Koops HS, et al. 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. Melanoma Res. 1999;9:491-502.

27. Cornett WR, McCall LM, Petersen RP, et al. Randomized multicenter trial of hyperthermic isolated limb perfusion with melphalan alone compared with melphalan plus tumor necrosis factor: American College of Surgeons Oncology Group Trial Z0020. J Clin Oncol. 2006;24:4196-201.

28. Rossi CR, Pasquali S, Mocellin S, et al. Long-term results of melphalan-based isolated limb perfusion with or without low-dose TNF for in-transit melanoma metastases. Ann Surg Oncol. 2010;17:3000-7.

29. Hoekstra HJ, Veerman K, van Ginkel RJ. Isolated limb perfusion for in-transit melanoma metastases: melphalan or TNF-melphalan perfusion? J Surg Oncol. 2014;109:338-47.

30. Di Filippo F, Giacomini P, Rossi CR, et al. Prognostic factors influencing tumor response, locoregional control and survival, in melanoma patients with multiple limb in-transit metastases treated with TNFalpha-based isolated limb perfusion. In Vivo. 2009;23:347-52.

31. Deroose JP, Grunhagen DJ, van Geel AN, et al. Long-term outcome of isolated limb perfusion with tumour necrosis factor-alpha for patients with melanoma in-transit metastases. Br J Surg. 2011;98:1573-80.

32. Olofsson R, Mattsson J, Lindner P. Long-term follow-up of 163 consecutive patients treated with isolated limb perfusion for in-transit metastases of malignant melanoma. Int J Hyperthermia. 2013;29:551-7.

33. Deroose JP, Eggermont AM, van Geel AN, et al. 20 years experience of TNF-based isolated limb perfusion for in-transit melanoma metastases: TNF dose matters. Ann Surg Oncol. 2012;19:627-35.

34. Kroon HM, Coventry BJ, Giles MH, et al. Australian Multicenter Study of Isolated Limb Infusion for Melanoma. Ann Surg Oncol. 2016;23:1096-103.

35. Noorda EM, Vrouenraets BC, Nieweg OE, et al. Isolated limb perfusion for unresectable melanoma of the extremities. Arch Surg. 2004;139:1237-42.

36. Kroon HM, Huismans A, Waugh RC, et al. Isolated limb infusion: technical aspects. J Surg Oncol. 2014;109:352-6.

37. Wong J, Chen YA, Fisher KJ, Zager JS. Isolated limb infusion in a series of over 100 infusions: a single-center experience. Ann Surg Oncol. 2013;20:1121-7.

38. Dossett LA, Ben-Shabat I, Olofsson Bagge R, Zager JS. Clinical response and regional toxicity following isolated limb infusion compared with isolated limb perfusion for in-transit melanoma. Ann Surg Oncol. 2016;23:2330-5.

39. Moreno-Ramirez D, de la Cruz-Merino L, Ferrandiz L, et al. Isolated limb perfusion for malignant melanoma: systematic review on effectiveness and safety. Oncologist. 2010;15:416-27.

40. Deroose JP, Grunhagen DJ, Eggermont AM, Verhoef C. Repeated isolated limb perfusion in melanoma patients with recurrent in-transit metastases. Melanoma Res. 2015;25:427-31.

41. Wieberdink J, Benckhuysen C, Braat RP, et al. Dosimetry in isolation perfusion of the limbs by assessment of perfused tissue volume and grading of toxic tissue reactions. Eur J Cancer Clin Oncol. 1982;18:905-10.

42. Bonvalot S, Laplanche A, Lejeune F, et al. Limb salvage with isolated perfusion for soft tissue sarcoma: Could less TNF-alpha be better? Ann Oncol. 2005;16:1061-8.

43. Deroose JP, Eggermont AM, van Geel AN, Verhoef C. Isolated limb perfusion for melanoma in-transit metastases: developments in recent years and the role of tumor necrosis factor alpha. Curr Opin Oncol. 2011;23:183-8.

44. van Etten B, van Geel AN, de Wilt JH, Eggermont AM. Fifty tumor necrosis factor-based isolated limb perfusions for limb salvage in patients older than 75 years with limb-threatening soft tissue sarcomas and other extremity tumors. Ann Surg Oncol. 2003;10:32-7.

45. Koops HS, Vaglini M, Suciu S, et al. 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. J Clin Oncol. 1998;16:2906-12.

46. Olofsson Bagge R, Mattsson J, Hafstrom L. Regional hyperthermic perfusion with melphalan after surgery for recurrent malignant melanoma of the extremities-long-term follow-up of a randomised trial. Int J Hyperthermia. 2014;30:295-8.

47. van Zeijl MC, van den Eertwegh AJ, Haanen JB, Wouters MW. (Neo)adjuvant systemic therapy for melanoma. Eur J Surg Oncol. 2016 Jul 11. [Epub ahead of print]

48. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507-16.

49. Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380:358-65.

50. Robert C, Karaszewska B, Schachter J, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015;372:30-9.

51. Larkin J, Ascierto PA, Dreno B, et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N Engl J Med. 2014;371:1867-76.

52. Long GV, Stroyakovskiy D, Gogas H, et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N Engl J Med. 2014;371:1877-88.

53. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711-23.

54. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517-26.

55. Robert C, Long GV, Brady B, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015;372:320-30.

56. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373:23-34.

57. Andtbacka RH, Kaufman HL, Collichio F, et al. Talimogene laherparepvec improves durable response rate in patients with advanced melanoma. J Clin Oncol. 2015;33:2780-8.

58. Puzanov I, Milhem MM, Minor D, et al. Talimogene laherparepvec in combination with ipilimumab in previously untreated, unresectable stage IIIB-IV melanoma. J Clin Oncol. 2016;34:2619-26.