A phase Ib/II randomized double-blind placebo controlled trial evaluating the effect of Nivolumab for patients with in-transit melanoma metastases treated with isolated limb perfusion (NivoILP trial)

Approximately 5-10% of patients with recurrence of malignant melanoma develop
lymphatic dissemination manifested as intransit metastasis (1). The initial
treatment option is surgical excision,however, when there are numerous lesions
or short intervals between the appearances of new lesions, other treatment
modalities must be considered.

The technique of isolated limb perfusion (ILP) was pioneered in the 1950s by
Creech and Krementz (2). Early pharmacokinetic studies using
melphalan in the ILP setting showed that high peak perfusate concentrations
were achieved, and that these levels could be about 20 to 100 times higher than
the peak levels achieved with the usual intravenous doses of melphalan (3). In
1969, Stehlin combined extreme hyperthermia (46.1°C) together with ILP to
potentiate the effect of melphalan (4). The method has since then been refined
and current results for ILP with melphalan (ILP) show an overall response rates
(ORR) between 65% and 100%, with a complete response (CR) rate between 25% to
76% (5). For patients with bulky melanoma metastasis, melphalan might be
combined with tumour necrosis factor-alpha (TNF-alpha, Beromun®) [2].

Most chemotherapeutic agents, including melphalan, induce tumour cell death by
apoptosis, a process that has long been regarded as immunologically "silent"
(6). However, recent evidence suggest that some anticancer drugs, such as
anthracyclines and the alkylating drug
cyclophosphamide, induce an immunogenic type of apoptosis that stimulates the
engulfment of apoptotic bodies by dendritic cells (DC) and the activation of
cytotoxic CD8 T cells through a process known as "cross-priming" (7, 8).

This type of immunogenic cell death is characterized by a series of events that
include preapoptotic surface translocation of calreticulin (sCRT), which serves
as an "eat me" signal for phagocytes, and the release of high-mobility group
box1 protein (HMGB1) in the extracellular milieu, whose binding to TLR4 on
dendritic cells (DCs) triggers adaptive antitumor responses (9, 10).
Hyperthermia is further known to induce heat shock proteins (HSPs), which play
an important role as 'endogenous danger signals' in the immune surveillance
system. Extracellular HSPs released from heat-stressed cells can stimulate
professional antigen-presenting cells, followed by cytokine release and
expression of cell surface molecules. In addition to such activity stimulating
innate immunity, extracellular HSPs can promote the cross-presentation of
HSP-bound tumor-peptide antigens to MHC class I molecules in dendritic cells,
leading to efficient induction of tumour-specific cytotoxic T-lymphocytes (11,
12).

During ILP the high local concentrations of melphalan will induce a local
apoptosis of tumour cells, while immune cells in non-perfused regions of the
body are spared. After reconstitution of the normal circulation, a massive
release of tumour-derived components, including tumourspecific
antigens and immunogenic HSPs and HMGB1-molecules, will follow. When these
components come in contact with antigen-presenting cells, such as dendritic
cells (DCs), this may result in the activation of these cells and in the
induction of tumor-specific T cell responses. Post-perfusion, the
tumour-containing limb may become *recolonized* by immigrating immune cells,
including DCs and cytotoxic lymphocytes, such as CD8+ T cells and NK cells,
that may contribute in reducing the tumour mass. Indeed, following a single
perfusion, tumours often decrease gradually in size during several months,
suggesting an immune- mediated mechanism of action, in addition to the direct
cytotoxic effects of melphalan.

In previous pilot studies, we have analysed immune cell populations in blood
before and after ILP. There was a drastic increase in the frequency of CD11b+
DCs in blood 4 weeks after ILP, which returned to baseline levels 12 weeks
after ILP (in manuscript). Furthermore, in four out of twelve patients (33%)
treated with ILP there was a striking increase of Melan-A specific CD8+ T cells
in blood 4 weeks after ILP, while the levels returned to baseline 12 weeks
after ILP (Figure 3A in protocol) (13). Melanoma patients with high absolute
counts of peripheral blood CD8+ T cells, high
numbers of CD45RA CD8+ T cells and high expression of the activation marker
HLA-DR prior to ILP, were more likely to achieve a complete clinical response
(CR) (Figure 3B) (13). Similar results were found in a follow up study, with 43
ILP treated patients with in-transit melanoma
metastases (14).

Though these results need to be further validated in larger prospective
studies, they provide indirect support to the concept of immunogenic cell death
after ILP in human melanoma, and point towards CD8+ T cells as a relevant
effector cell population.

Programmed death-1 (PD-1) is an immune checkpoint receptor that down regulates
T cell antitumour activity upon interaction with its ligand programmed cell
death 1 ligand (PD-L1). Blocking antibodies that prevent the interaction
between PD-1 and PD-L1 have been shown to reactivate T cells and restore
anti-tumour immunity, with a resulting prolonged survival for patients with
metastatic melanoma. PD-L1 may be expressed both by melanoma cells and by
myeloid immune cells (15).

Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is used as a first
line treatment for inoperable or metastatic melanoma. Not all patients respond
to anti-PD-1 treatment, and preexisting CD8+ T cells in the tumour appear
critical for clinical response. An evolving concept is
therefore to combine anti-PD-1 treatment with other immunomodulating therapies
that activate CD8+ T cells.

1. Pawlik TM, Ross MI, Johnson MM, Schacherer CW, McClain DM, Mansfield PF, et
al. Predictors and natural history of in-transit melanoma after sentinel
lymphadenectomy. Ann Surg Oncol. 2005;12(8):587-96.
2. Creech O, Jr., Krementz ET, Ryan RF, Winblad JN. Chemotherapy of cancer:
regional perfusion utilizing an extracorporeal circuit. Ann Surg.
1958;148(4):616-32.
3. Minor DR, Allen RE, Alberts D, Peng YM, Tardelli G, Hutchinson J. A clinical
and pharmacokinetic study of isolated limb perfusion with heat and melphalan
for melanoma. Cancer. 1985;55(11):2638-44.
4. Stehlin JS, Jr. Hyperthermic perfusion with chemotherapy for cancers of the
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Nieto-Garcia A. Isolated limb perfusion for malignant melanoma: systematic
review on effectiveness and safety. Oncologist. 2010;15(4):416-27.
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al. Molecular characteristics of immunogenic cancer cell death. Cell Death
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8. Schiavoni G, Sistigu A, Valentini M, Mattei F, Sestili P, Spadaro F, et al.
Cyclophosphamide synergizes with type I interferons through systemic dendritic
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9. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et
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Toll-like receptor 4-dependent contribution of the immune system to anticancer
chemotherapy and radiotherapy. Nat Med. 2007;13(9):1050-9.
11. Tulapurkar ME, Asiegbu BE, Singh IS, Hasday JD. Hyperthermia in the febrile
range induces HSP72 expression proportional to exposure temperature but not to
HSF-1 DNAbinding activity in human lung epithelial A549 cells. Cell Stress
Chaperones. 2009;14(5):499-508.
12. Murapa P, Gandhapudi S, Skaggs HS, Sarge KD, Woodward J

This study has been transitioned to CTIS with ID 2023-509265-21-00 check the CTIS register for the current data.

To evaluate safety and the effect of isolated limb perfusion together with
nivolumab as a way to increase efficacy and give further insights in early
immunological mechanisms. In the first phase Ib part, 20 patients will be
enrolled and followed for a minimum of 3 months. An independent data safety
monitoring board (DSMB) will continuously review safety and judge the
seriousness of the events and also recommend the study to stop if
necessary. The following safety objectives will be evaluated:
- Serious adverse events
- Adverse events

If the DSMB do not find safety issues, the trial will continue as a phase II
trial. (Objectives see below)

The study is a randomized placebo controlled double blind trial. Patients will
be randomized to ILP with nivolumab or ILP with placebo. Active follow-up for 3
years.
Arm A: ILP + Nivolumab 480mgx1
Arm B: ILP + Placebo x1

Arm A: ILP + Nivolumab 480mgx1
Arm B: ILP + Placebo x1

Numerous studies have been conducted and are ongoing with immune-checkpoint
inhibitors in patients with skin melanoma. The treatment is already approved
for melanoma in the form of ipilimumab, nivolumab and pembrolizumab and has
been shown to have a very good clinical
effect with prolonged survival.

The half-life of nivolumab in this dose is approximately 4 weeks, and unlike
previous studies where repeated doses are every 3 to 4 weeks, only one dose of
drug will be given. Thus, the adverse reaction profile is expected to be very
low, since adverse reactions usually occur after repeated injections with the
drug. The serious side effects seen so far after prolonged treatment are fever,
fatigue, vomiting, stomach pain, stomach upset, loss of appetite, loose or
watery stools, shortness of breath, low blood levels, back pain, weakness, red
blood cell counts and even immune side effects such as hyperthyroidism,
hypothyroidism, infusion reactions and immunological reactions in colon, liver,
skin, lungs, pituitary gland, kidneys, muscles, pancreas and eyes.

The adverse event profile after ILP is well known, and mainly causes local
effects in the perfused limb, with swelling and redness as major symptoms. In
rare cases this can evolve into a compartment syndrome that may require
fasciotomy.

Combinatorial treatment with ILP (using melphalan and/or tasonermin) and
nivolumab has not previously been performed, however, adverse events profiles
are not overlapping. Nivolumab treatment causes immune-related side effects,
while the ILP treatment causes local symptoms
caused by the toxic effects of hyperthermia and chemotherapy.

There is a risk of leakage during ILP, and leakage is monitored closely using a
continuous isotope measurement. The upper limit for tolerable leakage according
to the product information for tasonermin is 10%, and if that limit is reached
the treatment should be stopped. Therefore,
the hypothetically highest systemic exposure to both melphalan and tasonermin
is 15mg (10% of maximal dose 150 mg) and 0.4mg (10% of 4 mg), respectively. In
the largest series reporting leakage, including 438 ILPs with melphalan showed
a cumulative systemic leakage after ILP of
0.9 per cent (95 per cent confidence interval 0.7-1.1 per cent) (16). Any
potential leakage is verified before any drug is given, and at Sahlgrenska
University Hospital the leakage rates during ILP is in the majority of cases
(>95%) between 0% and 1%, and no patient in the modern era had a leakage more
than 10% (unpublished data). Based on a typical dose of 50-100 mg melphalan and
a 1% leakage rate, the systemic dose is rather 0.5-1mg of melphalan and based
on a maximal dose of 4 mg tasonermin the systemic dose is 0.04 mg.

For a disease with metastatic malignant melanoma, where today's most effective
treatment with ILP only receives complete response in about 60% of the
patients, it is considered very important to evaluate the possibility of new
treatment regimens. In view of this situation, the benefit-risk
balance is considered beneficial for a clinical evaluation of combination
therapy with nivolumab and ILP.