Cancer Treatment Reviews (2006) 32, 524– 531

available at www.sciencedirect.com

journal homepage: www.elsevierhealth.com/journals/ctrv

ANTI-TUMOUR TREATMENT

Targeted therapies in melanoma

Paola Queirolo *, Mirko Acquati

Department of Medical Oncology A, National Institute for Cancer Research, Largo Rosanna Benzi 10, Genova 16132, Italy

Received 18 May 2006; revised 13 July 2006; accepted 18 July 2006

KEYWORDS Summary In the last decade the incidence of melanoma has been rising. Despite this, survival
Melanoma; remains substantially constant because early diagnosis of thin lesions has increased. By con-
Monoclonal antibody; trast, metastatic melanoma continues to have a poor prognosis and it still represents a chal-
Protein-Kinase; lenge for oncologists. Response rates with single agent dacarbazine range from 10% to 25%
Targeted therapy with median survival of 8 months. The advent of new drugs with specific mechanisms of action
could help to improve the poor results of traditional therapies. In this review, we focused on
the novel agents that entered clinical trials in melanoma patients. We show the results of some
clinical trials with target-oriented drugs in melanoma patients. Moreover pre-clinical data and
rationale for use in melanoma was explained. Trials with protein-kinase inhibitors, anti-CTLA-4
agents, pro-apoptotic oligonucleotides and anti-angiogenic agents were reviewed. Combina-
tions with chemotherapeutic agents, immunotherapy and vaccine therapy were also analyzed.
c 2006 Elsevier Ltd. All rights reserved.

Introduction novel drugs such as fotemustine and temozolomide were

Chemotherapeutic agents have a limited activity in meta-
static melanoma. Response rates with single agent dacar-
bazine (DTIC) range from 10% to 25% with a median
survival of 8 months.1 Polychemotherapy and biochemo-
therapy do not seem to improve survival. In fact, a four-
drug regimen like Dartmouth (cisplatin, BCNU, DTIC and
tamoxifen) was not superior to single agent DTIC while
proving more toxic.2 On the other hand, the addition of
cytokines, interleukin-2 and interferon alfa, to chemother-
apy regimens improved response rate and progression-free
survival but without any benefit on median survival.3 Also
* Corresponding author. Tel.: +39 010 5600668; fax: +39 010
5600850.
E-mail address: paola.queirolo@istge.it (P. Queirolo).
not better than DTIC.4,5 Therefore, new approaches are
needed. Cytotoxic chemotherapy is not considered a tar-
geted therapy, because it is not specific and it is limited
by toxicity. In the last decade researchers have focused
on the explanation of an altered signal network in cancer
cell. The identification of pathways involved in growth reg-
ulation, which are important for cancer biology, allowed to
act with specificity at molecular level. The understanding
of EGFR and angiogenesis pathways, and the development
of agents directed on molecular targets, has led to very
surprising results, in particular for breast6 and colorectal
cancers.7 Many agents such as monoclonal antibodies
(mAb), antisense oligonucleotides and tyrosine-kinase
inhibitors are now available for clinical use and many more
are under investigation. Here we reviewed the progress in
the field of melanoma.


0305-7372/$ - see front matter c 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ctrv.2006.07.009
Targeted therapies in melanoma
Methods
Research strategy and selection criteria
Data for this review were identified by searching medical
databases, including PubMed, from 1990 to 2006. Only
papers published in English were included. For the search
we used terms such as ‘‘melanoma’’, ‘‘neoangiogenesis’’,
‘‘apoptosis’’, ‘‘monoclonal antibodies’’, ‘‘protein-kinases’’
and relevant subheadings. Also we retrieved references
cited in review articles. Moreover the past 5 years of
abstracts from the American Society of Clinical Oncology
were reviewed.
Protein-kinase inhibitors
Protein kinases transmit signals to the nucleus through
intracellular cascade. The activity of kinases involved in
transmission of proliferation signals in cancer cells often
become more intense. Kinase inhibitors are generally small
molecules designed to act with specificity on intracellular
protein-kinases. In particular two oral drugs, sorafenib and
imatinib mesylate, and an intravenous drug, temsirolimus,
were tested in melanoma patients.
Sorafenib (BAY 43-9006)
Mitogen-activated protein-kinase pathways have been found
to be altered in various malignancies.8 Serine-threonine
kinases of the RAF family play an important role in the signal
transduction, resulting in the activation of ERK 1/2 and
consequently cell’s proliferation.9 Mutations of B-RAF have
been frequently described in melanoma cell lines.10 In
particular, a missense mutation, causing a valine to glu-
tamic acid substitution in the ATP-binding site (V599E) and
thus constitutional activation, accounts for 90% of B-RAF
mutations.11 About 70% of melanomas exhibit B-RAF altera-
tions, therefore it is a suitable target for antitumor ther-
apy.12 Sorafenib (BAY 43-9006), a bi-aryl urea, is a small
molecule originally synthesized for B-RAF and C-RAF inhibi-
tion. It targets the ATP-binding site of the kinase and, at low
concentrations, it inhibits in vitro both wild-type and
mutant B-RAF. Moreover, sorafenib also inhibits other
tyrosine-kinase receptors involved in angiogenesis and
tumor progression, including vascular endothelial growth-
factor receptor (VEGFR)-2, VEGFR-3, c-kit and platelet-
derived growth-factor b (PDGFR-b). Wilhelm et al. demon-
strated a significant inhibition of neovascularization in
xenograft models, showing a broad-spectrum antitumor
activity.13 Preclinical studies showed a significant retarda-
tion in the growth of human melanoma tumor xenografts
with BAY 43-9006.14 A phase-I study established the maxi-
mum tolerated dose (MTD) as single agent at 400 mg twice
daily, the most common toxicities being gastrointestinal
(mainly diarrhea), dermatological (skin rash/hand-foot
syndrome) and fatigue.15 A phase-II trial was conducted at
the Royal Marsden hospital in patients with refractory
stage-IV melanoma.16 Single agent BAY 43-9006 was admin-
istered at 400 mg bid for 12 weeks. At tumor reassessment,
responders continued therapy until disease progression or
unacceptable toxicity, whereas patients with stable disease
525
(SD) were randomized to continue therapy or take placebo.
Twenty patients were recruited and results showed a
modest activity (1 partial response (PR) and 3 SD), with
favorable safety profile. The association of BAY 43-9006
may results in an enhancement of chemotherapy effect. A
combination phase I/II study with carboplatin and paclitaxel
and escalating doses of BAY 43-9006 was performed. BAY
43-9006 was administered at three dose levels from day 2
to 19 of a 21-day cycle, in association with carboplatin
(AUC 6) and paclitaxel (225 mg/m2) on day 1.17 Thirty-five
metastatic-melanoma patients were enrolled and no
changes in the pharmacokinetics values of the chemothera-
peutic agents were seen. A promising response rate of 31%
was yielded, also with 54% of patients experiencing a SD.
Based on these results an ECOG phase-III trial (E2603) was
planned to assess the efficacy of carboplatin and paclitaxel
plus BAY 43-9006 versus the same chemotherapeutic
regimen plus placebo. DTIC is the most active chemothera-
peutic drug in melanoma. A phase-I trial was conducted to
set the MTD in association with DTIC 1000 mg/m2 every
three weeks.18 Continuous administration of 400 mg bid
was safe. BAY 43-9006 exhibits very interesting activity in
melanoma patients, in particular it seems to enhance
antitumor response when added to chemotherapy with a
good tolerability profile. Results of phase-III trials will
define the real impact of this agent in melanoma patients.
Imatinib mesylate
Imatinib mesylate (STI-571), an oral tyrosine-kinase inhibi-
tor of c-Kit, PDGFR and BCR-ABL, has shown striking activity
in GIST and chronic myeloid leukaemia.19,20 C-Kit is a trans-
membrane receptor with tyrosine-kinase intracellular do-
main. When stem-cell factor binds c-Kit, it causes a
dimerization and consequently an autophosphorylation,
activating a down-stream signal that promotes prolifera-
tion. Imatinib blocks the ATP domain, preventing the bind-
ing of ATP. Subsequent phosphorylations and signal
transduction are avoided. Expression of c-Kit was observed
in nearly 50% of early stage melanomas, but c-Kit expression
is progressively reduced as the cells take on a more highly
metastatic phenotype.21 In contrast PDGFR is highly repre-
sented on melanoma cells, also at a more advanced stage.22
Although imatinib inhibits PDGFR phosphorylation in mice
model, it does not block the growth of melanoma cells.23
In contrast Redondo et al. showed that imatinib was able
to suppress B16F10 melanoma-cell growth.24 A phase-II trial
of 400 mg bid imatinib was performed in metastatic mela-
noma patients with at least a 25% cell expression of c-Kit.
Only one patient had high expression of c-Kit (75% of mela-
noma cells) and in this patient a near complete response
was achieved. Unfortunately the other patients pro-
gressed.25 In an attempt to improve results, a phase-I com-
bination trial was undertaken by Schuchter et al.26
Temozolomide in addition to daily imatinib was adminis-
tered at three dose levels. Therapy was well tolerated with
no unexpected toxicities. Preliminary results showed no
responses, but only stabilization of disease as best result.
The hypothesis that the benefit of treatment with imatinib
could be restricted to patients whose melanoma cells have
a high expression of c-Kit, has been tested in a multi-center
phase-II trial, involving 18 refractory melanoma patients.
526
Immunohistochemical analysis revealed that 75%, 58%, and
67% of patients, whose specimens were available, had a
positive stain respectively for CD117/c-Kit, PDGFR-a
and PDGFR-b. Despite these data, no patient achieved a re-
sponse. Moreover 38.9% of patients required a dose reduc-
tion for toxicity.27 Altogether, these results do not
support at present a role of imatinib in melanoma patients,
but further trials on very selected populations could explain
which patients benefit of such therapy.
Temsirolimus
Temsirolimus (CCI-779) is a novel potent inhibitor of the so-
called mammalian target of rapamycin (mTor). mTor is a
serine-threonine kinase that promotes phosphorylation of
S6K1 and the eukaryotic initiation factor 4E-binding pro-
tein-1 (4E-BP1), which are involved in G1 phase progression.
Temsirolimus is a rapamycin analog that specifically binds to
FK506-binding protein 12 (FKBP12), forming a complex that
interacts with mTor and blocking the subsequent phosphor-
ylation cascade.28 A phase-II study of intravenous 250 mg
temsirolimus every week in 33 patients with metastatic mel-
anoma was undertaken by Margolin et al.29 Toxicities were
mild including mucositis, rash and hyperlipidemia. One pa-
tient had a PR, but the authors concluded that as single
agent temsirolimus was inactive in melanoma.
Agents acting on immunological cells
Melanoma is a highly immunological neoplasm, therefore
target therapies directed at the immune system may be
an effective tool to enhance responses to other active
agents. Two interesting targets are cytotoxic T lymphocyte
antigen 4 (CTLA-4) and toll-like receptor 9 (TLR9).
Anti-CTLA-4 mAb
T-cell activation requires two steps: stimulation via T-cell
receptor (TCR) and additional costimulatory signals. CD28
is the main costimulatory molecule and it is expressed on
T-cells surface. CTLA-4, a homologue of CD28, is a glycopro-
tein expressed on the surface of activated T-cells.30 In par-
ticular CTLA-4 seems to have an inhibitory function. It is
suggested that CTLA-4 has a role in down-regulating T-cell
responses. In fact, a decrease in IL-2 production and an ar-
rest in cell-cycle progression has been observed. Blockade
of CTLA-4 could be an effective strategy for the mainte-
nance of T-cell antitumor responses. Administration of
anti-CTLA-4 mAb into early-tumor-bearing mice resulted in
regression of growing tumors. Moreover, an enhancement
of antitumor cytotoxicity in addition to chemotherapy has
been reported.31 Some researchers focused on such therapy
either alone or in combination.
MDX-010 is a fully-human IgG1 mAb that targets human
CTLA-4 resulting in augmentation and prolongation of T-cell
antitumor response. A phase-I study in metastatic mela-
noma patients was performed.32 A 3 mg/kg single dose of
MDX-010 was administered to 17 patients. The only toxici-
ties noted were a mild rash and pruritus. Two PRs were ob-
served including a 50% reduction of a lung lesion. Hersh
et al. performed a phase-II study to assess the activity of
P. Queirolo, M. Acquati
MDX-010 alone or in combination with DTIC in stage IV un-
treated melanoma patients.33 MDX-010 was administered
at a dose of 3 mg/kg monthly for four cycles, alone (arm
A = 37 patients) or in combination (arm B = 35 patients) with
DTIC (250 mg/m2 for 5 days monthly). There were two PRs
in arm A and 1 CR and 4 PRs in arm B, suggesting more activ-
ity for the combination arm. Toxicities were mainly colitis,
diarrhea and skin rash. Of note, the authors also reported
two deaths possibly related to MDX-010. In the long-term
follow up phase of this study a further CR was yielded in
the combination arm and durable clinical objective re-
sponses were observed.34 Another approach is the combina-
tion of anti-CTLA-4 to vaccine therapy. The administration
to mice bearing tumor of anti-CTLA-4 mAb and a vaccine re-
sulted in melanoma rejection.35 In a trial by Phan et al., 14
patients with metastatic melanoma were treated with a
combination of MDX-010 and vaccination with two modified
HLA-A*0201-restricted peptides, from the gp100 melanoma-
associated antigen. Autoimmune events were reported (der-
matitis, colitis and hepatitis), suggesting a breaking of
peripheral tolerance against self-antigens. Anyway, cancer
regression was revealed for three patients and all patients
experienced T-cell reactivity against the immunizing pep-
tides.36 Moreover in a recent phase-I trial in resected
stage-III/IV melanoma the combination of MDX-010 with
multiple-peptide melanoma vaccination has been shown to
be safe and has resulted in interesting immune response
to peptide vaccination.37 In this study 19 patients were trea-
ted with intravenous MDX-010 at three dose levels (0.3 mg/
kg; 1 mg/kg; 3 mg/kg). The MTD was set at 1 mg/kg, be-
cause in the highest-dose cohort (3 mg/kg) grade-3 gastroin-
testinal toxicity was observed. Diarrhea and abdominal pain
were the dose-limiting toxicities. An autoimmune etiology
was suggested; in fact, a rectal biopsy revealed a CD4+ T-
cell infiltrate. Furthermore, uveitis and rash were reported.
The authors noted that patients who developed autoim-
mune symptoms experienced longer relapse-free survival.
Similar results in terms of toxicity and activity in metastatic
melanoma were obtained with another human anti-CTLA-4
mAb, CP-675,206.38 The dose for further phase-II trials
was set at 10 mg/kg, but the pharmacokinetic analysis sug-
gested a relationship between clinical responses and plasma
concentration of CP-675,206. For this reason an accurate
exploration of higher doses could be suggested. Also, clini-
cal activity (5 PRs and 8 SD) and a decrease of the T-regula-
tory cells that are involved in the suppression of immune
response was demonstrated by Reuben et al.39 These data
on anti-CTLA-4 strategy suggest a role in the enhancement
of antitumor response and a possible synergistic effect with
anticancer vaccines. The relationship between autoimmu-
nity phenomena and anti-tumor response requires further
exploration, because it is suggested that development of
autoimmunity could be a surrogate endpoint for clinical
response.
CpG 7909
A non-antisense oligodeoxynucleotide, CpG 7909, was de-
signed to selectively target TLR9. Through agonism with
TLR9, CpG 7909 activates plasmacytoid dendritic cells
(pDC) and B cells. Moreover it is a strong activator of both
Targeted therapies in melanoma
innate and specific immunity. Intralesional administration in
melanoma metastases revealed anti-tumor activity.40 A
phase-I/II study of single agent CpG 7909 was undertaken
by Wagner et al.41 Weekly subcutaneous injection of 6 mg
was administered to 20 patients until disease progression.
Two PRs were obtained with three patients achieving a
SD. Moreover, the evaluation of peripheral-blood mononu-
clear cells revealed an activation of pDC. Based on these
encouraging results, a phase-II trial with DTIC was de-
signed.42 Patients were randomized into four arms, one
arm with DTIC alone (850 mg/m2), two arms with CpG
7909 alone at different doses (10 mg and 40 mg) and one
arm with CpG 7909 at 40 mg in combination with DTIC.
CpG 7909 was administered every week, whereas DTIC was
given every three weeks. At the preliminary analysis there
were 4 PRs in the combination arm, 2 PRs in the DTIC arm
and 1 PR in the 10-mg CpG 7909 arm. Adverse events in-
cluded injection site reactions, fever, arthralgias and ana-
phylaxis. Also, CpG 7909 was tested in combination with
vaccines. The addition of CpG 7909 to a MAGE-3 vaccine
in MAGE-3 positive patients was safe.43 Immunological re-
sponse to CpG 7909 and vaccination with Melan A/MART-1
was also reported to be strong in HLA-A2+ melanoma pa-
tients. An expansion of antigen specific CD8+ T cells was
also shown.44 These data represent a basis for future re-
searches either in combination with chemotherapy, immu-
notherapy or vaccine therapy.
Pro-apoptotic therapy
Apoptosis is mediated by a sequence of events that result in
cell death. The action of classic anticancer drugs on tumor-
cell death is dependent on induction of apoptosis. In cancer
cell there is often an imbalance in favor of antiapoptotic
signals, which confers cells a resistance to chemotherapeu-
tic drugs and radiation. Bcl-2, a family of proteins, play an
important role in cancer-cell survival. In fact, Bcl-2 inhibits
apoptosis by preventing the release of cytocrome c from
mitochondria, thus preventing the activation of caspase-9
and caspase-3.45 Overexpression of Bcl-2 has been observed
in many type of solid neoplasms and hematologic malignan-
cies, including breast carcinoma, small-cell lung cancer,
lymphoma, multiple myeloma and acute myeloid leukae-
mia. High levels of Bcl-2 protein were also found in mela-
noma. Nearly 90% of melanomas present overexpression
of this anti-apoptotic protein.46 Strategies to down-regu-
late levels of Bcl-2 protein have been developed.
Oblimersen
Oblimersen sodium (G3139) is an antisense oligonucleotide
that targets Bcl-2 at mRNA level. It has an 18-nucleotide se-
quence that is complementary to the first six codons of the
Bcl-2 mRNA. When oblimersen binds to Bcl-2 mRNA, it forms
an heteroduplex, which enables RNase H to cleave the
mRNA sequence, degrading Bcl-2 message.47 In xenotrans-
plantation models for human melanoma, a downregulation
of Bcl-2 concentration has been observed with the adminis-
tration of oblimersen and DTIC.48 Based on these preclinical
results, a phase-I-II trial was performed by Jansen et al. in
patients with metastatic melanoma.49 Oblimersen was
527
administered by continuous intravenous infusion from days
1 to 14 at dose levels ranging from 0.6 mg/kg/die to
6.5 mg/kg/die and DTIC was administered at a dose of
200 mg/m2 from day 5 to day 9. In addition, in the cohorts
of patients receiving 5.6 mg/kg and 6.5 mg/kg, oblimersen
was given by subcutaneous injection from days 1 to 7 twice
daily combined with 800 mg/m2 DTIC on day 5. Although
evaluation of toxicity was the primary end-point, one com-
plete and two partial responses were observed. Addition-
ally, two durable minor responses and two disease
stabilization were noted. Toxicities included fever, liver-
function abnormalities, skin rash and lymphopenia. A
phase-III trial in chemotherapy-naı̈ve melanoma patients
was completed.50 Seven-hundred seventy-one patients were
randomized to receive DTIC alone (1000 mg/m2 every three
weeks) or DTIC plus oblimersen by intravenous continuous
infusion for five days at 7 mg/kg/die. Primary endpoint
was not met; in fact, overall survival (OS) was not statisti-
cally significant between the two groups (9.1 months for
the combination group vs 7.9 months for DTIC alone),
although response rate was significantly in favor of the
oblimersen arm (11.7% vs. 6.8% p = 0.019) and a better pro-
gression-free survival was observed (74 vs. 49 days for both
p = 0.0003). Grade 3/4-neutropenia and thrombocytopenia
were increased by the addition of oblimersen but without
increasing the clinical consequences. In the updated analy-
sis presented at the ASCO meeting 2005, OS had still not
reached statistical significance, but for the patient’ sub-
group with LDH value 62x ULN there was a significant ben-
efit for the combination therapy (10.2 vs. 8.7 months
p = 0.02). Moreover, the addition of oblimersen resulted in
more durable responses (7% vs. 3% p = 0.02).51 Oblimersen
at present is the only target agent tested in a phase-III trial
in melanoma. Results are promising but a longer follow-up
and further trials are needed to set the efficacy of this
agent.
Anti-angiogenic agents
Neoangiogenesis has an important role in sustaining tumor
growth and in the process of metastatization. The develop-
ment of new vessels is a key step for neoplastic progression.
Several pro-angiogenic factors like vascular endothelial
growth factor (VEGF), basic fibroblastic growth factor
(bFGF) and transforming growth factor b (TGF-b) are se-
creted by cancer cells during hypoxic conditions.52 Among
these, the most important factor is VEGF type A. It stimu-
lates angiogenesis by binding to VEGFR-1 and VEGFR-2 ex-
pressed on the surface of endothelial cells.53
Bevacizumab
Bevacizumab is a recombinant humanized monoclonal anti-
body designed to specifically bind VEGF. The inhibition of
biologic activity of VEGF translates into reduction of micro-
vascular growth and regression of existing vessels that feed
tumor.54 Bevacizumab improves the delivery of chemother-
apy by altering tumor vasculature and reducing the elevated
interstitial pressure in tumors. Increased intratumoral
microvessel density correlates to tumor aggressiveness in
melanoma. Moreover VEGF is overexpressed in melanoma
528
and strongly correlates to poor prognosis.55 Anti-angioge-
netic therapy could be beneficial in patients with mela-
noma. A phase-II trial of bevacizumab (15 mg/kg
intravenously every two weeks) with or without daily subcu-
taneous interferon alpha at a dose of 1 million units was car-
ried out. Sixteen patients with metastatic melanoma were
enrolled. A complete response with disappearance of supra-
clavicular and mediastinal adenopathy was observed. More-
over, a patient achieved a PR. The responses were all in
patients receiving the two drugs. Four prolonged stabiliza-
tion of disease were also reported.56
SU5416
Inhibition of VEGF activity is also possible by acting at tyro-
sine-kinase receptor level. SU5416 is a potent inhibitor of
VEGFR-1 tyrosine kinase (flk-1). In a phase-II trial in pre-
treated melanoma patients, SU5416 was administered intra-
venously at 145 mg/mq twice weekly after an adequate pre-
medication against anaphylaxis. Among 31 patients, only
one patient experienced a PR. Anyway, evaluation by dy-
namic contrast magnetic resonance imaging showed a sig-
nificant decrease in tumor perfusion. Toxicities included
lymphopenia and hyperglycemia.57
MEDI-522
Another target for anti-angiogenetic therapy is integrin alp-
havbeta3 (avb3). Its overexpression in melanoma cells is re-
lated to invasive growth and metastatic pattern.58
Moreover, integrin avb3 promotes an upregulation of bFGF,
a well known pro-angiogenic factor.59 MEDI-522 is a novel
humanized monoclonal antibody designed to target integrin
avb3 and it was tested in a phase-II trial. Patients with met-
astatic melanoma were enrolled and randomized to DTIC
1000 mg/m2 every three weeks plus MEDI-522 8 mg/kg/
week (55 patients) or MEDI-522 alone (57 patients). In the
combination arm the response rate approached 13%
whereas in the other arm no response was obtained. Median
survival was promising with the combination arm approach-
ing 9.3 months and 11.8 months for single agent treatment.
There were two deaths possibly attributed to MEDI-522.60
PI-88
PI-88 is a new anti-angiogenetic agent. Due to heparan sul-
fate mimicry, PI-88 competitively binds both to angiogenic
growth factors such as FGF and VEGF, thus inhibiting angio-
genesis, and to heparanase, therefore preventing degrada-
tion of the extracellular matrix (ECM), a main step in the
metastatization process.61 Moreover, an increase of tissue
factor pathway inhibitor (TFPI), an antiangiogenic and
anti-metastatic agent, was reported.62 A phase-II trial of
250 mg/day of PI-88 was initiated in pre-treated patients.
PI-88 was administered subcutaneously for four days every
week.63 Grade-3/4 toxicities were thrombocytopenia,
thrombosis and an episode of brain hemorrhage. Disease
control rate (PRs+SD) was 36%.
Anti-angiogenetic agents exert promising clinical activ-
ity, in particular by increasing growth control. Combination
trials with chemotherapy are warranted.
P. Queirolo, M. Acquati
Other agents
Bortezomib
Ubiquitination pathway is essential for maintaining protein
homeostasis. Protein ubiquitination permits the recognition
by proteasome. Proteasome can proceed to the degradation
of polyubiquitinated proteins through several peptidases. In
cancer cells critical proteins such as p53 and other tumor
suppressing proteins could be regulated by the ubiquitin–
proteasome system.64 A constitutive activation of the nucle-
ar factor jB (NFjB) was reported in melanoma. NFjB
prevents cells from apoptosis and promotes proliferation.
One strategy to block the activity of NFjB is by acting on
26S subunit of proteasome. In fact, the inhibitor of NFjB,
IjB, is a target for the proteasome system and its degradation
promotes translocation of NFjB into the nucleus with subse-
quent activation of its target genes. Bortezomib (PS-341), a
low-molecular-weight dipeptide, inhibits the activity of pro-
teasome by binding to the site of chymotryptic-like en-
zymes.65 A preclinical study by Katayoun et al. suggested
good activity by combining temozolomide and bortezomib.66
On the contrary, a clinical phase-II trial of bortezomib alone
in metastatic melanoma showed negative results. In fact, the
study, planned to accrue 45 patients, was early stopped at
the interim analysis with 27 patients enrolled, because of
lack of clinical activity. No responses were observed. The
authors concluded that single agent bortezomib adminis-
tered intravenously twice weekly for two weeks is ineffec-
tive.67 Anyway, based on the preclinical data, combination
studies with chemotherapeutic agents are required for fur-
ther evaluation of its activity in melanoma.
Marimastat
A family of zinc-dependent enzymes, matrix metallopro-
teinases (MMPs), are involved in the degradation of ECM.
Proteolysis of the components of ECM is a step required
for both local and metastatic spreads of the tumor. More-
over, MMPs induce angiogenesis and activation of growth
factors.68 Marimastat (BB2516) is an oral inhibitor of MMPs
that blocks their action through mimicry of the collagen
site, thus preventing degradation and consequently tumor
spread.69 Melanoma cells have been shown to induce an
up-regulation of MMPs.70 A phase-II study in refractory mel-
anoma was performed by the NCIC-CTG (National Cancer
Institute of Canada Clinical Trials Group).71 Twenty-nine pa-
tients were enrolled in this trial and the first 18 were trea-
ted with 100 mg twice daily of oral Marimastat. Due to
musculo-skeletal toxicities (mainly arthralgia, myalgia)
and fatigue, the subsequent eleven patients received
10 mg twice daily. Evaluation of disease revealed two PRs
and five SD, showing activity in melanoma. Data were inter-
esting because only a control growth was expected whereas
also a tumor shrinkage was observed.
Conclusions
The intrinsic resistance of melanoma to conventional che-
motherapy has led researchers to evaluate new approaches.
Targeted therapies in melanoma 529

Table 1 Trials with target oriented drugs in melanoma

Drug Phase No. pts Responses Ref.
Sorafenib II 20 1 PR, 3 SD [16]
Sorafenib + CBDCA/paclitaxel I/II 35 11 PRs, 19 SD [17]
Imatinib mesylate II 31 1 PR [25]
Temsirolimus II 33 1 PR [29]
II [34]
MDX-010 Arm A 37 2 PRs, 4 SD
MDX-010 + DTIC Arm B 35 2 CRs, 4 PRs, 4 SD
CP-675,206 I/II 26 5 PRs, 8 SD [39]
II 184 [42]
CpG 7909 LD 0 OR
CpG 7909 HD 1 PR
CpG 7909 + DTIC 4 PRs
DTIC 2 PRs
III [51]
Oblimersen+DTIC Arm 1 386 48 ORs, 113 SD
DTIC Arm 2 385 26 ORs, 106 SD
Bevacizumab+/ IFNa II 16 2 PRs, 4 SD [56]
SU5416 II 31 1 PR, 1 SD [57]
II [60]
MEDI-522 Arm 1 57 0 PR, 26 SD
MEDI-522+DTIC Arm 2 55 7 PRs, 22 SD
PI-88 II 44 2 PRs, 3 SD [63]
Bortezomib II 27 0 PR, 6 SD [67]
Marimastat II 29 2 PRs, 5 SD [71]
Abbreviations: OR, objective response; PR, partial response, SD, stable disease.

Table 2 Future and ongoing phase III trials across USA and Europe
Drug Randomization Treatment line End-point
Sorafenib Arm 1: CBDCA/paclitaxel + sorafenib First line OS
Arm 2: CBDCA/paclitaxel + placebo
Sorafenib Arm 1: CBDCA/Paclitaxel + Sorafenib Progression after DTIC or TMZ PFS
Arm 2: CBDCA/paclitaxel + placebo
CP-675,206 Arm 1: CP-675,206 First line OS
Arm 2: DTIC or TMZ
MDX-010 Arm 1: DTIC + MDX-010 First line PFS
Arm 2: DTIC + placebo
MDX-010 Arm 1: MDX-010 Progression after DTIC, TMZ or IL-2 OS
Arm 2: MDX-1379 vaccine
Arm 3: MDX-010 + MDX-1379 vaccine
Abbreviations: TMZ, temozolomide; DTIC, dacarbazine; OS, overall survival; PFS, progression-free-survival.

Molecular biology and better knowledge of cancer cells al-
lowed the development of targeted drugs. Many trials have
been performed, testing either single agent or combination
therapies (Table 1). Unfortunately most of these are phase-
I/II studies, thus designed to evaluate safety and activity.
Only with well-designed phase-III trials will it be possible
to assess the real efficacy of agents oriented to the molec-
ular target (Table 2). In fact, also biochemotherapy in
phase-II studies72,73 seemed to have an impact on mela-
noma, but this effect disappeared in subsequent phase-III
trials.74,75 Moreover, it will be important to select patients
that will benefit from the administration of these new anti-
melanoma weapons. Novel surrogate clinical endpoints,
such as autoimmunity for anti-CTLA-4 mAb, could be useful
to predict clinical response. In the near future, targeted
therapies are going to join chemotherapy, immunotherapy
and vaccines as the oncologist’s tools in the treatment of
melanoma.
References
1. Eggermont AMM, Kirkwood JM. Re-evaluating the role of
dacarbazine in metastatic melanoma: what have we learned
in 30 years? Eur J Cancer 2004;40:1825–36.
2. Chapman PB, Einhorn LH, Meyers ML, et al. Phase III multi-
center randomized trial of the Dartmouth regimen versus
530
dacarbazine in patients with metastatic melanoma. J Clin
Oncol 1999;17:2745–51.
3. Eton O, Legha SS, Bedikian AY, et al. Sequential biochemo-
therapy versus chemotherapy for metastatic melanoma: results
from a phase III randomized trial. J Clin Oncol
2002;20:2045–52.
4. Avril MF, Aamdal S, Grob JJ, et al. Fotemustine compared with
dacarbazine in patients with disseminated malignant mela-
noma: a phase III study. J Clin Oncol 2004;22:1118–25.
5. Middleton MR, Grob JJ, Aaronson N, et al. Randomized phase III
study of temozolomide versus dacarbazine in the treatment of
patients with advanced metastatic malignant melanoma. J Clin
Oncol 2000;18:158–66.
6. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trast-
uzumab after adjuvant chemotherapy in HER2-positive breast
cancer. N Engl J Med 2005;353:1659–72.
7. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab
plus irinotecan, fluorouracil, and leucovorin for metastatic
colorectal cancer. N Engl J Med 2004;350:2335–42.
8. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene
in human cancer. Nature 2002;417:949–54.
9. Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf
kinase family. Cell Signal 2003;15:463–9.
10. Satyamoorthy K, Li G, Gerrero MR, et al. Constitutive mitogen-
activated protein kinase activation in melanoma is mediated by
both BRAF mutations and autocrine growth factor stimulation.
Cancer Res 2003;63:756–9.
11. Sharma A, Trivedi NR, Zimmerman MA, Tuveson DA, Smith CD,
Robertson GP. Mutant V599EB-Raf regulates growth and vascu-
lar development of malignant melanoma tumors. Cancer Res
2005;65:2412–21.
12. Brose MS, Volpe P, Feldman M, et al. BRAF and RAS mutations
in human lung cancer and melanoma. Cancer Res
2002;62:6997–7000.
13. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits
broad spectrum anti-tumor activity and targets raf/MEK/ERK
pathway and receptor tyrosine kinases involved in tumor
progression and angiogenesis. Cancer Res 2004;64:7099–109.
14. Karasarides M, Chiloeches A, Hayward R, et al. B-RAF is a
therapeutic target in melanoma. Oncogene 2004;23:6292–8.
15. Strumberg D, Richly H, Hilger RA, et al. Phase I clinical and
pharmacokinetic study of the Novel Raf kinase and vascular
endothelial growth factor receptor inhibitor BAY 43-9006 in
patients with advanced refractory solid tumors. J Clin Oncol
2005;23:965–72.
16. Ahmad T, Marais R, Pyle L, et al. BAY 43-9006 in patients with
advanced melanoma: The Royal Marsden experience. Proc Am
Soc Clin Oncol 2004;22. [abstr. 7506].
17. Flaherty KT, Brose M, Schucter L, et al. Phase I/II trial of BAY
43-9006, carboplatin (C) and paclitaxel (P) demonstrates
preliminary antitumor activity in the expansion cohort of
patients with metastatic melanoma. Proc Am Soc Clin Oncol
2004;22. [abstr. 7507].
18. Eisen T, Ahmad T, Gore ME, et al. Phase I trial of BAY 43-9006
(sorafenib) combined with dacarbazine (DTIC) in metastatic
melanoma patients. Proc Am Soc Clin Oncol 2005;23. [abstr.
7508].
19. Demetri GD, von Mehren B, Blanke CD, et al. Efficacy and
safety of imatinib mesylate in advanced gastrointestinal stro-
mal tumors. N Engl J Med 2002;347:472–80.
20. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a
specific inhibitor of the BCR-ABL tyrosine kinase in chronic
myeloid leukemia. N Engl J Med 2001;344:1031–7.
21. Janku F, Tomancova V, Novotny J, et al. Expression of c-Kit
was found in more than 50% of early stages malignant
melanoma. A retrospective study of 261 patients. Proc Am
Soc Clin Oncol 2003;22. [abstr 2864].
P. Queirolo, M. Acquati
22. Shen SS, Zhang PS, Eton O, Prieto VG. Analysis of protein
tyrosine kinase expression in melanocytic lesions by tissue
array. J Cutan Pathol 2003;30:539–47.
23. McGary EC, Onn A, Mills L, et al. Imatinib mesylate inhibits
platelet-derived growth factor receptor phosphorylation of
melanoma cells but does not affect tumorigenicity in vivo. J
Invest Dermatol 2004;122:400–5.
24. Redondo P, Lloret P, Andreu J, Inoges S. Imatinib mesylate in
cutaneous melanoma. J Invest Dermatol 2004;123:1208–9.
25. Eton O, Billings L, Kim K, et al. Phase II trial of imatinib
mesylate (STI-571) in metastatic melanoma (MM). Proc Am Soc
Clin Oncol 2004;22. [abstr 7528].
26. Schuchter L, Flaherty K, Davidson R, et al. Phase I trial of
imatinib and temozolomide in patients with metastatic mela-
noma. Proc Am Soc Clin Oncol 2004;22. [abstr 7572].
27. Ugurel S, Hildenbrand R, Zimpfer A, et al. Lack of clinical
efficacy of imatinib in metastatic melanoma. Br J Cancer
2005;92:1398–405.
28. Meric-Bernstam F, Mills GB. Mammalian target of rapamycin.
Semin Oncol 2004;31:10–7.
29. Margolin K, Longmate J, Baratta T, et al. CCI-779 in metastatic
melanoma. Cancer 2005;104:1045–8.
30. Egen JG, Kuhns MS, Allison JP. CTLA-4: new insights into its
biological function and use in tumor immunotherapy. Nat
Immunol 2002;3:611–8.
31. Mokyr MB, Kalinichenko T, Gorelik L, Bluestone JA. Realization
of the therapeutic potential of CTLA-4 blockade in low-dose
chemotherapy-treated tumor-bearing mice. Cancer Res
1998;58:5301–4.
32. Tchekmedyian S, Glasby J, Korman A, Keler T, Deo Y, Davis TA.
MDX-010 (human anti-CTLA4): a phase I trial in malignant
melanoma. Proc Am Soc Clin Oncol 2002;21. [abstr. 56].
33. Hersh EM, Weber J, Powderly J, et al. A phase II, randomized
multi-center study of MDX-010 alone or in combination with
dacarbazine (DTIC) in stage IV metastatic malignant melanoma.
Proc Am Soc Clin Oncol 2004;22. [abstr. 7511].
34. Fischkoff SA, Hersh E, Weber J, et al. Durable responses and long-
term progression-free survival observed in a phase II study of MDX-
010 alone or in combination with dacarbazine (DTIC) in metastatic
melanoma. Proc Am Soc Clin Oncol 2005;23. [abstr. 7525].
35. van Elsas A, Sutmuller RP, Hurwitz AA, et al. Elucidating the
autoimmune and antitumor effector mechanisms of a treat-
ment based on cytotoxic T lymphocyte antigen-4 blockade in
combination with a B16 melanoma vaccine: comparison of
prophylaxis and therapy. J Exp Med 2001;194:481–9.
36. Phan GQ, Yang JC, Sherry RM, et al. Cancer regression and
autoimmunity induced by cytotoxic T lymphocyte-associated
antigen 4 blockade in patients with metastatic melanoma. Proc
Natl Acad Sci USA 2003;100:8372–7.
37. Sanderson K, Scotland R, Lee P, et al. Autoimmunity in a phase
I trial of a fully human anti-cytotoxic T-lymphocyte antigen-4
monoclonal antibody with multiple melanoma peptides and
Montanide ISA 51 for patients with resected stages III and IV
melanoma. J Clin Oncol 2005;23:741–50.
38. Ribas A, Camacho LH, Lopez-Berestein G, et al. Antitumor
activity in melanoma and anti-self responses in a phase I trial
with the anti-cytotoxic T lymphocyte-associated antigen 4
monoclonal antibody CP-675,206. J Clin Oncol 2005;23:8968–77.
39. Reuben M, Lee BN, Li C, et al. Biologic and immunomodulatory
events after CTLA-4 blockade with ticilimumab in patients with
advanced malignant melanoma. Cancer 2006;106:2437–44.
40. Trefzer U, Kors C, Pelzer K, Walden P, Sterry W, O’Keefe E.
Preliminary results of a phase I trial of intralesional injection of
CPG 7909 in patients with basal cell carcinoma or melanoma.
Proc Am Soc Clin Oncol 2002;21. [abstr. 1902].
41. Wagner SN, Pashenkov M, Goess G, et al. TLR9-targeted CpG
immunostimulatory treatment of metastatic melanoma: A
Targeted therapies in melanoma
phase II trial with CpG 7909 (ProMune). Proc Am Soc Clin Oncol
2004;22. [abstr 7513].
42. Wagner S, Weber J, Redman B, et al. CPG 7909, a TLR9 Agonist
Immunomodulator in Metastatic Melanoma: A Randomized
Phase II Trial Comparing Two Doses and in Combination with
DTIC. Proc Am Soc Clin Oncol 2005;23. [abstr 7526].
43. Kruit WH, van Ojik H, Portielje J, Verloes R, Delire M, Stoter G.
Phase I/II study with CpG 7909 as adjuvant to vaccination with
MAGE-3 protein in patients with MAGE-3 positive tumors. Proc
Am Soc Clin Oncol 2002;21. [abstr 1854].
44. Speiser DE, Lienard D, Rufer N, et al. Rapid and strong human
CD8+ T cell responses to vaccination with peptide, IFA, and CpG
oligodeoxynucleotide 7909. J Clin Invest 2005;115:739–46.
45. Kim R, Emi M, Tanabe K, Toge T. Therapeutic potential of
antisense Bcl-2 as a chemosensitizer for cancer therapy. Cancer
2004;101:2491–502.
46. Selzer E, Schlagbauer-Wadl H, Okamoto I, Pehamberger H,
Potter R, Jansen B. Expression of Bcl-2 family members in
human melanocytes, in melanoma metastases and in melanoma
cell lines. Melanoma Res 1998;8:197–203.
47. Klasa RJ, Gillum AM, Klem RE, Frankel SR. Oblimersen Bcl-2
antisense: facilitating apoptosis in anticancer treatment. Anti-
sense Nucleic Acid Drug Dev 2002;12:193–213.
48. Jansen B, Schlagbauer-Wadl H, Brown BD, et al. Bcl-2 antisense
therapy chemosensitizes human melanoma in SCID mice. Nat
Med 1998;4:232–4.
49. Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitization
of malignant melanoma Bcl-2 antisense therapy. Lancet
2000;356:1728–33.
50. Millward MJ, Bedikian AY, Conry RM, Gore ME, Pehamberger HE,
Sterry W. Randomized multinational phase 3 trial of dacarba-
zine (DTIC) with or without Bcl-2 antisense (oblimersen sodium)
in patients (pts) with advanced malignant melanoma (MM):
Analysis of long-term survival. Proc Am Soc Clin Oncol 2004;22.
[abstr 7505].
51. Kirkwood JM, Bedikian AY, Millward MJ, et al. Long-term
survival results of a randomized multinational phase 3 trial of
dacarbazine (DTIC) with or without Bcl-2 antisense (oblimersen
sodium) in patients (pts) with advanced malignant melanoma
(MM). Proc Am Soc Clin Oncol 2005;23. [abstr 7506].
52. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic
switch. Nat Rev Cancer 2003;3:401–10.
53. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ,
Holash J. Vascular-specific growth factors and blood vessel
formation. Nature 2000;407:242–8.
54. Kerbel R, Folkman J. Clinical translation of angiogenesis
inhibitors. Nat Rev Cancer 2002;2:727–39.
55. Ugurel S, Rappl G, Tilgen W, Reinhold U. Increased serum
concentration of angiogenic factors in malignant melanoma
patients correlates with tumor progression and survival. J Clin
Oncol 2001;19:577–83.
56. Carson WE, Biber J, Shah N, et al. A phase 2 trial of a
recombinant humanized monoclonal anti-vascular endothelial
growth factor (VEGF) antibody in patients with malignant
melanoma. Proc Am Soc Clin Oncol 2003;22. [abstr 2873].
57. Peterson AC, Swiger S, Stadler WM, Medved M, Karczmar G,
Gajewski TF. Phase II study of the Flk-1 tyrosine kinase inhibitor
SU5416 in advanced melanoma. Clin Cancer Res 2004;10:
4048–54.
58. Albelda SM, Mette SA, Elder DE, Stewart RM, Damjanovich S,
Herlyn M, et al. Integrin distribution in malignant melanoma:
association of the beta 3 subunit with tumor progression.
Cancer Res 1990;50:6757–74.
59. Dome B, Raso E, Dobos J, et al. Parallel expression of
alphaIIbbeta3 and alphavbeta3 integrins in human melanoma
531
cells upregulates bFGF expression and promotes their angio-
genic phenotype. Int J Cancer 2005;116:27–35.
60. Hersey P, Sosman J, O’Day S, et al. A phase II, randomized,
open-label study evaluating the antitumor activity of MEDI-522,
a humanized monoclonal antibody directed against the human
alpha v beta 3 (avb3) integrin, ± dacarbazine (DTIC) in patients
with metastatic melanoma (MM). Proc Am Soc Clin Oncol
2005;23. [abstr 7507].
61. Joyce JA, Freeman C, Meyer-Morse N, et al. A functional
heparan sulfate mimetic implicates both heparanase and
heparan sulfate in tumor angiogenesis and invasion in a mouse
model of multistage cancer. Oncogene 2005;24:4037–51.
62. Tobu M, Ma Q, Iqbal O, et al. Comparative tissue factor
pathway inhibitor release potential of heparins. Clin Appl
Thromb Hemost 2005;11:37–47.
63. Thomson DB, Gonzalez R, Millward M, et al. A phase II trial of
PI-88, a novel antiangiogenic and antimetastatic heparanase
inhibitor, in patients with advanced melanoma. Proc Am Soc
Clin Oncol 2005;23. [abstr 7559].
64. Rajkumar SV, Richardson PG, Hideshima T, Anderson KC.
Proteasome inhibition as a novel therapeutic target in human
cancer. J Clin Oncol 2005;23:630–9.
65. Adams J, Palombella VJ, Sausville EA, et al. Proteasome
inhibitors: a novel class of potent and effective antitumour
agents. Cancer Res 1999;59:2615–22.
66. Amiri KI, Horton LW, LaFleur BJ, Sosman JA, Richmond A.
Augmenting chemosensitivity of malignant melanoma tumors
via proteasome inhibition: implication for bortezomib (VEL-
CADE, PS-341) as a therapeutic agent for malignant melanoma.
Cancer Res 2004;64:4912–8.
67. Markovic SN, Geyer SM, Dawkins F, et al. A phase II study of
bortezomib in the treatment of metastatic malignant mela-
noma. Cancer 2005;103:2584–9.
68. Brown PD, Giavazzi R. Matrix metalloproteinase inhibition: a
review of anti-tumour activity. Ann Oncol 1995;6:967–74.
69. Ramnath N, Creaven PJ. Matrix metalloproteinase inhibitor.
Curr Oncol Rep 2004;6:96–102.
70. Loffek S, Zigrino P, Angel P, Anwald B, Krieg T, Mauch C. High
invasive melanoma cells induce matrix metalloproteinase-1
synthesis in fibroblasts by interleukin-1 alpha and basic fibro-
blast growth factor-mediated mechanisms. J Invest Dermatol
2005;124:638–43.
71. Quirt I, Bodurth A, Lohmann R, et al. Phase II study of
marimastat (BB-2516) in malignant melanoma: a clinical and
tumor biopsy study of the National Cancer Institute of Canada
Clinical Trials Group. Invest New Drugs 2002;20:431–7.
72. Legha SS, Ring S, Eton O, et al. Development of a biochemo-
therapy regimen with concurrent administration of cisplatin,
vinblastine, dacarbazine, interferon alfa, and interleukin-2 for
patients with metastatic melanoma. J Clin Oncol
1998;16:1752–9.
73. Atkins MB, O’Boyle KR, Sosman JA, et al. Multiinstitutional
phase II trial of intensive combination chemoimmunotherapy
for metastatic melanoma. J Clin Oncol 1994;12: 1553–60.
74. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Prospec-
tive randomized trial of the treatment of patients with
metastatic melanoma using chemotherapy with cisplatin,
dacarbazine, and tamoxifen alone or in combination with
interleukin-2 and interferon alfa-2b. J Clin Oncol 1999;17:
968–75.
75. Del Vecchio M, Bajetta E, Nova P, et al. Multicenter phase III
randomized trial of polychemotherapy (CVD regimen) versus
the same chemotherapy (CT) plus subcutaneous interleukin-2
and interferon-{alpha}2b in metastatic melanoma. Ann Oncol
2006;17:571–7.