Targeting Oncogenic Drivers and the Immune System

in Melanoma
Grant A. McArthur and Antoni Ribas
See accompanying article on page 482
Grant A. McArthur, Peter MacCallum
Cancer Centre, East Melbourne; Univer-
sity of Melbourne, Parkville; St.
Vincents Hospital, University of
Melbourne, Fitzroy, Victoria, Australia;
and Antoni Ribas, University of Califor-
nia at Los Angeles (UCLA) and the
Jonsson Comprehensive Cancer Center
at UCLA, Los Angeles, CA.
Published online ahead of print at
www.jco.org on December 17, 2012.
Supported by project Grant No.
APP1002655 and a Research Fellow-
ship from the National Health and
Medical Research Council of Australia,
by the Victorian Cancer Agency, and by
the Cancer Council of Victoria Sir
Edward Weary Dunlop Clinical
Research Fellowship (G.A.M.); also
supported by The Seaver Institute, the
Louise Belley and Richard Schnarr
Fund, the Wesley Coyle Memorial
Fund, the Garcia-Corsini Family Fund,
the Bila Alon Hacker Memorial Fund,
the Fred L. Hartley Family Foundation,
the Ruby Family Foundation, the Jons-
son Cancer Center Foundation, the Eli
and Edythe Broad Center of Regenera-
tive Medicine and Stem Cell Research
at University of California, Los Angeles
(UCLA), and the California Institute of
Technology of California-UCLA Joint
Center for Translational Medicine (A.R.).
Authors disclosures of potential con-
icts of interest and author contribu-
tions are found at the end of this
article.
Corresponding author: Grant A. McArthur,
Division of Cancer Medicine and Research,
Peter MacCallum Cancer Centre, St.
Andrews Place, East Melbourne, Victoria
3002, Australia; e-mail: grant.mcarthur@
petermac.org.
2012 by American Society of Clinical
Oncology
0732-183X/13/3104-499/$20.00
DOI: 10.1200/JCO.2012.45.5568
A B S T R A C T
Melanoma is one of the most common cancers in Western countries but has deed the trend of
reductions in age-adjusted mortality observed in most other cancers in recent years. Biologically,
melanoma is characterized by a high propensity to metastasize at low tumor volumes necessitat-
ing the need for effective drug therapies to support efforts in prevention and early detection for
reducing mortality. Efforts to study the clinical biology of melanoma have led to a new
understanding of the disease, with genomic studies identifying several targetable oncogenes, in
particular the protein kinases BRAF and KIT. Biologic studies have also identied a variety of
immunologic targets, including the programmed death 1 (PD-1) and cytotoxic T-cell lymphocyte
associated antigen 4 (CTLA-4) inhibitory molecules expressed on T lymphocytes. After several
decades of clinical trials that failed to demonstrate improvement in overall survival in patients with
advanced melanoma, small molecule inhibitors of BRAF or MEK and inhibition of CTLA-4 can
improve survival in patients with advanced disease. These early clinical studies have provided a
great opportunity to improve mortality in melanoma with the signicant potential of combinations
of signaling inhibitors or signaling inhibitors combined with immunologic agents, particularly when
used in the adjuvant setting, and to transform the care of patients with this most challenging
of cancers.
J Clin Oncol 31:499-506. 2012 by American Society of Clinical Oncology
INTRODUCTION
Melanoma is the fth most common cancer in the
UnitedStates
1
andthe fourthmost commoninAus-
tralia.
2
Whereas the age-adjusted rates of cancer
deaths have beenfalling for most malignancies, mel-
anoma has deed the trend with no improvements
over the last two decades (Fig 1). Indeed the rates of
diagnosis are steadily increasing, particularly in
males older than age 60 years.
2,3
Given the clear role
of ultraviolet (UV) radiationas a carcinogeninmel-
anoma, public health strategies to reduce UV expo-
sure have begun to make inroads in preventing this
increase innewmelanoma diagnoses, particularly in
younger people.
2,3
Despite these efforts, the number
of deaths frommelanoma is not falling.
2,3
THE CLINICAL BIOLOGY OF MELANOMA
Pioneering work by Curtin et al
4,5
demonstrated
that the anatomic site of primary melanoma is
associated with distinct features in the melanoma
genomethat appear toreect theUVexposureof the
anatomic location. For example, melanomas arising
on mucosal surfaces of the upper respiratory tract
or anogenital regions that are not exposed directly
to UV have low rates of BRAF mutations (3% to
14%)
4,6
and higher rates of KIT mutations (10% to
39%).
4,7
In contrast, melanomas on the trunk that
are associated with intermittent UV exposure
have the opposite pattern of mutations, with ap-
proximately 70% having mutations in BRAF or
NRAS
4,7,8
and KIT mutations being found in less
than 2%.
4,9
Interestingly, melanomas arising in
anatomic locations that reect chronic UV expo-
sure on the head and neck also show distinct
patterns of mutations.
8
Therefore, the histologic
classication of melanomas according to site of
origincutaneous nonacral, cutaneous acral, and
mucosal melanomaspredicts the frequency of
mutations that are associated with benet fromnew
targetedtherapies. What is less clear is whether this is
secondary to inherent biologic differences in mela-
nocytes at these sites or the direct effect of the UV
carcinogen on specic base pair substitutions in
these commonly mutated genes.
Clinical outcomes inmelanoma are directly re-
latedtoclassical TNMstaging. Thesedatahaveledto
JOURNAL OF CLINICAL ONCOLOGY
B I O L O G Y O F N E O P L A S I A
VOLUME 31 NUMBER 4 FEBRUARY 1 2013
2012 by American Society of Clinical Oncology 499
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
astagingsystembasedonanalyses of morethan30,000patients, which
provides a useful clinical tool for deciding on patient follow-up, sur-
veillance techniques, and adjuvant therapies.
10
One biologic property
of melanoma that distinguishes this malignancy from others is the
propensity for metastatic tumor spreadat small tumor sizes (staging is
measuredinmillimeters inmelanoma, but inmost other histologies, it
is measured in centimeters). For example, a 2.01- to 4-mm-thick
melanoma that is ulcerated has a 32% chance of becoming lethal at 5
years.
10
Intriguingly, melanomas and normal melanocytes express
high levels of transcriptional regulators of a highly invasive migratory
cellular phenotype, notably, the transcription factor SLUG.
11
This
developmental property of melanocytes may explainthe propensity of
melanoma to metastasize at such small tumor volumes and has im-
portant implications for the relative importance of therapeutic at-
tempts to improve survival.
This biologic property of melanomas has led to intensive
efforts to nd drug therapies that eradicate melanoma cells that
have metastasized to distant sites. Unlike other malignancies in
which DNA-damaging agents have improved survival in both the
advanced and adjuvant setting, three decades of clinical trials re-
sulting in more than 1,500 publications have not resulted in any
traditional cytotoxic agents that could improve overall survival in
patients with advanced melanoma. Dacarbazine, approved by the
US Food and Drug Administration in 1975, has been the mainstay
of cytotoxic therapy, yet it has never been demonstrated to improve
overall survival. Multicenteredclinical trials that comparedacarbazine
with temozolomide, cisplatin based-regimens, nitrosoureas such as
fotemustine, and combinations of biologic agents such as interferon
andinterleukin-2 have not shownbenets inoverall survival.
12-16
The
reason for the failure of melanoma to respond to classic cytotoxic
therapies sufciently to improve survival is not clear, but it may in-
volve inherent resistance tothe inductionof apoptosis or the ability to
repair andsurvive DNAdamage that may relate tothe highmutagenic
environment of UV exposure during the early stages of melanoma
development. These frustrating and negative results with cytotoxic
agents set the scene for investigation of novel therapies based on the
genome biology of melanoma andthe ability of melanoma torespond
to manipulation of the immune system.
GENOMIC LANDSCAPE OF MELANOMA
Recent efforts to understand the molecular pathogenesis of mela-
noma by detailed characterization of the melanoma genome have
provided a detailed roadmap of the underlying drivers of the dis-
ease. Initial studies
17,18
showed mutations in classic oncogenes
such as RAS proteins
19-21
and mutations and deletion of classic
tumor suppressors such as CDKN2A, which encodes the cyclin-
dependent kinase 4 (CDK4) inhibitor p16INK4A. Following a
screen for mutations in protein kinases, Davies et al
22
identied
mutations in BRAF in 50% of melanomas that have helped dene
some clear patterns of genomic change in melanoma as summa-
rized in Figure 2. First and foremost, approximately 65% of mela-
nomas have mutations affecting the RAS/RAF/MEK/ERK
pathway.
23-25
Second, genetic studies characterizing somatic mu-
tations in melanoma cells and germline mutations in familial mel-
anoma identied frequent genetic events activating CDK4, a
central mediator of progression from the G1 to S phases of the cell
cycle
26,27
involving cyclin D1 (CCND1), CDK4 itself, or CDKN2A
in more than 50% of melanomas.
5,28
Third, mutation or, most
frequently, deletion of the PTEN tumor suppressor is found in ap-
proximately 30%of patients with melanoma and notably in approxi-
mately 40%of BRAF-mutant melanomas.
28,29
However, mutation or
amplication of other components of the PTEN/PI3K/AKT/mTOR
pathway appear infrequent in melanoma.
30,31
Finally, mutations or
deletion of p53, or amplication of the p53 inhibitor MDM2, are
infrequent in melanoma.
32-34
However, a recent study
35
has iden-
tied high levels of expression, but not gene amplication, of the
p53 inhibitor MDM4 in melanoma that may explain in part the
resistance of melanoma cells to cytotoxic drugs that induce apo-
ptosis by activating p53.
The advent of next-generation sequencing techniques is now
allowing a systematic analysis of sequence variants and copy num-
ber changes across the entire melanoma genome. One clear con-
clusion of the early data is that melanoma contains signicantly
more sequence variations compared with most other cancers. For
example, melanomas have 15 times more mutations per mb of
DNA than colorectal cancer and four times more than lung
cancer.
36
Because a high proportion of these mutations are
cytosine-to-thymine and guanine-to-adenine substitutions, typi-
cal of UV-induced thymine dimers, it is highly likely that the high
rate of sequence variants in melanoma is as a result of the role of
UV radiation as the principal carcinogen in the disease.
37
These
observations may also have clinical importance because the degree
of genetic heterogeneity between cells in melanoma may mean that
resistance to systemic therapies might develop at a higher rate
compared with other cancers. These next-generation sequencing
studies have identied several recurrent mutations in known and
novel genes, including kinases such as EPHA3 and ERBB4,
38
sig-
naling molecules such as MAP3K5, MAP3K9,
39
and PREX2 a reg-
ulator of PTEN and the PI3K pathway.
40
Importantly, to date,
none of these are found at frequencies greater than 15%. These
ongoing efforts, particularly when coupled with functional studies
in melanocytes, melanoma cell lines, and mouse models of mela-
noma may go on to dene a new series of therapeutic targets in
the disease.
All cancers, Australia
All cancers, USA
Melanoma, Australia
Melanoma, USA
A
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Fig 1. Age-adjusted total cancer and melanoma mortality in the United States
(USA) and Australia, 1982 to 2007.
2,2a
McArthur and Ribas
500 2012 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY
TARGETING ONCOGENIC DRIVERS IN MELANOMA
The nding that approximately 65% of melanomas contain muta-
tions that activate the RAS/RAF/MEK/ERK pathway made the RAS/
RAF/MEK/ERK pathway a prime therapeutic target in melanoma.
Early results were disappointing with the type II RAF inhibitor
sorafenib
41
and the MEK inhibitors selumetinib
42
and PD0325901,
43
showing low rates of response independent of BRAF or NRAS muta-
tion status. However, it was not clear that these agents were able to
sufciently inhibit the pathway. Moreover, prospective selection of
patients with mutations that activate the pathway was not performed,
thus preventing the robust assessment of their efcacy in BRAF- and
NRAS-mutant melanoma. The recent development of type I BRAF
inhibitors that show high potency for the active form of the kinase as
seen with the BRAF V600E mutation and the development in parallel
of MEKinhibitors that allowmore sustainable inhibition of the path-
way have led to dramatic clinical results.
The rst type I BRAF inhibitor to enter clinical development,
vemurafenib, has consistently shown more than 50% conrmed re-
sponse rates in phase I, II, III, and postmarketing clinical trials.
44-46
The molecule potently inhibits the RAF/MEK/ERKpathway in mela-
noma tissue at the maximum-tolerated dose, resulting in profound
inhibition of glucose uptake as detected by [
18
F]uorodeoxyglucose-
positron emission tomography.
47,48
Complete responses are uncom-
mon, occurring in approximately 5% of patients
44-46
; however, this
agent dramaticallyimproves overall survival inpatients withadvanced
melanoma containing the BRAF V600E mutation when compared
withdacarbazine.
46
Medianoverall survival inpatients withadvanced
melanoma treated with single-agent vemurafenib is 14 to 16
months,
45,46
a signicant improvement over the mediansurvival seen
with cytotoxic agents. Nonetheless, progression occurs in the vast
majority of patients with a median progression-free survival of 5 to 7
months, indicating there is a clear needtodevelopfurther strategies to
improve clinical outcomes.
45,46
Asecond type I BRAF inhibitor, dab-
rafenib, has also entered clinical development and, like vemurafenib,
induces conrmed responses in more than 50%of patients.
49
There has been a concerted effort to investigate the mecha-
nisms of acquired resistance to vemurafenib. These studies have
been highly successful and have resulted in the identication of
some clear mechanisms of resistance. Unlike BCR-ABL-, KIT-, or
EGFR-mutant cancers, mutations in the target that prevent drug
binding have not been found, possibly because any structural change
in the kinase that prevents drug binding also reduces activity of the
mutant kinase. Strikingly, more than 50% of patients progressing on
vemurafenib reactivate the RAF/MEK/ERK pathway
50,51
by either
acquiring mutations in NRAS
52,53
that promote formation of active
BRAF:CRAF heterodimers to phosphorylate MEK,
53-55
by amplica-
tion of BRAF itself,
56
by expression of splice-variants of BRAF that
formactive BRAF dimers,
57
by overexpression of the MEK-activating
kinase COT1,
58
or possibly by mutations inMEK.
59,60
However, some
patients do not seem to reactivate the RAF/MEK/ERK pathway and
instead, the pathway is bypassed because of activation of receptor
tyrosine kinases, including PDGFR,
52
IGF1R,
61
FGFR3,
62
and
MET
63,64
with associated activation of the PI3Kpathway.
Interestingly, although RAF inhibitors are generally well tol-
erated, vemurafenib, dabrafenib, and the type II inhibitors
sorafenib and XL281 induce hyperproliferative phenotypes in the
epidermis resulting in keratoderma, keratotosis piliaris, induction
of papillomas, and low-grade squamous neoplastic lesions with
features of keratoacanthomas and squamous cell carcino-
mas.
44,49,65,66
The hyperproliferative phenotypes are believed to be
the result of paradoxical activation of the pathway
53-55
in which the
BRAF inhibitor promotes MEKactivation in normal cells that have
activation of upstream receptors or signaling molecules.
65,67,68
Consistent with this hypothesis, molecular studies have shown
frequent HRAS mutations in the neoplastic lesions suggesting that
MEK
ERK
ETS
CCND1
CDK4
NRAS 15%
BRAF 50%
50%
Melanoma Cell
T lymphocyte
Antigen-Presenting
Cell
PD-L1
PD-1
B7
CTLA-4
TCR
Antigen
MHC
-
-
RTK
p21
PUMA p53
MDM4
70%
TCR
Antigen
MHC
mTOR
PI3K
AKT
S6K 4EBP1
PTEN 40%
CDKN2A Fig 2. Therapeutic biology of melanoma.
Schematic representation of frequent
genomic changes in melanoma. Numbers
represent percentage of patients who
have altered protein expression or muta-
tion. Melanoma cells interact with T lym-
phocytes, where the activity of the T cells
is controlled by a series of regulatory
molecular interactions, including those be-
tween B7 and cytotoxic T-cell lymphocyte
associated antigen 4 (CTLA-4) and between
programmed death 1 (PD-1) and its main
ligand, PD-L1. CCND1, cyclin D1; CDK4,
cyclin-dependent kinase 4; CDKN2A,
p16INK4A inhibitor of CDK4; MHC, major his-
tocompatibility molecule; TCR, T-cell receptor.
New Approaches to Melanoma
www.jco.org 2012 by American Society of Clinical Oncology 501
the RAF inhibitors induce the formation of BRAF:CRAF het-
erodimers to activate the RAF/MEK/ERK pathway and drive pro-
liferation.
67,68
These studies also suggest that the RAF inhibitors
could promote proliferation of other neoplastic lesions, particu-
larly those with RAS mutations, indicating that ongoing safety
studies to monitor for development of secondary neoplasms are a
high priority.
Recently, several MEK inhibitors with more favorable pharma-
cokinetic and pharmacodynamic properties compared with the early
MEK inhibitors have entered clinical development. Trametinib has
shown conrmed response rates of more than 20% in BRAF-mutant
melanoma, and a recent phase III trial has shown substantial
progression-free and overall survival benet compared with dacarba-
zine or paclitaxel.
69
Like most MEK inhibitors, dose is limited by
diarrhea andskinrash, andsome patients developocular toxicity with
reversible central serous retinopathy.
70,71
It is intriguing to speculate
that, given that MEK is the major substrate of BRAF, if higher doses
could be tolerated, response rates would approach those seen with
type I BRAF inhibitors. It is perhaps the absence of paradoxical acti-
vation with MEK inhibitors that limits maximum-tolerated dose of
these agents. Importantly, emerging data show that the combination
of BRAF and MEK inhibitors can reduce the single-agent toxicity of
each agent, probably because the MEK inhibitors reduce the down-
streamconsequences of paradoxical activation and, in turn, the para-
doxical activationreduces theconsequences of MEKinhibition.
71
This
combination approach is particularly promising because it may in-
hibit the pathway following the acquisition of some of the genomic
events that induce resistance to single-agent type I BRAF inhibitors
and more profoundly inhibit the pathway in melanoma, with BRAF
mutations potentially increasing progression-free survival.
72
The oncogenes KIT and NRAS are also possible drug targets in
melanoma. Melanomas with KIT mutations and/or amplication of
KITmay respondtoKITinhibitors,
73-76
andregistrationstudies of the
KIT inhibitors nilotinib and masitinib are under way. In contrast to
BRAF, the early results of trials of KIT inhibitors such as imatinib in
KIT-mutant melanoma appear to be more complex because some
melanomas do not respond despite containing KIT mutations,
73,74
suggesting that KIT even when mutated is not always a driver of
melanoma proliferation and survival. Interestingly, patients with the
most common KIT mutations in melanoma at position L576 and
K642 may obtain greatest benet from this therapeutic approach.
73
NRASis mutatedinapproximately 15%of melanoma and, like BRAF,
induces an oncogene-dependent state in melanoma cells.
22,77
Several
approaches are underway to attempt to target NRAS, including the
combination of PI3Kand MEKinhibition and MEKinhibition alone
(reviewed in Kelleher and McArthur
78
). However, it is unclear
whether these approaches will inhibit the MEKpathway sufciently to
achieve clinical benet at the maximum-tolerated doses, and alterna-
tive approaches may be necessary.
IMMUNOTHERAPIES COMING OF AGE
Melanoma has long been considered an immunogenic cancer on the
basis of anecdotal spontaneous responses and a low frequency of
tumor responses when treated with a variety of immune-stimulating
agents. The inherent immunogenicity of melanoma may be related to
its higher mutation rate compared with other cancers resulting it its
being mostly a carcinogen-induced cancer.
79
The high frequency of
point mutations in melanoma predicts that there may be plenty of
unique neoantigens resulting from mutated proteins, which can be
presented by major histocompatibility molecules and recognized by
the immune systemonthe cancer cells.
80
Inaddition, vitiligo is a clear
example of spontaneous breaking of tolerance to self-tissues through
an immune response to melanocytes resulting from T lymphocytes
recognizing mainly melanocyte-restricted pigmentation pathway
proteins. This ability to break tolerance to lineage-specic proteins
may result in the generation of immune responses to melanomas
being made easier.
On the basis of observations of melanoma immunogenicity,
multiple approaches have beenusedover the past 100 years toharness
animmune response tomelanoma. Initial attempts were basedonthe
intratumoral injection of inactivated bacteria such as BCG to induce
inammation and hopefully result in melanoma antigen cross-
presentation. Other approaches included inactivated tumor cells ad-
ministered as vaccines, in some cases, after genetic modication to
increase their immunogenicity. Intratumoral injectionof viruses with
genetic alterations to improve immunogenicity has also been tested.
Other attempts at turning on an immune response to melanoma by
using active immunotherapy strategies have included the administra-
tion of dendritic cell vaccines, a series of immune-stimulating cyto-
kines such as interferons and interleukins, or the administration of
immune-activating monoclonal antibodies to activate signaling from
costimulatory molecules such as CD40, CD137, or OX40 (Fig 3).
These approaches have resulted in occasional tumor responses, usu-
ally in less than 10% of the treated population, with the main feature
being that once they happen, they tend to be durable and can last
for years.
81
Approaches aimed at turning on an immune response to cancer
have been limited by the multiple mechanisms by which the immune
system avoids overactivation against self-antigens. The seminal work
by Krummel and Allison
81a
on blocking the negative immune regula-
tory role of the cytotoxic T-cell lymphocyteassociated antigen 4
(CTLA-4) provided a different approach to turning on the immune
system to cancer. There are several breaks to an immune response
recognizing self-antigens, and CTLA-4 is a negative costimulatory
molecule that competes with the positive costimulatory receptor
CD28 on activated T cells. Inhibiting CTLA-4 with blocking mono-
clonal antibodies allows T cells to proliferate and maintain activation
(Fig 2). This approachhas beenexploitedinthe clinic by using mono-
clonal antibodies that block CTLA-4. The fully human immunoglob-
ulin G1 (IgG1) antiCTLA-4 antibody ipilimumab demonstrated for
the rst time an improvement in overall survival in patients with
metastatic melanoma in two randomized clinical trials, one compar-
ingipilimumabwithapeptidevaccineandtheother comparingit with
dacarbazine.
82,83
These reproducible benets were most notable inthe
10%difference inoverall survival betweenthe ipilimumab-containing
arms and the control arms at 2 to 3 years of follow-up, suggesting that
ipilimumab induces a low-frequency but durable benet in treating
patients with metastatic melanoma. This is at the risk of 15% to 20%
incidence of clinically signicant inammatory and autoimmune ad-
verseeffects, most frequentlytothegut andendocrineglands (thyroid,
pituitary). Another antiCTLA-4 antibody has been tested in mela-
noma, the fully humanIgG2 tremelimumab. However, a randomized
McArthur and Ribas
502 2012 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY
trial did not demonstrate an improvement in overall survival over a
dacarbazine/temozolomide control group, which could be the result
of unintended crossover to ipilimumab in the control arm (Ribas,
manuscript submitted for publication).
The programmeddeath1(PD-1) receptor is another negative regu-
lator of T cells. As opposed to CTLA-4, PD-1 is expressed on chronic
antigenexposure, andits ligands canbe expressedby tumor cells directly.
Therefore, it is likelythat blockingPD-1allows amoredirect activationof
cancer-specic T cells.
84
The initial clinical experience with antibodies
blocking bothPD-1 andits mainligand, PD-L1 suggests that this may be
the case. Administration of nivolumab, a fully human IgG4 antibody
(formerlyBMS-936558andMDX1106)blockingPD-1, resultedinobjec-
tive responses in approximately 30% of patients across a wide dose
range in phase I testing, most responses being durable beyond 1 year.
The incidence of serious (grade 3 to 4) adverse effects was at a range
similar to that with CTLA-4blocking antibodies, but most were less
clinically signicant with the exception of pneumonitis, which led to
death as a result of toxicity.
85
Blocking the ligand PD-L1 with the fully
humanIgG4antibodyBMS-936559resultedinaslightlylower frequency
of objective tumor responses and also fewer adverse effects in phase I
testing.
86
Theseinitial experiences, togetherwiththecurrent initial clinical
testing of a series of other antibodies and blocking constructs to the
PD-1/PD-L1axis, provideanimpetus for thecontinuedclinical testingof
these highly active immunotherapies for melanoma.
Theresult of all of thetumor immunotherapyapproaches shouldbe
intratumoral inltrationbyactivatedTcellswithcancer-speciccytotoxic
activity. Adoptive cell transfer (ACT) therapies are based on generating
large quantities of activated T cells in the laboratory and infusing them
back intopatients. The Tcells for ACTcanbe harvestedfromthe tumor,
which is enriched for T cells with tumor antigen specicities but is non-
functional because of the immune-suppressive tumor microenviron-
ment. These tumor-inltrating lymphocytes (TILs) harvested from
tumors can be expanded and fully activated ex vivo. Their further in
vivo expansion on reinfusion into patients is facilitated by condition-
ing the patient rst with a chemotherapy regimen that partially de-
pletes endogenous lymphocytes, providing a proliferative advantage
for the reinfused TILs.
87
Activated T cells for ACT can also be ex-
panded from peripheral blood on repetitive ex vivo antigen expo-
sure,
88
by genetic modication that provides T cells with transgenic
T-cell receptors specic for melanoma antigens, or with chimeric
antigenreceptors generatedby the fusionof tumor-specic antibodies
with intracellular signaling molecules to activate T cells (Fig 3).
89
These ACTapproaches are advancing through their clinical tests, and
they currently have the most reproducible durable response rate in
melanoma with TIL-based ACT.
90
THE FUTURE OF MELANOMA TREATMENT WITH
COMBINATION THERAPIES
The increasingly rened understanding of howto therapeutically tar-
get oncogenic melanoma signaling and induce cytotoxic T-cell im-
mune responses tomelanoma, together withthe clinical availability of
highly targeted agents, provides great promise to combinatorial ap-
proaches. MEK inhibitors, when combined with BRAF inhibitors,
result in dual blockade of the oncogenic BRAF
V600
signaling through
the MAPK pathway.
91
At the same time, this combination may pre-
vent the development of some of the acquired resistance mechanisms
to single-agent BRAF inhibitors that are mediated by the reactiva-
tion of MEK signaling.
52
This combination is rapidly advancing
throughclinical tests, andthe early results are encouraging. Combina-
tions of BRAF inhibitors with inhibitors of the other major oncogenic
signal transduction pathway (the PI3K/AKT/mTOR pathway) could
prevent or treat nonMAPK-reactivating acquired resistance
Active immunotherapy
ACT immunotherapy
OX40
CD137
CD40
PD1
CTLA4
TCR or CAR
genetic engineering T-cell cloning
Peptide vaccine
DC vaccine
Genetic vaccine
IL-2
IFN
IL-15
IL-21
Fig 3. Immunotherapeutic approaches to
treating melanoma. Active immunother-
apy with cytokines, vaccines, and antibod-
ies targeting positive regulatory molecules
(CD137, CD40, and OX40) and negative
regulatory molecules (programmed death
1 [PD-1] and cytotoxic T-cell lymphocyte
associated antigen 4 [CTLA-4]). Adoptive
cell transfer (ACT) therapies by T-cell clon-
ing or genetically engineered T cells with
T-cell receptors (TCRs) or chimeric antigen
receptors (CARs) to melanoma antigens.
DC, dendritic cell; IFN, interferon; IL-2,
interleukin 2.
New Approaches to Melanoma
www.jco.org 2012 by American Society of Clinical Oncology 503
mechanisms,
61,92-94
and this combination will be tested in the clinic.
However, it is unlikely that the wide therapeutic window afforded by
the combination of BRAF and MEK inhibitors will be maintained.
Other combinatorial targeted therapies building on the success of
BRAF inhibitors that may enter clinical testing include epigenetic
therapies and apoptosis-enhancing agents with the goal of increasing
cell death induced by BRAF inhibitors. Since no drug currently has
demonstrated ability to directly inhibit activating NRAS
Q61
muta-
tions, combination of targeted therapies that block downstream
NRAS oncogenic signaling may provide benets to patients (eg, the
combinedinhibitionof MEKwithPI3Kor AKTinhibitors). However,
these combinations are also likely to have a narrow therapeutic win-
dow since these two pathways are of great importance for multiple
physiologic cellular functions.
Immunotherapy combinations also provide great promise for ad-
vancing melanoma treatment. Immune checkpoint blockade with
CTLA-4orPD-1canbecombinedwithimmune-stimulatingapproaches
that were previously limited by negative immune regulatory control.
Doubleimmunecheckpoint blockadewithipilimumabandnivolumabis
currentlyinphaseIclinical testing, andif theexpectedincreasedincidence
of autoimmune adverse effects is manageable, it may provide a pow-
erful combination for unleashing immune responses to melanoma.
The CTLA-4 and PD-1 blocking approaches are also amenable to
combination with ACT approaches with the goal of maintaining the
cytotoxic functions of adoptively transferred antitumor lymphocytes.
Combination of highly targeted BRAF inhibitors with immuno-
therapy may allowmergingthe best features of bothmodes of therapy:
the highfrequency tumor responses of BRAF inhibitors andthe dura-
ble tumor responses with immunotherapies.
95,96
Preclinical and early
clinical evidence suggest that such combinations are feasible since
BRAF inhibitors do not negatively impact lymphocyte functions,
97,98
and Tcells have been found to inltrate regressing tumors in patients
treated with BRAF inhibitors.
99
Combinations of the new active therapies for melanoma with
standard forms of therapy are also likely to be tested in the clinic.
Ipilimumab in combination with radiation therapy may induce an
abscopal effect, wherein the localized cytotoxic effects of radiation
therapy could function as an endogenous vaccination to target the
immune response to nonirradiated metastatic lesions.
100
Combina-
tions of immunotherapies withcytotoxic chemotherapies, commonly
known as biochemotherapy, have long been pursued in patients with
advanced melanoma. After a long series of randomized clinical trials
with negative results,
101
it is unlikely that such combinations benet
patients unless there is animprovedapproachtotheir sequencing and
a way to avoid how cytotoxics could negate immune responses to
cancer.
102
Combining chemotherapy with BRAF inhibitors may have
a similar lack of mechanistic potential since BRAF inhibitors are po-
tent blockers of cell cycle progression, which is usually required for
cytotoxic chemotherapy to kill cancer cells. However, such a combi-
nationmay be warrantedinthe setting of acquiredresistance toBRAF
inhibitors, where melanoma cells progress through the cell cycle de-
spite continued BRAF
V600
blockade.
PROSPECTS FOR REDUCING MELANOMA DEATHS
Melanoma is a malignancy characterizedby a highpotential tometas-
tasize at a relatively small size of the primary tumor. Althoughpreven-
tion and early detection of primary melanomas are vital for reducing
the burden of the disease, we suggest that because the high metastatic
potential has a large impact on the number of melanoma deaths,
systemic therapies that can signicantly extend survival will be re-
quired. There are unprecedented opportunities for achieving this vi-
sion. The high genomic complexity of melanoma likely will create
challenges to extending survival with targeted therapies alone in the
advanced disease setting because of the propensity of the disease to
cause acquired resistance. However, the combination therapies that
enhance the death of melanoma cells by engaging an antitumor im-
mune response while also inhibiting oncogene function may over-
come these barriers. Importantly, the opportunity to treat patients
at high risk of relapse in the adjuvant setting may also break down
these barriers. These opportunities create a new outlook for treat-
ing this disease that has for so long beenone of the major challenges
in oncology.
AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
Although all authors completed the disclosure declaration, the following
author(s) and/or an authors immediate family member(s) indicated a
nancial or other interest that is relevant to the subject matter under
consideration in this article. Certain relationships marked with a U are
those for which no compensation was received; those relationships marked
with a C were compensated. For a detailed description of the disclosure
categories, or for more information about ASCOs conict of interest policy,
please refer to the Author Disclosure Declaration and the Disclosures of
Potential Conicts of Interest section in Information for Contributors.
Employment or Leadership Position: None Consultant or Advisory
Role: Grant A. McArthur, Bristol-Myers Squibb (U), GlaxoSmithKline
(U), Millennium Pharmaceuticals (U), Plexxikon (U), Roche-Genentech
(U); Antoni Ribas, Bristol-Myers Squibb (C), GlaxoSmithKline (C),
Millennium Pharmaceuticals (C), Novartis (C), Roche-Genentech (C)
Stock Ownership: None Honoraria: None Research Funding: Grant A.
McArthur, Millennium Pharmaceuticals, Novartis, Pzer Expert
Testimony: None Other Remuneration: None
AUTHOR CONTRIBUTIONS
Conception and design: Grant A. McArthur, Antoni Ribas
Administrative support: Antoni Ribas
Data analysis and interpretation: Grant A. McArthur, Antoni Ribas
Manuscript writing: All authors
Final approval of manuscript: All authors
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