Mechanisms of Disease Melanoma

The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 51
review article
mechanisms of disease
Melanoma
Arlo J. Miller, M.D., Ph.D., and Martin C. Mihm, Jr., M.D.
From the Dermatopathology Unit, Massa-
chusetts General Hospital, and Harvard
Medical School — both in Boston. Ad-
dress reprint requests to Dr. Mihm at the
Department of Dermatopathology, Massa-
chusetts General Hospital, 55 Fruit St.,
Warren 827, Boston, MA 02114.
N Engl J Med 2006;355:51-65.
Copyright © 2006 Massachusetts Medical Society.
A
lthough melanoma accounts for only 4 percent of all derma-
tologic cancers, it is responsible for 80 percent of deaths from skin cancer;
only 14 percent of patients with metastatic melanoma survive for five years.
1

The intractability of advanced melanoma shows how much we have to learn about
the changes that facilitate the vertical growth and deep invasion of melanoma and
about the mechanisms that block the effectiveness of chemotherapy.
The Clark model of the progression of melanoma emphasizes the stepwise trans-
formation of melanocytes to melanoma (Fig. 1). The model depicts the proliferation
of melanocytes in the process of forming nevi and the subsequent development of
dysplasia, hyperplasia, invasion, and metastasis.
2
Numerous molecular events, many
of them revealed by genomic
3
and proteomic
4
methods, have been associated with
the development of melanoma. But rather than catalogue all the molecular lesions
in this tumor, we will focus on connections between molecular pathways and risk
factors for melanoma, the different steps of neoplastic transformation, and the
patterns of molecular changes in melanoma (Fig. 2).
Environmental and Genetic Interactions
Risk Factors
The strongest risk factors for melanoma are a family history of melanoma, multiple
benign or atypical nevi, and a previous melanoma. Immunosuppression, sun sensi-
tivity, and exposure to ultraviolet radiation are additional risk factors. Each of these
risk factors corresponds to a genetic predisposition or an environmental stressor
that contributes to the genesis of melanoma. Each factor is understood to various
degrees at a molecular level. For example, 25 to 40 percent of the members of
melanoma-prone families have mutations in cyclin-dependent kinase inhibitor 2A
(CDKN2A)
5
(Table 1 lists all genes mentioned in this article), and a few rare kindreds
have mutations in cyclin-dependent kinase 4 (CDK4). There is a rational basis for a
link between susceptibility to melanoma and a mutation in CDKN2A or CDK4 since
both are tumor-suppressor genes. They will be discussed later in the context of
disease progression. In addition, sensitivity to ultraviolet light is associated with a
polymorphic genetic determinant that affects susceptibility to melanoma, thereby
highlighting an important genetic–environmental interaction.
Photosensitivity, Tanning, and Melanoma
The effect of exposure to ultraviolet light is governed by variations in particular
genes (polymorphisms) that affect both the defensive response of the skin to ultra-
violet light and the risk of melanoma. Ultraviolet radiation causes genetic changes
in the skin, impairs cutaneous immune function, increases the local production of
growth factors, and induces the formation of DNA-damaging reactive oxygen species
that affect keratinocytes and melanocytes.
6,7
The tanning response is a defensive
measure in which melanocytes synthesize melanin and transfer it to keratinocytes,
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 52
where it absorbs and dissipates ultraviolet energy.
7

Clinically, variations in pigmentation and the tan-
ning response to ultraviolet light are associated
with variations in susceptibility to melanoma.
8,9
At the molecular level, exposure to ultraviolet
light increases skin pigmentation, in part through
the action of α-melanocyte–stimulating hormone
(α-MSH) on its receptor, the melanocortin recep-
tor 1 (MC1R) (Fig. 3). Binding of the hormone to
the receptor stimulates intracellular signaling in
melanocytes, and this signaling increases the ex-
pression of enzymes involved in the production
of melanin. Light-skinned and redheaded people
often carry germ-line polymorphisms in the MC1R
gene
10,11
that reduce the activity of the receptor.
12

Such polymorphisms increase the risk of mel-
anoma considerably.
13
In light-skinned people,
therefore, the basis of increased susceptibility to
melanoma is a genetic impairment in the produc-
tion of melanin, the main defense of melanocytes
against ultraviolet radiation.
Although the tanning response to ultraviolet
radiation appears dose-dependent, the nature of
the exposure is also a factor. Melanoma occurs
most frequently after intermittent exposure to the
sun and in people with frequent sunburns. Epi-
demiologic observations suggest that chronic or
low-grade exposures to ultraviolet light induce
protection against DNA damage, whereas intense,
intermittent exposures cause genetic damage.
7
A Molecular Model
of Melanoma Progression
The Clark model (Fig. 1) describes the histologic
changes that accompany the progression from
normal melanocytes to malignant melanoma.
2

We will relate these histologic changes to particu-
lar gene mutations (Table 1) in melanoma and dis-
cuss how these mutations affect molecular sig-
naling to contribute to the progression from
normal melanocytes to melanoma (Fig. 2).
Hyperplasia and Nevus Formation
In the Clark model, the first phenotypic change
in melanocytes is the development of benign nevi,
which are composed of neval melanocytes (Fig. 1).
The control of growth in these cells is disrupted,
yet the growth of a nevus is limited — a nevus
rarely progresses to cancer.
2
The absence of pro-
gression is probably due to oncogene-induced
cell senescence, in which growth that is stimulat-
ed by the activation of oncogenic pathways is lim-
ited.
14
At a molecular level, abnormal activation
of the mitogen-activated protein kinase (MAPK)
signaling pathway (also called extracellular-related
kinase [ERK]) stimulates growth in melanoma
cells (Fig. 4A).
15-17
Activation of this pathway is the
result of somatic mutations of N-RAS, which are
associated with about 15 percent of melanomas,
or BRAF, which are associated with about 50 per-
cent of melanomas. These mutations, which occur
exclusively of each other, cause constitutive acti-
vation of the serine–threonine kinases in the
ERK–MAPK pathway.
18-20
BRAF mutations occur at a similar frequency
in benign nevi and in primary and metastatic
melanomas.
21
Since most nevi cease proliferation
and remain static for decades, these similar fre-
quencies suggest that nevi must acquire addition-
al molecular lesions to free themselves of growth
restraints and become malignant. Experiments in
model systems support this hypothesis. In zebra-
fish, melanocyte-specific expression of a mutant
BRAF protein causes an ectopic proliferation of
melanocytes, analogous to human nevi.
22
In these
fish, the combination of a BRAF mutation and
inactivation of the tumor-suppressor gene p53
causes melanocytes to become malignant.
22
In
human melanocytes, mutant BRAF protein induc-
es cell senescence by increasing the expression
of the cell-cycle inhibitor of kinase 4A (INK4A).
23

INK4A limits hyperplastic growth caused by a
BRAF mutation. The arrest of the cell cycle caused
by INK4A can, however, be overcome by mutations
in INK4A itself, as well as other cell-cycle factors.
In vitro, depletion of BRAF and N-RAS from
melanoma cells suppresses their growth.
24-26

Small molecules that inhibit BRAF are being
tested clinically (BAY 43-9006) but have had only
limited success as single agents.
27
In mice, the
Figure 1 (facing page). The Clark Model (Hematoxylin
and Eosin).
Melanocytes progress through a series of steps toward
malignant transformation. The frequency of both the
progression of nevi toward becoming malignant and
the regression of nevi is unknown. The model empha-
sizes the histopathological changes that occur in the
progression of melanoma. Normal melanocytes pro-
gressively develop a malignant phenotype through the
acquisition of various phenotypic features. The partic-
ular histologic features characterizing each step of pro-
gression are the visible manifestations of underlying
genetic changes.
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 53
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 54
growth of melanomas with BRAF mutations can
be suppressed by the inhibition of the down-
stream MEK enzymes, providing a possible target
for treatment.
28
Cytologic Atypia and Tumor-Suppressor
Genes
The Clark model suggests that the next step toward
melanoma is the development of cytologic atypia
in dysplastic nevi, which may arise from preex-
isting benign nevi or as new lesions. The molecu-
lar abnormalities at this stage of progression
affect cell growth, DNA repair, and the suscepti-
bility to cell death. In 25 to 40 percent of cases of
familial melanoma,
6
a genetic defect inactivates
CDKN2A, a single gene that encodes two tumor-
suppressor proteins, p16
INK4A
and p19
ARF29,30
; in
25 to 50 percent of nonfamilial melanoma,
31,32
a
different tumor-suppressor gene, phosphatase and
tensin homologue (PTEN) (Fig. 3), is inactivated
by mutation.
33,34
In murine models of melanoma,
mutation of either CDKN2A or PTEN alone fails to
cause melanoma, but when combined with each
other or with mutations in other genes,
35
mela-
nomas do arise. Mutation of CDKN2A or PTEN is
only one molecular step on the path to the devel-
opment of melanoma, but it is unclear precisely
when such mutations occur. The increased sus-
ceptibility to melanoma that is associated with
loss of the germ-line CDKN2A gene suggests that
this genetic lesion increases the probability that
dysplastic nevi will become malignant or increas-
es the rate of the development of new melanoma
without a precursor.
CDKN2A
The G1–S checkpoint that governs the commitment
of a cell to DNA replication during the S phase
(synthesis of DNA) is a site where many pathways
that control cell division converge
36,37
(Fig. 4B).
In some familial and sporadic cases of melano-
ma,
36,37
the CDKN2A locus is lost by homozygous
deletion of a portion of chromosome 9.
36-38
One
of the genes in this locus encodes INK4A,
(p16
INK4A
), a protein that blocks the cell cycle at
the G1–S checkpoint by inhibiting cyclin-depen-
dent kinases. INK4A (an inhibitor of CDK4) sup-
presses the proliferation of cells with damaged
DNA or activated oncogenes and also acts when
cells are old or crowded.
39
Mice lacking INK4A
appear normal but are abnormally sensitive to
carcinogens and prone to the development of
tumors.
40
The development of melanoma in such
mice requires mutations in other genes, such as
an activating mutation in H-RAS, an upstream
component in MAPK signaling, which triggers
MEK signaling.
41
Genes that encode CDK4 and
cyclin D1 (CCND1) encode proteins that act down-
stream of INK4A, and they are also mutated in
some melanomas. These targets of INK4A func-
tion together as part of a complex that promotes
the progression of the cell cycle by phosphorylat-
ing retinoblastoma (Rb) protein, a cell-cycle reg-
ulator. Rare melanoma kindreds carry germ-line
mutations in CDK4 that disrupt cell-cycle control
by preventing the molecular interaction that allows
INK4A to repress CDK4.
42
Mice that carry the hu-
man CDK4 mutation are prone to melanoma when
exposed to various carcinogens.
43
The D-type cyclin CD1 may have an oncogenic
role in acral melanoma, in which amplification of
the CCND1 gene and overexpression of cyclin CD1
protein occur more frequently than in melanoma
at other sites.
44
Inhibition of CCND1 (with anti-
sense CCND1) causes apoptosis of human mela-
noma xenografts implanted in immunodeficient
mice, without an apparent effect on normal mela-
nocytes.
Alternative splicing of various exons within
CDKN2A yields two distinct tumor-suppressor pro-
teins, INK4A and alternate reading frame (ARF)
(Fig. 3).
39
The ARF gene (also called p14
ARF
) de-
rives its name from the use of an alternative
reading frame of the exons it shares with INK4A.
ARF functions as a tumor suppressor by arrest-
ing the cell cycle or promoting cell death after
DNA damage or when various oncogenes or loss
of Rb stimulate aberrant cell proliferation. ARF
participates in the core regulatory process that
controls levels of the p53 protein. It acts through
the mouse double minute 2 (MDM2) protein,
which triggers the ubiquination of p53, thereby
instigating its destruction in the proteosome.
ARF binds to MDM2, sequestering it from p53
and in this way causes p53 to accumulate; p53
then arrests the cell cycle at the G2–M site, allow-
ing for repair of damaged DNA or the induction
of apoptosis.
45,46
In cells, ARF deficiency abro-
gates oncogene-induced senescence and increases
susceptibility to transformation.
47
In vitro, im-
mortalization of cells often occurs with the loss
of either ARF or p53.
48
In animals, ARF deficiency
shortens the time required for the development
of melanoma after exposure to ultraviolet light;
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 55
when both gene products of CDKN2A (INK4A and
ARF) are deficient, the latent period is even short-
er.
49
These data suggest how ARF facilitates the
progression of melanoma and indicate that the
low frequency of p53 mutations in melanoma is
partly related to loss of ARF, which renders the
p53 pathway inactive.
39
PTEN, AKT, and Cell Death
A second chromosomal region that is frequently
affected by homozygous deletion in melanoma
and other cancers is the PTEN locus on chromo-
some 10.
33,34,50
PTEN encodes a phosphatase that
attenuates signaling by a variety of growth factors
that use phosphatidylinositol phosphate (PIP
3
) as
an intracellular signal. In the presence of such
growth factors, intracellular levels of PIP
3
rapidly
increase. This increase triggers the activation of
protein kinase B (PKB, also called AKT) by phos-
phorylation (Fig. 3). Activated AKT phosphory-
lates and inactivates proteins that suppress the
cell cycle or stimulate apoptosis, thereby facilitat-
ing the proliferation and survival of cells. PTEN
normally keeps PIP
3
levels low; in its absence,
Figure 2. Biologic Events and Molecular Changes in the Progression of Melanoma.
At the stage of the benign nevus, BRAF mutation and activation of the mitogen-activated protein kinase (MAPK) pathway occur. The cy-
tologic atypia in dysplastic nevi reflect lesions within the cyclin-dependent kinase inhibitor 2A (CDKN2A) and phosphatase and tensin
homologue (PTEN) pathways. Further progression of melanoma is associated with decreased differentiation and the decreased expres-
sion of melanoma markers regulated by microphthalmia-associated transcription factor (MITF). The vertical-growth phase and meta-
static melanoma are notable for striking changes in the control of cell adhesion. Changes in the expression of the melanocyte-specific
gene melastatin 1 (TRPM1) correlate with metastatic propensity, but the function of this gene remains unknown. Other changes include
the loss of E-cadherin and increased expression of N-cadherin, αVβ3 integrin, and matrix metalloproteinase 2 (MMP-2).
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 56
T
a
b
l
e

1
.

I
m
p
o
r
t
a
n
t

G
e
n
e
s

i
n

M
e
l
a
n
o
m
a
.
P
a
t
h
w
a
y
G
e
n
e

o
r

P
r
o
t
e
i
n
*
F
u
n
c
t
i
o
n
C
h
a
n
g
e
s

i
n

M
e
l
a
n
o
m
a
R
A
S

a
n
d

M
A
P
K
N
-
R
A
S
O
n
c
o
g
e
n
e
S
p
o
r
a
d
i
c

a
c
t
i
v
a
t
i
n
g

m
u
t
a
t
i
o
n

a
t

G
1
3
R
B
R
A
F
O
n
c
o
g
e
n
e
S
p
o
r
a
d
i
c

a
c
t
i
v
a
t
i
n
g

m
u
t
a
t
i
o
n

a
t

c
o
d
o
n

V
6
0
0
E

i
n

n
e
v
i

a
n
d

m
e
l
a
n
o
m
a
M
i
t
o
g
e
n
-
a
c
t
i
v
a
t
e
d

p
r
o
t
e
i
n

k
i
n
a
s
e
–
e
x
t
r
a
c
e
l
l
u
l
a
r
-
r
e
l
a
t
e
d

k
i
n
a
s
e

(
M
E
K
)
S
i
g
n
a
l

t
r
a
n
s
d
u
c
t
i
o
n
U
p
-
r
e
g
u
l
a
t
e
d

i
n

r
a
d
i
a
l
-
g
r
o
w
t
h

a
n
d

v
e
r
t
i
c
a
l
-
g
r
o
w
t
h

p
h
a
s
e
s
E
x
t
r
a
c
e
l
l
u
l
a
r
-
r
e
l
a
t
e
d

k
i
n
a
s
e

1

o
r

2

(
E
R
K
1

o
r

E
R
K
2
)

o
r

m
i
t
o
g
e
n
-
a
c
t
i
v
a
t
e
d

p
r
o
t
e
i
n

k
i
n
a
s
e

(
M
A
P
K
)
S
i
g
n
a
l

t
r
a
n
s
d
u
c
t
i
o
n
I
n
c
r
e
a
s
e
d

a
c
t
i
v
i
t
y
I
N
K
4
A
,

C
D
K
,

a
n
d

R
b
C
y
c
l
i
n
-
d
e
p
e
n
d
e
n
t

k
i
n
a
s
e

i
n
h
i
b
i
t
o
r

2
A

o
r

i
n
h
i
b
i
t
o
r

o
f

k
i
n
a
s
e

4
A

(
C
D
K
N
2
A

o
r

I
N
K
4
A
)
T
u
m
o
r

s
u
p
p
r
e
s
s
o
r
–
n
e
g
a
t
i
v
e

r
e
g
u
l
a
t
o
r

o
f

c
e
l
l

p
r
o
l
i
f
-
e
r
a
t
i
o
n
G
e
r
m
-
l
i
n
e

m
u
t
a
t
i
o
n
s

i
n

s
o
m
e

f
a
m
i
l
i
a
l

m
e
l
a
n
o
m
a
s
;

s
p
o
r
a
d
i
c

d
e
l
e
t
i
o
n
s
,

p
r
o
m
o
t
e
r

i
n
a
c
t
i
v
a
t
i
o
n
,

l
o
s
s

o
f

h
e
t
e
r
o
z
y
g
o
s
i
t
y

i
n

m
a
n
y

m
e
l
a
n
o
m
a
s
C
y
c
l
i
n
-
d
e
p
e
n
d
e
n
t

k
i
n
a
s
e

4

(
C
D
K
4
)
P
r
o
m
o
t
e
r

o
f

c
e
l
l

p
r
o
l
i
f
e
r
a
t
i
o
n
P
r
o
t
e
i
n

i
n
s
e
n
s
i
t
i
v
e

t
o

i
n
h
i
b
i
t
i
o
n

b
y

I
N
K
4
A

d
u
e

t
o

r
a
r
e

f
a
m
i
l
i
a
l

g
e
r
m
-
l
i
n
e

m
u
t
a
t
i
o
n
s

a
t

R
2
4
C


C
y
c
l
i
n

D
1

(
C
C
N
D
1
)
P
r
o
m
o
t
e
r

o
f

c
e
l
l

p
r
o
l
i
f
e
r
a
t
i
o
n
S
p
o
r
a
d
i
c

a
m
p
l
i
f
i
c
a
t
i
o
n

i
n

a
c
r
a
l

m
e
l
a
n
o
m
a
R
e
t
i
n
o
b
l
a
s
t
o
m
a

(
R
b
)
T
u
m
o
r

s
u
p
p
r
e
s
s
o
r
–
n
e
g
a
t
i
v
e

r
e
g
u
l
a
t
o
r

o
f

c
e
l
l

p
r
o
l
i
f
-
e
r
a
t
i
o
n
P
h
o
s
p
h
o
r
y
l
a
t
i
o
n

l
e
a
d
s

t
o

p
r
o
g
r
e
s
s
i
o
n

f
r
o
m

G
1

t
o

S
A
R
F

a
n
d

p
5
3
A
l
t
e
r
n
a
t
e

r
e
a
d
i
n
g

f
r
a
m
e

(
A
R
F
)
T
u
m
o
r

s
u
p
p
r
e
s
s
o
r
,

d
e
g
r
a
d
e
s

M
D
M
2
G
e
r
m
-
l
i
n
e

m
u
t
a
t
i
o
n
s

i
n

s
o
m
e

f
a
m
i
l
i
a
l

m
e
l
a
n
o
m
a
s
;

s
p
o
r
a
d
i
c

d
e
l
e
t
i
o
n
s
,

p
r
o
m
o
t
e
r

i
n
a
c
t
i
v
a
t
i
o
n
,

i
n

m
a
n
y

m
e
l
a
n
o
m
a
s
T
u
m
o
r

p
r
o
t
e
i
n

5
3

(
p
5
3
)
T
u
m
o
r

s
u
p
p
r
e
s
s
o
r

t
h
a
t

i
n
d
u
c
e
s

a
p
o
p
t
o
s
i
s

a
n
d

s
u
p
-
p
r
e
s
s
e
d

p
r
o
l
i
f
e
r
a
t
i
o
n

a
f
t
e
r

D
N
A

d
a
m
a
g
e
E
x
p
r
e
s
s
i
o
n

u
s
u
a
l
l
y

p
r
e
s
e
n
t

i
n

m
e
l
a
n
o
m
a
M
o
u
s
e

d
o
u
b
l
e

m
i
n
u
t
e

2

(
M
D
M
2
)
T
a
r
g
e
t
e
r

o
f

p
5
3

f
o
r

u
b
i
q
u
i
n
a
t
i
o
n

a
n
d

d
e
s
t
r
u
c
t
i
o
n
U
p
-
r
e
g
u
l
a
t
e
d

i
n

p
r
e
s
e
n
c
e

o
f

A
R
F

m
u
t
a
t
i
o
n
B
C
L
-
2
–
a
s
s
o
c
i
a
t
e
d

X

p
r
o
t
e
i
n

(
B
A
X
)
I
n
d
u
c
e
r

o
f

c
e
l
l

d
e
a
t
h
V
a
r
i
a
b
l
e

b
u
t

u
s
u
a
l
l
y

d
o
w
n
-
r
e
g
u
l
a
t
e
d
P
T
E
N

a
n
d

A
K
T
P
h
o
s
p
h
a
t
a
s
e

a
n
d

t
e
n
s
i
n

h
o
m
o
l
o
g
u
e

(
P
T
E
N
)
T
u
m
o
r

s
u
p
p
r
e
s
s
o
r
,

r
e
p
r
e
s
s
e
s

P
I
3
K
S
p
o
r
a
d
i
c

d
e
l
e
t
i
o
n

o
f

c
h
r
o
m
o
s
o
m
a
l

r
e
g
i
o
n
P
h
o
s
p
h
a
t
i
d
y
l
i
n
o
s
i
t
o
l

3

k
i
n
a
s
e

(
P
I
3
K
)
S
i
g
n
a
l
i
n
g

m
o
l
e
c
u
l
e

f
o
r

m
a
n
y

g
r
o
w
t
h

f
a
c
t
o
r
s
A
c
t
i
v
e

i
n

p
r
e
s
e
n
c
e

o
f

P
T
E
N

m
u
t
a
t
i
o
n
P
r
o
t
e
i
n

k
i
n
a
s
e

B

(
A
K
T

o
r

P
K
B
)
O
n
c
o
g
e
n
e

t
h
a
t

i
s

a
c
t
i
v
a
t
e
d

b
y

P
I
3
K
,

l
e
a
d
i
n
g

t
o

i
n
c
r
e
a
s
e
d

c
e
l
l

s
u
r
v
i
v
a
l
A
m
p
l
i
f
i
e
d

i
n

s
o
m
e

m
e
l
a
n
o
m
a
s
B
C
L
-
2

a
n
t
a
g
o
n
i
s
t

o
f

c
e
l
l

d
e
a
t
h

(
B
A
D
)
I
n
d
u
c
e
r

o
f

c
e
l
l

d
e
a
t
h
V
a
r
i
a
b
l
e

b
u
t

o
f
t
e
n

d
o
w
n
-
r
e
g
u
l
a
t
e
d
F
o
r
k
h
e
a
d

r
e
c
e
p
t
o
r

(
F
K
H
R
)
G
r
o
w
t
h

s
u
p
r
e
s
s
i
o
n
A
c
t
i
v
a
t
e
d

i
n

r
e
s
p
o
n
s
e

t
o

P
I
3

p
a
t
h
w
a
y

The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 57
M
S
H

a
n
d

M
I
T
F
P
r
o
-
o
p
i
o
m
e
l
a
n
o
c
o
r
t
i
n

o
r

α
-
m
e
l
a
n
o
c
y
t
e
–
s
t
i
m
u
l
a
t
i
n
g

h
o
r
m
o
n
e

(
P
O
M
C

o
r

α
-
M
S
H
)
S
i
g
n
a
l
i
n
g

m
o
l
e
c
u
l
e

i
m
p
o
r
t
a
n
t

i
n

p
i
g
m
e
n
t
a
t
i
o
n
I
n
c
r
e
a
s
e
d

m
e
l
a
n
o
m
a

v
e
r
t
i
c
a
l
-
g
r
o
w
t
h

p
h
a
s
e
M
e
l
a
n
o
c
o
r
t
i
n

r
e
c
e
p
t
o
r

1

(
M
C
1
R
)
R
e
c
e
p
t
o
r

f
o
r

α
-
M
S
H
P
o
l
y
m
o
r
p
h
i
c

g
e
n
e

a
f
f
e
c
t
i
n
g

h
a
i
r

a
n
d

s
k
i
n

c
o
l
o
r

a
n
d

r
e
s
p
o
n
s
e

t
o

u
l
t
r
a
v
i
o
l
e
t

r
a
d
i
a
t
i
o
n
A
d
e
n
y
l
a
t
e

c
y
c
l
a
s
e

(
A
C
)
P
r
o
d
u
c
e
r

o
f

c
y
c
l
i
c

A
M
P
U
p
-
r
e
g
u
l
a
t
e
d
c
A
M
P

r
e
s
p
o
n
s
e

e
l
e
m
e
n
t
–
b
i
n
d
i
n
g

p
r
o
t
e
i
n

(
C
R
E
B
)
T
r
a
n
s
c
r
i
p
t
i
o
n

f
a
c
t
o
r
U
p
-
r
e
g
u
l
a
t
e
d
;

a
f
f
e
c
t
s

M
I
T
F

a
n
d

m
e
l
a
n
o
c
y
t
e

d
i
f
f
e
r
e
n
t
i
a
t
i
o
n
M
i
c
r
o
p
h
t
h
a
l
m
i
a
-
a
s
s
o
c
i
a
t
e
d

t
r
a
n
s
c
r
i
p
t
i
o
n

f
a
c
t
o
r

(
M
I
T
F
)
T
r
a
n
s
c
r
i
p
t
i
o
n

f
a
c
t
o
r
S
p
o
r
a
d
i
c

a
m
p
l
i
f
i
c
a
t
i
o
n

o
f

c
h
r
o
m
o
s
o
m
a
l

r
e
g
i
o
n
T
y
r
o
s
i
n
a
s
e

(
T
Y
R
)
P
i
g
m
e
n
t

s
y
n
t
h
e
s
i
s
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n
T
y
r
o
s
i
n
a
s
e
-
r
e
l
a
t
e
d

p
r
o
t
e
i
n

1

(
T
Y
R
P
1
)
P
i
g
m
e
n
t

s
y
n
t
h
e
s
i
s
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n
D
o
p
a
c
h
r
o
m
e

t
a
u
t
o
m
e
r
a
s
e

(
D
C
T
)
P
i
g
m
e
n
t

s
y
n
t
h
e
s
i
s
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n
M
e
l
a
n
-
A

(
M
L
A
N
A
)
A
n
t
i
g
e
n

r
e
c
o
g
n
i
z
e
d

b
y

m
e
l
a
n
-
A

a
n
d

m
e
l
a
n
o
m
a

a
n
t
i
-
g
e
n

r
e
c
o
g
n
i
z
e
d

b
y

T
-
c
e
l
l
s

1

(
M
A
R
T
1
)

a
n
t
i
b
o
d
i
e
s
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n
S
i
l
v
e
r

h
o
m
o
l
o
g
u
e

(
S
I
L
V
)
A
n
t
i
g
e
n

r
e
c
o
g
n
i
z
e
d

b
y

H
M
B
-
4
5

a
n
t
i
b
o
d
y
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n
M
e
l
a
s
t
a
t
i
n

1

(
T
R
P
M
1
)
U
n
k
n
o
w
n
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n

i
n

m
e
t
a
s
t
a
t
i
c

m
e
l
a
n
o
m
a
B
C
L
-
2
C
e
l
l

s
u
r
v
i
v
a
l
V
a
r
i
a
b
l
e

u
p
-
r
e
g
u
l
a
t
i
o
n

i
n

v
a
r
i
o
u
s

p
h
a
s
e
s

o
f

m
e
l
a
n
o
m
a
C
e
l
l

a
d
h
e
s
i
o
n
W
i
n
g
l
e
s
s
-
t
y
p
e

m
a
m
m
a
r
y

t
u
m
o
r

v
i
r
u
s

i
n
t
e
g
r
a
t
i
o
n
-
s
i
t
e

f
a
m
i
l
y

(
W
N
T
)
P
r
o
t
o
o
n
c
o
g
e
n
e
,

s
e
c
r
e
t
e
d

g
r
o
w
t
h

f
a
c
t
o
r

t
h
a
t

i
n
a
c
t
i
-
v
a
t
e
s
G
S
K
3
-
B
P
a
t
h
w
a
y

u
p
-
r
e
g
u
l
a
t
e
d
G
l
y
c
o
g
e
n

s
y
n
t
h
a
s
e

k
i
n
a
s
e

3
β

(
G
S
K
3
β
)
S
e
r
i
n
e
–
t
h
r
e
o
n
i
n
e

k
i
n
a
s
e

t
h
a
t

t
a
r
g
e
t
s

β
-
c
a
t
e
n
i
n

f
o
r

d
e
g
r
a
d
a
t
i
o
n
V
a
r
i
a
b
l
e
;

a
f
f
e
c
t
e
d

b
y

W
N
T

p
a
t
h
w
a
y
β
-
C
a
t
e
n
i
n
A
d
h
e
r
e
n
s

j
u
n
c
t
i
o
n

p
r
o
t
e
i
n
,

t
r
a
n
s
c
r
i
p
t
i
o
n
a
l

c
o
-
a
c
t
i
v
a
t
o
r
S
p
o
r
a
d
i
c

s
t
a
b
i
l
i
z
i
n
g

m
u
t
a
t
i
o
n
s
T
-
c
e
l
l

f
a
c
t
o
r
–
l
y
m
p
h
o
i
d
-
e
n
h
a
n
c
i
n
g

f
a
c
t
o
r

(
T
C
F
–
L
E
F
)
T
r
a
n
s
c
r
i
p
t
i
o
n

f
a
c
t
o
r
U
p
-
r
e
g
u
l
a
t
e
d
E
-
c
a
d
h
e
r
i
n
C
e
l
l
-
a
d
h
e
s
i
o
n

m
o
l
e
c
u
l
e
D
e
c
r
e
a
s
e
d

e
x
p
r
e
s
s
i
o
n

i
n

v
e
r
t
i
c
a
l
-
g
r
o
w
t
h

p
h
a
s
e
N
-
c
a
d
h
e
r
i
n
C
e
l
l
-
a
d
h
e
s
i
o
n

m
o
l
e
c
u
l
e
A
b
e
r
r
a
n
t

e
x
p
r
e
s
s
i
o
n

i
n

v
e
r
t
i
c
a
l
-
g
r
o
w
t
h

p
h
a
s
e
α
V
β

3
i
n
t
e
g
r
i
n
D
i
m
e
r

t
h
a
t

f
o
r
m
s

c
e
l
l
-
a
d
h
e
s
i
o
n

m
o
l
e
c
u
l
e
A
b
e
r
r
a
n
t

e
x
p
r
e
s
s
i
o
n

i
n

v
e
r
t
i
c
a
l
-
g
r
o
w
t
h

p
h
a
s
e
*

A
b
b
r
e
v
i
a
t
e
d

f
o
r
m
s

o
f

t
h
e

g
e
n
e

a
r
e

g
i
v
e
n

i
n

p
a
r
e
n
t
h
e
s
e
s
.

The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 58
levels of PIP
3
and active (phosphorylated) AKT in-
crease. Increased AKT activity prolongs cell sur-
vival through the inactivation of BCL-2 antago-
nist of cell death (BAD) protein and increases
cell proliferation by increasing CCND1 expres-
sion, and affects many other cell-survival and cell-
cycle genes through the activation of the forkhead
(FKHR) transcription factor.
32,51
AKT activity can
also be increased in cells by mutations that cause
the amplification and overexpression of the pro-
tein. Restoration of PTEN in cultured mouse me-
lanocytes decreases the ability of the cells to form
tumors.
52
In model systems, suppression of AKT3,
a member of the AKT family, reduces the survival
of melanoma cells and the growth of human mela-
nomas implanted in immunodeficient nude mice.
53

Figure 3. Microphthalmia-Associated Transcription Factor (MITF) and β-Catenin Pathways.
In the MITF pathway, MITF is regulated at both transcriptional and post-translational levels. The post-translational activation can occur
through the ERK component of the MAPK pathway. The chief transcriptional pathways that are activated by extracellular signals are the
melanocortin and WNT pathways. The melanocortin pathway regulates pigmentation through the MC1R. MC1R activates the cyclic AMP
(cAMP) response-element binding protein (CREB). Increased expression of MITF and its activation by phosphorylation (P) stimulate the
transcription of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerase (DCT), which produce melanin;
melan-A, silver homologue, and melastatin 1 (TRPM1) are melanoma markers; inhibitor of kinase 4A (INK4A) leads to cell-cycle arrest,
and BCL-2 suppresses apoptosis. In the β-catenin pathway, β-catenin plays a central role in cell adhesion and cell signaling. Signals from
WNT ligands block the breakdown of β-catenin. When WNT proteins bind the G-protein–coupled receptor (called frizzled), they inacti-
vate the kinase GSK3β, an enzyme that phosphorylates β-catenin and targets it for destruction in the proteosome. Then β-catenin accu-
mulates in the cytoplasm and translocates to the nucleus, where it binds to LEF–TCF transcription factors and increases the expression
of several genes, including MITF, the cell-cycle mediator cyclin D1 (CCND1), and matrix metalloproteinase 7 (MMP-7).
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 59
As compared with normal melanocytes, increased
levels of the active form of AKT were found in the
radial-growth phase.
53
MITF and Melanocy te
Differentiation
Clark proposed that many nevi regress through
differentiation and that the failure of differentia-
tion is necessary for dysplasia.
2
The normal pro-
cess of melanocyte differentiation requires exit
from the cell cycle and the expression of genes
that encode proteins necessary for the produc-
tion of pigment — two processes that are de-
regulated in melanoma. The microphthalmia-
associated transcription factor (MITF) regulates
the development and differentiation of melano-
cytes
54
and maintains melanocyte progenitor cells
in adults.
55,56
MITF in Development
Mice lacking functional MITF are albino because
they lack melanocytes, whereas those with par-
tial MITF function have premature graying owing
to the death of melanocytes. These experiments
show that MITF is important in the differentiation
and maintenance of melanocytes.
57,58
MITF ap-
pears to contribute to melanocyte survival by in-
creasing the expression of the BCL-2 gene, a key
antiapoptotic factor.
59
In mice, deficiencies of both
MITF and BCL-2 cause gray hair due to a loss of
differentiated melanocytes. The loss of melano-
cytes is due to the apoptosis of melanocyte pro-
genitor cells in the hair follicle.
55
In melanoma
cell lines, a reduction in BCL-2 protein also causes
cell death, suggesting that the survival of malig-
nant melanocytes depends on BCL-2.
60
MITF in Differentiation
MITF functions in a key pathway leading to me-
lanocyte pigmentation (Fig. 3). Intracellular sig-
naling induced by α-MSH acting on MC1R in-
creases MITF expression, which in turn increases
the transcription of genes underlying melanin
synthesis: tyrosinase, tyrosinase-related-protein 1,
and dopachrome tautomerase.
61
MITF also regu-
lates the transcription of the melanocyte-specific
genes silver homologue (SILV)
62,63
and melan-A
(MLANA),
62
whose immunohistochemical detec-
tion points to the diagnosis of melanoma. In addi-
tion, MITF causes cell-cycle arrest by the induction
of INK4A.
64
MITF in Melanoma
Decreased or absent pigmentation and decreased
or absent expression of SILV and MLANA accom-
pany the progression from nevus to melanoma.
Tumors that are deficient in these proteins have
a poor prognosis.
65-68
Expression of the mela-
statin 1 (TRPM1) gene, whose function is unknown,
is also controlled by MITF.
69
Melanomas that are
deficient in melastatin have a poor prognosis.
70

The mechanism of decreased expression of these
genes is a puzzle because MITF is present in nearly
all melanomas.
71-73
Although MITF causes differentiation and cell-
cycle arrest in normal melanocytes, melanoma
cells do not have these characteristics. Recently,
a large-scale search for genomic changes in mela-
noma with the use of high-density single-nucleo-
tide polymorphisms (SNPs) found an increased
copy number (4 to 119 copies per cell) of a region
of chromosome 3 that includes the MITF locus.
74

This increase was accompanied by the increased
expression of MITF protein. The overexpression
of both MITF and BRAF could transform primary
cultures of human melanocytes, implicating MITF
as an oncogene. Notably, MITF amplification oc-
curs most frequently in tumors that have a poor
prognosis and is associated with resistance to
chemotherapy.
74
Interference with MITF function
increased the chemosensitivity of a melanoma
cell line, making MITF a potential target for
treatment.
Cell Adhesion and Invasion
Local invasion and metastatic spread are respon-
sible for the morbidity and mortality in melano-
ma. In the Clark model, invasive characteristics
appear in the vertical-growth phase, when mela-
noma cells not only penetrate the basement mem-
brane but also grow intradermally as an expand-
ing nodule (Fig. 2). Metastatic melanoma develops
when tumor cells dissociate from the primary
lesion, migrate through the surrounding stroma,
and invade blood vessels and lymphatics to form
a tumor at a distant site.
75
Clinically, the absolute
depth of local invasion, measured directly by histo-
pathologic analysis (the Breslow index), is the
principal prognostic factor and primary criterion
in melanoma staging.
76
Invasion and spread of
melanoma are related to alterations in cell adhe-
sion. Normally, cell adhesion controls cell migra-
tion, tissue organization, and organogenesis,
77

The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 60
but disturbances in cell adhesion contribute to
tumor invasion, tumor–stroma interactions, and
tumor-cell signaling.
Cadherins
Cadherins are multifunctional transmembrane
proteins that sustain cell-to-cell contacts, form
connections with the actin cytoskeleton, and in-
fluence intracellular signaling. The extracellular
domain of cadherins binds to like cadherins on
other cells in regions of cell contacts called adhe-
rens junctions. Cadherins are divided into three
subtypes: E (epithelial), present in polarized epi-
thelial cells in the epidermis, including melano-
cytes and keratinocytes; P (placental); and N (neu-
ral), found in mesenchymal cells in the dermis.
The intracellular domain is associated with a large
protein complex that includes β-catenin and forms
structural links with bundles of actin filaments.
Several signaling pathways cause β-catenin to
dissociate from the cell adhesion complex and
transduce signals to the nucleus (Fig. 3). One of
these pathways is called the wingless-type mam-
mary tumor virus integration-site family (WNT)
pathway. WNTs are secreted proteins with impor-
tant functions in development, especially in neu-
ral crest cells like melanocytes. When WNT pro-
teins bind their receptors, they inactivate the
kinase GSK3β, an enzyme that phosphorylates
β-catenin and targets it for destruction in the pro-
teosome.
78,79
Tyrosine phosphorylation of β-catenin
disrupts the association between E-cadherin and
β-catenin,
80
allowing β-catenin to translocate to
the nucleus, where it binds to lymphoid enhancer
factor–T-cell factor (LEF–TCF). Mutations in the
β-catenin gene can stabilize the β-catenin pro-
tein
81
or increase its nuclear localization.
82-84
In-
creased levels of nuclear β-catenin increase the
expression of MITF
85
and CCND1,
86
and these in
turn increase the survival and proliferation of
melanoma cells. Alterations in cadherin expres-
sion affect the interaction of melanoma cells with
the environment and alter β-catenin signaling.
E-cadherin expression occurs in melanocytes and
keratinocytes in the epidermis and causes mela-
nocytes to associate with keratinocytes.
87
In turn,
contacts with undifferentiated keratinocytes from
the basal-cell layer inhibit melanocyte prolif-
eration, suppress the expression of melanoma
Figure 4 (facing page). MAPK and PTEN Pathways and the CDKN2A Tumor-
Suppressor Locus.
Panel A shows the pathway associated with N-RAS, BRAF, and mitogen-
activated protein kinase (MAPK). MAPKs are involved in signaling from
numerous growth factors and cell-surface receptors. There are many vari-
ations in the components of particular cascades from various cell-surface
receptors. Typically, adapter proteins (not shown) link the growth-factor
receptor to RAS proteins, including N-RAS. When activated, RAS proteins
phosphorylate (P) the mitogen-activated protein kinase (MEK) kinases,
which then act on extracellular-related kinase (ERK) kinases. ERK kinases
phosphorylate many targets in the cytoplasm and interact with other path-
ways, including phosphatidylinositol 3 kinase (PI3K) and MITF. ERK kinases
translocate to the nucleus, where they activate transcription factors that
promote cell-cycle progression and proliferation by increasing the transcrip-
tion of many genes, including CD1. In survival signaling associated with
phosphatase and tensin homologue (PTEN) and AKT, also known as pro-
tein kinase B, PTEN inhibits growth-factor signaling by inactivating phos-
phatidylinositol triphosphate (PIP
3
) generated by PI3K. A variety of growth
factors (PDGF, NGF, and IGF-1) bind to their respective receptor tyrosine
kinases and activate PI3K. The activated molecule converts the plasma
membrane lipid phosphatidylinositol 4,5-bisphosphonate to PIP
3
. PIP
3
acts
as a second messenger, leading to the phosphorylation and activation of
AKT. AKT is itself a kinase that phosphorylates protein substrates that af-
fect the cell cycle, growth, and survival. Often, these AKT targets are inacti-
vated by phosphorylation. PTEN attenuates this pathway through dephos-
phorylation and inactivation of PIP
3
, suppressing signaling from growth
factors by blocking the activation of AKT. In Panel B, CDKN2A encodes two
distinct tumor-suppressor genes; separate first exons that are spliced into
alternate reading frames (ARF) of the second and third exons permit the
expression of two different proteins from the same genetic locus. The gene
has 4 exons. Transcription of messenger RNA (mRNA) can be initiated at
either E1B or E1A, and the initiation site determines which gene the locus
will express. RNA that is transcribed from either exon is spliced with the re-
maining two exons, E2 and E3, to produce mRNA for either INK4A or ARF.
However, ARF uses a different reading frame of the exon 2 and 3 codons.
In the cell-cycle progression involving INK4A, ARF, and retinoblastoma pro-
tein (Rb), a family of cyclins and cyclin-dependent kinases (CDKs) regulate
progression through the cell cycle, and a family of CDK inhibitors opposes
this action. In particular, the two phases of the G1–S checkpoint are gov-
erned primarily by cyclin D associated with cyclin-dependent kinases 4 and
6 (CDK4 and CDK6) at its early phase and cyclin A or E associated with
CDK2 at the later restriction phase. INK4A encodes a cyclin-dependent ki-
nase inhibitor that inhibits CDK4 and CDK6. Ordinarily, these two kinases
associate with D-type cyclins and drive the cell cycle by phosphorylating
Rb, releasing it from its inhibitory interaction with the E2F transcription
factor, thereby allowing the expression of E2F-related genes and progres-
sion from G1 to S. The absence of INK4A leads to unopposed CDK4 or
6 activity and increased cell-cycle activity. In response to DNA damage,
mouse double minute 2 (MDM2) protein binds to the transcriptional acti-
vation domain of protein 53 (p53), blocking p53-mediated gene regulation
while simultaneously leading to p53 ubiquination, nuclear export, and pro-
teosomal degradation. ARF opposes this action by sequestering MDM2.
This disruption of the MDM2–p53 interaction stabilizes p53 and increases
p53 activity. Depending on other events, p53 either activates DNA repair
and cell-cycle arrest or causes apoptosis and the formation of BCL-2–asso-
ciated X protein (BAX). In the absence of ARF, p53 levels are decreased and
the response to DNA damage is blunted.
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 61
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 62
markers, and cause melanocytes to become den-
dritic.
88
Progression from the radial-growth phase to
the vertical-growth phase of melanoma is marked
by the loss of E-cadherin and the expression of
N-cadherin
89-91
(Fig. 2).

N-cadherin is a character-
istic of invasive carcinomas and enables metastatic
spread by permitting melanoma cells to interact
with other N-cadherin–expressing cells, such as
dermal fibroblasts and the vascular endothelium.
87

Besides these changes in cell adhesion, decreased
E-cadherin expression
92
and aberrant N-cadherin
expression increase the survival of melanoma
cells by stimulating β-catenin signaling.
93,94
Integrins
The integrins mediate cell contacts with fibro-
nectin, collagens, and laminin, components of the
extracellular matrix.
95
Transition from radial to
vertical growth of melanoma is associated with
the expression of αVβ3 integrin.
96
This integrin
induces expression of matrix metalloproteinase 2,
an enzyme that degrades the collagen in basement
membrane.
97-99
In addition, αVβ3 integrin increas-
es expression of the prosurvival gene BCL-2
100
and
stimulates the motility of melanoma cells through
the reorganization of melanoma cytoskeleton.
101
These observations form a rationale for the de-
velopment of integrin antagonists to treat mela-
noma.
102
Patterns of Genetic Alteration
The genetic changes in melanoma can be seen as
particular combinations of molecular lesions that
interrupt a precise set of pathways, each with a
crucial role in the development of melanoma.
The MEK pathway can be activated by a mutation
in either NRAS or BRAF, and an NRAS mutation
can activate both the MEK and PTEN pathways.
Similarly, INK4A, CDK4, and CCND1 function in
a unique pathway that affects the cell cycle; a mu-
tation of INK4A has similar consequences as a
mutation of CCND1 or CDK4.
103-105
There are particular genetic changes in mela-
nomas in different sites, consistent differences
related to ultraviolet exposure on sites that are
chronically exposed (head and neck) or intermit-
tently exposed (chest and back) and in acral and
mucosal skin. For example, CCND1 amplification
occurs predominantly in acral regions,
44
whereas
activating mutations in BRAF occur most frequent-
ly in skin sites of intermittent sun exposure.
106
Modeling Melanoma
Progression
For many of the molecular lesions we have de-
scribed, animal models have provided validation.
A surprising new model is the zebrafish, in which
premalignant and malignant lesions can be cre-
ated by the expression of mutant BRAF with or
without p53 mutation.
22
This model is the only
currently tractable system in which genetic
screens can be performed for modifiers of mela-
noma.
Human melanomas that are grafted onto or
injected into nude mice allow measures of the
tumors’ metastatic potential and have allowed for
the testing of therapeutic interventions. Genetic
manipulation of mice has validated the contribu-
tion of many genetic alterations in melanoma, but
there are fundamental differences between mouse
and human skin. Mouse melanocytes occur in
hair follicles and the dermis, rather than in the
epidermis, as in humans. To circumvent this
problem, human melanocytes can be altered in
cell culture and combined with keratinocytes to
produce graft material. Using this system, the
inactivation of p53 and the simultaneous intro-
duction of activated N-RAS, CDK4, and telomer-
ase led to darkly pigmented grafts that became
grossly ulcerated and displayed histologic features
of melanoma, including vertical invasion.
107
This
experimental system provides a novel model to
test invasion and metastases of transformed hu-
man melanocytes in a host organism.
Supported by a grant (MCM202534) from the Cancer Research
Institute of New York and a grant (T32-GM07753, to Dr. Miller)
from the National Institute of General Medical Science. No other
potential conflict of interest relevant to this article was reported.
We are indebted to Drs. David E. Fisher, Adriano Piris, Jenni-
fer Y. Lin, and Jennifer C. Broder for their critical reading of the
manuscript, and to Dr. Claudio Clemente for contributing im-
ages for Figure 1.
References
Cancer facts & figures, 2003. Atlanta:
American Cancer Society, 2003.
Clark WH Jr, Elder DE, Guerry D IV,
Epstein MN, Greene MH, Van Horn M.
A study of tumor progression: the precur-
1.
2.
sor lesions of superficial spreading and
nodular melanoma. Hum Pathol 1984;15:
1147-65.
Curtin JA, Fridlyand J, Kageshita T,
et al. Distinct sets of genetic alterations
3.
in melanoma. N Engl J Med 2005;353:
2135-47.
Alonso SR, Ortiz P, Pollan M, et al.
Progression in cutaneous malignant mel-
anoma is associated with distinct expres-
4.
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 63
sion profiles: a tissue microarray-based
study. Am J Pathol 2004;164:193-203.
Aitken J, Welch J, Duffy D, et al. CD-
KN2A variants in a population-based sam-
ple of Queensland families with melano-
ma. J Natl Cancer Inst 1999;91:446-52.
Thompson JF, Scolyer RA, Kefford RF.
Cutaneous melanoma. Lancet 2005;365:
687-701.
Gilchrest BA, Eller MS, Geller AC,
Yaar M. The pathogenesis of melanoma
induced by ultraviolet radiation. N Engl J
Med 1999;340:1341-8.
MacKie RM. Risk factors for the de-
velopment of primary cutaneous malig-
nant melanoma. Dermatol Clin 2002;20:
597-600.
Marks R. Epidemiology of melanoma.
Clin Exp Dermatol 2000;25:459-63.
Naysmith L, Waterston K, Ha T, et al.
Quantitative measures of the effect of the
melanocortin 1 receptor on human pig-
mentary status. J Invest Dermatol 2004;
122:423-8.
Valverde P, Healy E, Jackson I, Rees JL,
Thody AJ. Variants of the melanocyte-
stimulating hormone receptor gene are
associated with red hair and fair skin in
humans. Nat Genet 1995;11:328-30.
Frandberg PA, Doufexis M, Kapas S,
Chhajlani V. Human pigmentation pheno-
type: a point mutation generates non-
functional MSH receptor. Biochem Biophys
Res Commun 1998;245:490-2.
Kennedy C, ter Huurne J, Berkhout M,
et al. Melanocortin 1 receptor (MC1R)
gene variants are associated with an in-
creased risk for cutaneous melanoma
which is largely independent of skin type
and hair color. J Invest Dermatol 2001;
117:294-300.
Braig M, Schmitt CA. Oncogene-
induced senescence: putting the brakes on
tumor development. Cancer Res 2006;66:
2881-4.
Welsh CF, Roovers K, Villanueva J, Liu
Y, Schwartz MA, Assoian RK. Timing of
cyclin D1 expression within G1 phase is
controlled by Rho. Nat Cell Biol 2001;3:
950-7.
Brunet A, Roux D, Lenormand P,
Dowd S, Keyse S, Pouyssegur J. Nuclear
translocation of p42/p44 mitogen-activat-
ed protein kinase is required for growth
factor-induced gene expression and cell
cycle entry. EMBO J 1999;18:664-74.
Lin AW, Barradas M, Stone JC, van
Aelst L, Serrano M, Lowe SW. Premature
senescence involving p53 and p16 is acti-
vated in response to constitutive MEK/
MAPK mitogenic signaling. Genes Dev
1998;12:3008-19.
Albino AP, Nanus DM, Mentle IR, et al.
Analysis of ras oncogenes in malignant
melanoma and precursor lesions: correla-
tion of point mutations with differentia-
tion phenotype. Oncogene 1989;4:1363-74.
Davies H, Bignell GR, Cox C, et al.
Mutations of the BRAF gene in human
cancer. Nature 2002;417:949-54.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Omholt K, Platz A, Kanter L, Ring-
borg U, Hansson J. NRAS and BRAF
mutations arise early during melanoma
pathogenesis and are preserved through-
out tumor progression. Clin Cancer Res
2003;9:6483-8.
Pollock PM, Harper UL, Hansen KS,
et al. High frequency of BRAF mutations
in nevi. Nat Genet 2003;33:19-20.
Patton EE, Widlund HR, Kutok JL, et
al. BRAF mutations are sufficient to pro-
mote nevi formation and cooperate with
p53 in the genesis of melanoma. Curr Biol
2005;15:249-54.
Michaloglou C, Vredeveld LC, Soen-
gas MS, et al. BRAFE600-associated se-
nescence-like cell cycle arrest of human
naevi. Nature 2005;436:720-4.
Eskandarpour M, Kiaii S, Zhu C, Cas-
tro J, Sakko AJ, Hansson J. Suppression of
oncogenic NRAS by RNA interference in-
duces apoptosis of human melanoma
cells. Int J Cancer 2005;115:65-73.
Hingorani SR, Jacobetz MA, Robert-
son GP, Herlyn M, Tuveson DA. Suppres-
sion of BRAF(V599E) in human melano-
ma abrogates transformation. Cancer Res
2003;63:5198-202.
Hoeflich KP, Gray DC, Eby MT, et al.
Oncogenic BRAF is required for tumor
growth and maintenance in melanoma
models. Cancer Res 2006;66:999-1006.
Lyons JF, Wilhelm S, Hibner B, Bollag
G. Discovery of a novel Raf kinase inhibi-
tor. Endocr Relat Cancer 2001;8:219-25.
Solit DB, Garraway LA, Pratilas CA, et
al. BRAF mutation predicts sensitivity to
MEK inhibition. Nature 2006;439:358-62.
Kamb A, Gruis NA, Weaver-Feldhaus J,
et al. A cell cycle regulator potentially in-
volved in genesis of many tumor types.
Science 1994;264:436-40.
Nobori T, Miura K, Wu DJ, Lois A,
Takabayashi K, Carson DA. Deletions of
the cyclin-dependent kinase-4 inhibitor
gene in multiple human cancers. Nature
1994;368:753-6.
Flores JF, Walker GJ, Glendening JM,
et al. Loss of the p16INK4a and p15INK4b
genes, as well as neighboring 9p21 mark-
ers, in sporadic melanoma. Cancer Res
1996;56:5023-32.
Wu H, Goel V, Haluska FG. PTEN sig-
naling pathways in melanoma. Oncogene
2003;22:3113-22.
Li J, Yen C, Liaw D, et al. PTEN, a pu-
tative protein tyrosine phosphatase gene
mutated in human brain, breast, and pros-
tate cancer. Science 1997;275:1943-7.
Steck PA, Pershouse MA, Jasser SA, et
al. Identification of a candidate tumour
suppressor gene, MMAC1, at chromosome
10q23.3 that is mutated in multiple ad-
vanced cancers. Nat Genet 1997;15:356-
62.
You MJ, Castrillon DH, Bastian BC, et
al. Genetic analysis of Pten and Ink4a/Arf
interactions in the suppression of tumori-
genesis in mice. Proc Natl Acad Sci U S A
2002;99:1455-60.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Kamb A, Shattuck-Eidens D, Eeles R,
et al. Analysis of the p16 gene (CDKN2) as
a candidate for the chromosome 9p mela-
noma susceptibility locus. Nat Genet 1994;
8:23-6.
Hussussian CJ, Struewing JP, Gold-
stein AM, et al. Germline p16 mutations
in familial melanoma. Nat Genet 1994;8:
15-21.
Pollock PM, Trent JM. The genetics of
cutaneous melanoma. Clin Lab Med 2000;
20:667-90.
Sharpless E, Chin L. The INK4a/ARF
locus and melanoma. Oncogene 2003;22:
3092-8.
Serrano M, Lee H, Chin L, Cordon-
Cardo C, Beach D, DePinho RA. Role of
the INK4a locus in tumor suppression
and cell mortality. Cell 1996;85:27-37.
Chin L, Pomerantz J, Polsky D, et al.
Cooperative effects of INK4a and ras in
melanoma susceptibility in vivo. Genes
Dev 1997;11:2822-34.
Zuo L, Weger J, Yang Q, et al. Germ-
line mutations in the p16INK4a binding
domain of CDK4 in familial melanoma.
Nat Genet 1996;12:97-9.
Sotillo R, Garcia JF, Ortega S, et al.
Invasive melanoma in Cdk4-targeted mice.
Proc Natl Acad Sci U S A 2001;98:13312-
7.
Sauter ER, Yeo UC, von Stemm A, et
al. Cyclin D1 is a candidate oncogene in
cutaneous melanoma. Cancer Res 2002;
62:3200-6.
Pomerantz J, Schreiber-Agus N, Liege-
ois NJ, et al. The Ink4a tumor suppressor
gene product, p19Arf, interacts with MDM2
and neutralizes MDM2’s inhibition of
p53. Cell 1998;92:713-23.
Harris SL, Levine AJ. The p53 path-
way: positive and negative feedback loops.
Oncogene 2005;24:2899-908.
Sharpless NE, Ramsey MR, Balasub-
ramanian P, Castrillon DH, DePinho RA.
The differential impact of p16(INK4a) or
p19(ARF) deficiency on cell growth and
tumorigenesis. Oncogene 2004;23:379-85.
Kamijo T, Zindy F, Roussel MF, et al.
Tumor suppression at the mouse INK4a
locus mediated by the alternative reading
frame product p19ARF. Cell 1997;91:649-
59.
Recio JA, Noonan FP, Takayama H, et
al. Ink4a/arf deficiency promotes ultravi-
olet radiation-induced melanomagenesis.
Cancer Res 2002;62:6724-30.
Guldberg P, Thor Straten P, Birck A,
Ahrenkiel V, Kirkin AF, Zeuthen J. Disrup-
tion of the MMAC1/PTEN gene by dele-
tion or mutation is a frequent event in
malignant melanoma. Cancer Res 1997;57:
3660-3.
Cantley LC, Neel BG. New insights
into tumor suppression: PTEN suppresses
tumor formation by restraining the phos-
phoinositide 3-kinase/AKT pathway. Proc
Natl Acad Sci U S A 1999;96:4240-5.
Stahl JM, Cheung M, Sharma A, Trive-
di NR, Shanmugam S, Robertson GP.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
The new engl and j ournal o f medicine
n engl j med 355;1 www.nejm.org july 6, 2006 64
Loss of PTEN promotes tumor develop-
ment in malignant melanoma. Cancer
Res 2003;63:2881-90.
Stahl JM, Sharma A, Cheung M, et al.
Deregulated Akt3 activity promotes de-
velopment of malignant melanoma. Can-
cer Res 2004;64:7002-10.
Hodgkinson CA, Moore KJ, Nakaya-
ma A, et al. Mutations at the mouse mi-
crophthalmia locus are associated with
defects in a gene encoding a novel basic-
helix-loop-helix-zipper protein. Cell 1993;
74:395-404.
Nishimura EK, Granter SR, Fisher DE.
Mechanisms of hair graying: incomplete
melanocyte stem cell maintenance in the
niche. Science 2005;307:720-4.
Widlund HR, Fisher DE. Microphtha-
lamia-associated transcription factor: a
critical regulator of pigment cell devel-
opment and survival. Oncogene 2003;22:
3035-41.
Lerner AB, Shiohara T, Boissy RE, Ja-
cobson KA, Lamoreux ML, Moellmann GE.
A mouse model for vitiligo. J Invest Der-
matol 1986;87:299-304.
Steingrimsson E, Moore KJ, Lamor-
eux ML, et al. Molecular basis of mouse
microphthalmia (mi) mutations helps ex-
plain their developmental and phenotypic
consequences. Nat Genet 1994;8:256-63.
McGill GG, Horstmann M, Widlund
HR, et al. Bcl2 regulation by the melano-
cyte master regulator Mitf modulates lin-
eage survival and melanoma cell viability.
Cell 2002;109:707-18.
Banerjee D. Genasense (Genta Inc).
Curr Opin Investig Drugs 2001;2:574-80.
Goding CR. Mitf from neural crest to
melanoma: signal transduction and tran-
scription in the melanocyte lineage.
Genes Dev 2000;14:1712-28.
Du J, Miller AJ, Widlund HR, Horst-
mann MA, Ramaswamy S, Fisher DE.
MLANA/MART1 and SILV/PMEL17/GP100
are transcriptionally regulated by MITF in
melanocytes and melanoma. Am J Pathol
2003;163:333-43.
Baxter LL, Pavan WJ. Pmel17 expres-
sion is Mitf-dependent and reveals cranial
melanoblast migration during murine de-
velopment. Gene Expr Patterns 2003;3:703-
7.
Loercher AE, Tank EMH, Delston RB,
Harbour JW. MITF links differentiation
with cell cycle arrest in melanocytes by
transcriptional activation of INK4A. J Cell
Biol 2005;168:35-40.
Hofbauer GF, Kamarashev J, Geertsen
R, Boni R, Dummer R. Melan A/MART-1
immunoreactivity in formalin-fixed par-
affin-embedded primary and metastatic
melanoma: frequency and distribution.
Melanoma Res 1998;8:337-43.
Salti GI, Manougian T, Farolan M,
Shilkaitis A, Majumdar D, Das Gupta TK.
Micropthalmia transcription factor: a new
prognostic marker in intermediate-thick-
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
ness cutaneous malignant melanoma. Can-
cer Res 2000;60:5012-6.
Seiter S, Monsurro V, Nielsen MB, et al.
Frequency of MART-1/MelanA and gp100/
PMel17-specific T cells in tumor metasta-
ses and cultured tumor-infiltrating lym-
phocytes. J Immunother 2002;25:252-63.
Takeuchi H, Kuo C, Morton DL, Wang
HJ, Hoon DS. Expression of differentia-
tion melanoma-associated antigen genes
is associated with favorable disease out-
come in advanced-stage melanomas. Can-
cer Res 2003;63:441-8.
Miller AJ, Du J, Rowan S, Hershey CL,
Widlund HR, Fisher DE. Transcriptional
regulation of the melanoma prognostic
marker melastatin (TRPM1) by MITF in
melanocytes and melanoma. Cancer Res
2004;64:509-16.
Duncan LM, Deeds J, Hunter J, et al.
Down-regulation of the novel gene mela-
statin correlates with potential for mela-
noma metastasis. Cancer Res 1998;58:
1515-20.
King R, Weilbaecher KN, McGill G,
Cooley E, Mihm M, Fisher DE. Microph-
thalmia transcription factor: a sensitive
and specific melanocyte marker for mela-
noma diagnosis. Am J Pathol 1999;155:731-
8.
Miettinen M, Fernandez M, Franssila
K, Gatalica Z, Lasota J, Sarlomo-Rikala M.
Microphthalmia transcription factor in the
immunohistochemical diagnosis of meta-
static melanoma: comparison with four
other melanoma markers. Am J Surg
Pathol 2001;25:205-11.
Granter SR, Weilbaecher KN, Quigley
C, Fisher DE. Role for microphthalmia
transcription factor in the diagnosis of
metastatic malignant melanoma. Appl Im-
munohistochem Mol Morphol 2002;10:47-
51.
Garraway LA, Widlund HR, Rubin MA,
et al. Integrative genomic analyses iden-
tify MITF as a lineage survival oncogene
amplified in malignant melanoma. Na-
ture 2005;436:117-22.
Haass NK, Smalley KS, Li L, Herlyn M.
Adhesion, migration and communication
in melanocytes and melanoma. Pigment
Cell Res 2005;18:150-9.
Balch CM, Soong SJ, Gershenwald JE,
et al. Prognostic factors analysis of 17,600
melanoma patients: validation of the
American Joint Committee on Cancer
melanoma staging system. J Clin Oncol
2001;19:3622-34.
Johnson JP. Cell adhesion molecules
in the development and progression of
malignant melanoma. Cancer Metastasis
Rev 1999;18:345-57.
Bienz M. Beta-catenin: a pivot between
cell adhesion and Wnt signalling. Curr
Biol 2005;15:R64-R67.
Gottardi CJ, Gumbiner BM. Adhesion
signaling: how beta-catenin interacts with
its partners. Curr Biol 2001;11:R792-R794.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
Brembeck FH, Schwarz-Romond T,
Bakkers J, Wilhelm S, Hammerschmidt
M, Birchmeier W. Essential role of BCL9-2
in the switch between beta-catenin’s ad-
hesive and transcriptional functions. Genes
Dev 2004;18:2225-30.
Rubinfeld B, Robbins P, El-Gamil M,
Albert I, Porfiri E, Polakis P. Stabilization
of beta-catenin by genetic defects in mel-
anoma cell lines. Science 1997;275:1790-
2.
Cowley GP, Smith ME. Cadherin ex-
pression in melanocytic naevi and malig-
nant melanomas. J Pathol 1996;179:183-7.
Rimm DL, Caca K, Hu G, Harrison
FB, Fearon ER. Frequent nuclear/cytoplas-
mic localization of beta-catenin without
exon 3 mutations in malignant melano-
ma. Am J Pathol 1999;154:325-9.
Sanders DS, Blessing K, Hassan GA,
Bruton R, Marsden JR, Jankowski J. Al-
terations in cadherin and catenin expres-
sion during the biological progression of
melanocytic tumours. Mol Pathol 1999;52:
151-7.
Widlund HR, Horstmann MA, Price
ER, et al. Beta-catenin-induced melanoma
growth requires the downstream target
Microphthalmia-associated transcription
factor. J Cell Biol 2002;158:1079-87.
Shtutman M, Zhurinsky J, Simcha I, et
al. The cyclin D1 gene is a target of the
beta-catenin/LEF-1 pathway. Proc Natl Acad
Sci U S A 1999;96:5522-7.
Hsu M, Andl T, Li G, Meinkoth JL,
Herlyn M. Cadherin repertoire determines
partner-specific gap junctional communi-
cation during melanoma progression.
J Cell Sci 2000;113:1535-42.
Valyi-Nagy IT, Hirka G, Jensen PJ,
Shih IM, Juhasz I, Herlyn M. Undifferenti-
ated keratinocytes control growth, mor-
phology, and antigen expression of normal
melanocytes through cell-cell contact. Lab
Invest 1993;69:152-9.
Danen EH, de Vries TJ, Morandini R,
Ghanem GG, Ruiter DJ, van Muijen GN.
E-cadherin expression in human melano-
ma. Melanoma Res 1996;6:127-31.
Hsu MY, Wheelock MJ, Johnson KR,
Herlyn M. Shifts in cadherin profiles be-
tween human normal melanocytes and
melanomas. J Investig Dermatol Symp
Proc 1996;1:188-94.
Scott RA, Lauweryns B, Snead DM,
Haynes RJ, Mahida Y, Dua HS. E-cadherin
distribution and epithelial basement mem-
brane characteristics of the normal human
conjunctiva and cornea. Eye 1997;11:607-
12.
Gottardi CJ, Wong E, Gumbiner BM.
E-cadherin suppresses cellular transfor-
mation by inhibiting beta-catenin signal-
ing in an adhesion-independent manner.
J Cell Biol 2001;153:1049-60.
Qi J, Chen N, Wang J, Siu CH. Trans-
endothelial migration of melanoma cells
involves N-cadherin-mediated adhesion and
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.
mechanisms of disease
n engl j med 355;1 www.nejm.org july 6, 2006 65
activation of the beta-catenin signaling
pathway. Mol Biol Cell 2005;16:4386-97.
Li G, Satyamoorthy K, Herlyn M.
N-cadherin-mediated intercellular inter-
actions promote survival and migration
of melanoma cells. Cancer Res 2001;61:
3819-25.
Kuphal S, Bauer R, Bosserhoff AK. In-
tegrin signaling in malignant melanoma.
Cancer Metastasis Rev 2005;24:195-222.
Danen EH, Ten Berge PJ, Van Muijen
GN, Van ’t Hof-Grootenboer AE, Brocker
EB, Ruiter DJ. Emergence of alpha 5 beta
1 fibronectin- and alpha v beta 3 vitronec-
tin-receptor expression in melanocytic tu-
mour progression. Histopathology 1994;24:
249-56.
Brooks PC, Stromblad S, Sanders LC,
et al. Localization of matrix metallopro-
teinase MMP-2 to the surface of invasive
cells by interaction with integrin alpha v
beta 3. Cell 1996;85:683-93.
Felding-Habermann B, Fransvea E,
O’Toole TE, Manzuk L, Faha B, Hensler
M. Involvement of tumor cell integrin al-
94.
95.
96.
97.
98.
pha v beta 3 in hematogenous metastasis
of human melanoma cells. Clin Exp Metas-
tasis 2002;19:427-36.
Hofmann UB, Westphal JR, Waas ET,
Becker JC, Ruiter DJ, van Muijen GN. Co-
expression of integrin alpha(v)beta3 and
matrix metalloproteinase-2 (MMP-2) co-
incides with MMP-2 activation: correla-
tion with melanoma progression. J Invest
Dermatol 2000;115:625-32.
Petitclerc E, Stromblad S, von Schal-
scha TL, et al. Integrin alpha(v)beta3 pro-
motes M21 melanoma growth in human
skin by regulating tumor cell survival.
Cancer Res 1999;59:2724-30.
Li X, Regezi J, Ross FP, et al. Integrin
alphavbeta3 mediates K1735 murine mel-
anoma cell motility in vivo and in vitro.
J Cell Sci 2001;114:2665-72.
Dechantsreiter MA, Planker E, Matha
B, et al. N-methylated cyclic RGD peptides
as highly active and selective alpha(V)beta(3)
integrin antagonists. J Med Chem 1999;
42:3033-40.
Tsao H, Zhang X, Fowlkes K, Haluska
99.
100.
101.
102.
103.
FG. Relative reciprocity of NRAS and
PTEN/MMAC1 alterations in cutaneous
melanoma cell lines. Cancer Res 2000;60:
1800-4.
Daniotti M, Oggionni M, Ranzani T,
et al. BRAF alterations are associated
with complex mutational profiles in ma-
lignant melanoma. Oncogene 2004;23:
5968-77.
Tsao H, Goel V, Wu H, Yang G,
Haluska FG. Genetic interaction between
NRAS and BRAF mutations and PTEN/
MMAC1 inactivation in melanoma. J Invest
Dermatol 2004;122:337-41.
Maldonado JL, Fridlyand J, Patel H,
et al. Determinants of BRAF mutations in
primary melanomas. J Natl Cancer Inst
2003;95:1878-90.
Chudnovsky Y, Adams AE, Robbins
PB, Lin Q, Khavari PA. Use of human tis-
sue to assess the oncogenic activity of
melanoma-associated mutations. Nat Gen-
et 2005;37:745-9.
Copyright © 2006 Massachusetts Medical Society.
104.
105.
106.
107.
JOURNAL EDITORIAL FELLOW
The Journal’s editorial office invites applications for a one-year
research fellowship beginning in July 2007 from individuals at any
stage of training. The editorial fellow will work on Journal projects
and will participate in the day-to-day editorial activities of the Journal
but is expected in addition to have his or her own independent
projects. Please send curriculum vitae and research interests
to the Editor-in-Chief, 10 Shattuck St., Boston, MA 02115
(fax, 617-739-9864), by October 1, 2006.
The New England Journal of Medicine
Downloaded from nejm.org by hani amalia on June 3, 2014. For personal use only. No other uses without permission.
Copyright © 2006 Massachusetts Medical Society. All rights reserved.