Melanoma

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Melanoma

• Jeff Sosman, MD
• Vanderbilt School of Medicine
• Department of Medicine
• Section of Hematology/Oncology

April 4, 2007
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Melanoma A Neural Crest Derived Cancer

• Neural Crest and melanoma- biology and genetics
• Regulation of melanoma functional characteristics
  by embryonic microenvironment
• Melanocyte Stem Cells and MITF
• Clinical Melanoma
• Melanoma Progression Model
• Melanoma as a malignancy with molecular defects
  to target therapeutically
• Future therapeutic targets and treatment

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Neural Crest Cells

• Arise along vertebrae axis into dorsal neural
  tube and migrate into surrounding tissue
• Give rise to bone, cartilage, neurons, glia of
  PNS, adrenal medulla, craniofacial tissue, and
  cardiac cells, and pigment cells (melanocytes)
• Follow stereotypical migratory pathways to form
  segregated streams of cells that emerge at
  specific locations along vertebrae axis
• Local inhibitory factor and cells inhibit
  migration into NCC-free zones and redirect them
  to streams

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Activation of Neural Crest Genetic Program in
Neuroectodermal Malignancies

• Neurofibroma, schwannoma, neuroblastoma,
  malignant nerve sheath tumor, melanoma,
  supratentorial PNET, medulloblastoma and Ewings
  Sarcoma
• Inappropriate activation of genetic network
  critical to neural crest development in Neural
  Crest malignancies
• Expression pattern of transcription corresponds
  to developmental progression
• Transcriptional factors and their targets-
  SOX10(sex-determining Y region box-10) (P0,
  MITF), AP-2a (ERBB2, ERBB3), PAX3 (MYOD, STX)
  target genes---P0, ERBB3, STX
• PAX3 target gene- c-met receptor for SF/HGF
  which is highly expressed in melanoma
• PAX7 restricted to mesoderm and poorly
  differentiated malignant tumors (not found in
  melanoma)
• Transcription factors play a role in
  epithelial-mesenchymal transformation (EMT)

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PAX3 (paired-box 3) overexpressed in melanoma

• Importance of transcriptional factors PAX3 and
  their (MITF, myoD, STX) target genes
• PAX3 leads to overexpression of c-met receptor
  (target gene) for SF/HGF which is highly
  expressed in melanoma
• Transgenic mice with c-met overexpression or
  ectopic SF/HGF develop melanoma
• Important in development of melanoblasts (MITF)
  and muscle (myoD)
• C-met may be critical in melanoma and
  rhabdomyosarcoma
• Other PAX3 target genes- IGFR1 and bcl-XL

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Wnt-5a in melanoma

• Important regulator of morphogenetic movements
  during embryonic development and proliferation of
  progenitor cells
• High expression as neural crest cells migrate to
  skin
• Overexpressionin melanoma cells changes
  morphology and actin redistribution and increase
  adhesion to ECM
• Wnt5a signal through PKC and increase
  phosphorylation of PKC associated with changes in
  cytoskeleton, cell adhesion, and motility
• Increase in melanoma invasiveness
• Association with melanoma aggressiveness in
  patients based on biopsies and stages and
  histology

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Wnt-5a Signaling through Frizzled 5
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Blue is negative IHC Orange is moderate IHC Red
is strong IHC Gray is heterogeneous IHC
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Microenvironment influence malignant phenotype

• Reverting (reprogramming) metastatic melanoma
  malignant phenotype
• Chick embryo implant melanoma cells do not form
  tumors but instead invade into an organized
  neural crest cell migratory pathway display
  neural crest features with morphology and
  population patterns of host peripheral tissues
• Melanoma cells do not migrate to regions normally
  avoided by NCC (NCC-free zones)
• Cells express both melanocyte and neuronal
  markers MART-1 and Tuj1- previously metastatic
  melanoma did not initially

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Embryonic progenitor - Malignant stem cells

• Cancer is derived from a subpopulation of
  pluripotent self renewing cells
• Fate specificed by interaction with autocrine and
  paracrine signals from surrounding cells and
  tissue
• Multipotent plasticity of progenitor cells
  respond to microenvironment factors and influence
  other cells thru epigenetic mechanisms
• Zebrafish embryos transplanted with labelled
  melanoma cells induce secondary ectopic
  structures (axial mesoderms)
• Key role for Nodal expression- and inhibition
  with Lefty block axial structures and also lead
  to more pigment and less aggressiveness
• Nodal expression in melanoma correlate to stage
  and aggressiveness metastaticgtVGPgtHGPgtmelanocytes
• Signalling ALK/4/5/7 then SMAD2/3- positive
  regulatory loop
• Association of invasiveness and induction of
  ectopic embryo structures

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Melanoblasts

• Migrate from neural crest (multi-potent) to
  dermis and then basal layer of epidermis
  especially at hair root bulb
• MITF and c-kit are critical for survival of
  melanoblasts
• Possibly through Bcl-2, Cdk2, CDK inhibitor p16
  and p21/Cip1
• In late stages of migration from dermis to
  epidermis, HGF, ET1 and 2, and basic FGF are
  likely to play a role
• In malignancies- rarely see mutations
  characteristic of UVR
• Suggest effect may be through keratinocytes

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Cell-cell communication to regulate growth and
differentiation

• Nodal- TGFb family member- receptor Cripto --
  type I and II activin-like kinase receptors (ALK)
  ---- activate SMAD2 or SMAD3 which bind to SMAD4
• Nodal regulate tumorigenicity and melanocytic
  differentation/phenotype- possibly through NOTCH
• Potent embryonic morphogen
• Nodal signaling leads to down-regulation of
  VE-Cadherin and keratin and increase invasiveness
• Role of TGFb family in EMT

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Melanoma model Primary Human Melanocytes
Transduced with SV40ER hTERT RasG12V /- HGF/SF
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Table 1 Primary tumor formation and secondary
metastasis
 

BJ-STR
MEC-STR
Mel-STV
Mel-STR
21/21
21/21 (S), 20/20 (O)
0/12
93/93
Primary tumor



0/21
1/21 (S), 0/21 (O)
NA
85/93
Any organ
Metastasis
0/21
1/21 (S), 0/21 (O)
NA
85/93
Lung
0/21
0/21 (S,O)
NA
25/93
Liver
0/21
0/21 (S,O)
NA
21/93
Spleen
0/21
0/21 (S,O)
NA
11/93
Small bowel
0/21
0/21 (S,O)
NA
27/93
Lymph nodes

Melanoma has ability to metastasize to a much
greater extent than other lineages (mammary and
fibroblast). Characteristic due to lineage and
origin

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Snail family member Slug is expressed in
melanoma cell lines, Expression correlates with
CD44, ET-3 and ERBB3 expressionKnockdown of Slug
leads to minimal change in tumor growth, but
marked decreased in metastases
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MITF- micropthalmia transcription factorMaster
regulator of melanocytes

• Regulate pigment expression through control of
  expression of group of enzymes
• Regulate TBX2 p19, CDK2, BCL-2, HIF1a, c-MET
• Levels of MITF can decrease from melanocyte
  nevus, dysplastic nevus, RGP, VGP, Metastatic
  melanoma
• This may relate to expression of TRPM1 levels
• Commonly downregulated in melanoma
• However, 10-20 have amplification of MITF and
  poorer prognosis

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ET-1
PAX3, SOX10, TCF,MITF
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Melanoma Progression Model
Critical transition
Aberrant differentiation and nuclear atypia
Aberrant differentiation
Up to 50 of sporadic melanoma arise without a
clinical precursor lesion
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Cell-cell communication to regulate growth and
differentiation
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Melanocyte- Keratinocyte Communication

• Melanocyte transports pigment containing
  melanosomes (lysosome-like structures) via
  dendrites to number of keratinocytes
  (epidermal/melanin unit)
• Melanocyte growth and invasion controlled within
  unit of epidermis by keratinocyte
• Melanomocytes aligned at the basal layer of
  epidermis
• Escape from regulation by
• Downregulate receptors for communication with
  keratinocytes (E-cadherin, P-cadherin, desmoglein
• Upregulate receptor of melanocyte- melanocyte,
  melanocyte-fibroblast (N-CAN, Mel-CAM, ZO-1)
• Deregulation of morphogens such as NOTCH
  receptors and ligands
• Loss of anchorage to basement membrane-
  alteration in cell adhesion- molecules to matrix
• Increase elaboration of metalloproteinases (MMPs)

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Downregulate receptors for communication with
keratinocytes (E-cadherin)

• E-cadherin plays a major role in adhesion
• Snail and Twist, transcription factors
  downregulate E-cadherin expression
• Twist upregulate mesenchymal markers (N-cadherin,
  vimentin, and fibronectin
• Twist has role in Epithelial-mesenchymal
  transformation
• Loss of regulation of melanocyte from
  keratinocyte
• Hepatocyte growth factor (HGF), Platelet derived
  growth factor (PDGF), and Endothelin-1 (ET-1)
• Melanocyte growth- FGF2, SCF, and ET-3 stimulate
• Increase in integrins aV,b3 and a1b1 and
  activation of MMP

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Epithelial-Mesenchymal Transformation (EMT
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Melanoma- A disease on the Rise

• gt60,000 cases in the US in 2006
• gt8000 deaths due to melanoma
• High incidence in individuals who are 45-50 years
  of age at the peak of their productivity
• Very curable if diagnosed at early stage
• lt1.0 mm Breslow thickness
• lt level IV Clark
• Radial Growth Phase (RGP), without vertical
  growth phase (VGP)
• Later stages have very poor prognosis
• Stage IIC gt4 mm ulcerated 50 5 year survival
• Stage III- Lymph node involvement- 15-60 5-year
  survival (size and number of metastases)
• Stage IV- median survival 6-10 months and 5 year
  survival ltlt5

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Lifetime Risk of Developing Cutaneous Melanoma
in the US
174
1100
1150
1250
1600
11500
1935
1960
1980
1985
1993
2000
Rigel DS, Carucci JA. CA Cancer J Clin.
200050215236.
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The ABCDEs of Melanoma Diagnosis
Asymmetry
One half of the lesion is shaped differently than
the other
The border of the lesion is irregular, blurred,
or ragged
Border
Color
Inconsistent pigmentation, with varying shades
of brown and black
gt6 mm, or a progressive change in size
Diameter
Evolution
History of change in the lesion
Photos courtesy of the American Cancer Society.
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Morphologic Types of Melanoma
Type Frequency Features
Superficial 60-70 Flat during early phase
notching,spreading scalloping, areas of
regression Nodular 15-30 Darker and thicker
than superficial spreading, rapid onset
commonly blue-black or blue-red (5
amelanotic) Lentigo 5 Enlarge slowly usually
large, flat, tan maligna or
brown Acral Uncommon On soles, palms, beneath
nail beds lentiginous Asians (46), usually
large, tan or brown irregular Blacks (70)
border subungual melanoma more common in
older, dark-skinned people Desmoplastic 1.7 Rare,
locally aggressive, occur primarily on head
and neck in elderly
Data from Lotze MT, et al. Cutaneous Melanoma.
In DeVita VT Jr,. et al, eds. Cancer
Principles Practice of Oncology. 6th ed.
Philadelphia, PA Lippincott-Raven 2001.
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Prognostic Factors for Primary MelanomaPatients
Staged after Node Dissection
Variable P Risk Ratio 95 CI Nodal status
lt.00001 2.239 1.913-2.621 Thickness lt.00001
1.583 1.433-1.749 Ulceration
lt.00001 1.938 1.674-2.242 Site
lt.00001 1.483 1.281-1.716 Age
.0002 1.095 1.044-1.147 Sex .1705
0.900 0.774-1.046 Level of invasion .9082 1.007
0.896-1.131
J Clin Oncol. 2001193622-3634.
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AJCC Staging Criteria T Stage5-Year Survival
Ulceration
Depth


lt1.0 mm 1.01-2.0 mm 2.01-4.0 mm gt4.0 mm
95 89 78 67
91 77 63 45
10-year survival was 10-15 less in each
category J Clin Oncol. 2001193635-3648.
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5-Year Survival in Stage III Melanoma
Microscopic
Macroscopic
J Clin Oncol. 2001193622-3634.
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MOLECULAR TARGETS IN MELANOMAB-RAF
MUTATED IN 59 of melanoma cell lines 80 of
melanoma cell cultures 66 of melanoma
metastases 1796 mutation T?A Or V600E
Gain of function mutation with transforming
activity, blocking it, prevents tumor formation
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Molecular alterations in melanoma
Sites for pathway blockade
-KIT
15
50-65
N-RAS GTP
Overexpression
RAS GDP
C-RAF
25-50
PTEN
x
Frequent loss
Loss of expression
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Curtin J et al. J Clin Oncol 2006
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Amplifications and Mutations of KIT in Melanoma
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RGP VGP

• Loss of Function
• c-KIT
• p16 (CDKN2)
• p53
• E-cadherin
• AP-2

• Gain of Function
• MCAM
• IL-8/MMP-2
• IL-10
• Integrins
• Thrombin Receptor (PAR-1)
• CREB/ATF-1

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Progression of Melanoma Biology

• Rare to find mutations in p53, RAS, and p16INK4A
• B-RAF mutation very early in process
• Observed in benign and dysplastic nevi
• Constitutive activation of MAPK pathway
• Critical loss of p16INK4A and p19ARF or CDK4
  function to regulate cell cycle and apoptosis and
  p53
• PTEN loss with activation of Akt/PI-3K
• Activation of STAT3 and NFkB signaling
• Loss of expression of c-kit, AP-2, MITF, p16,
  E-cadherin
• Expression of MCAM, PAR-1 integrin AvB3,
  IL-8/CREB transcription

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Morphologic Types of Melanoma
Type Frequency Features
Superficial 60-70 Flat during early phase
notching,spreading scalloping, areas of
regression Nodular 15-30 Darker and thicker
than superficial spreading, rapid onset
commonly blue-black or blue-red (5
amelanotic) Lentigo 5 Enlarge slowly usually
large, flat, tan maligna or
brown Acral Uncommon On soles, palms, beneath
nail beds lentiginous Asians (46), usually
large, tan or brown irregular Blacks (70)
border subungual melanoma more common in
older, dark-skinned people Desmoplastic 1.7 Rare,
locally aggressive, occur primarily on head
and neck in elderly
Data from Lotze MT, et al. Cutaneous Melanoma.
In DeVita VT Jr,. et al, eds. Cancer
Principles Practice of Oncology. 6th ed.
Philadelphia, PA Lippincott-Raven 2001.
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C-kit mutations/ amplifications (30)
C-kit mutations/ amplifications (35)
C-kit mutations/ amplifications (40)
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PLX4032- kinase inhibitor B-raf
mutant specific
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Melanoma signaling pathways to target
Hsp-90 inhibitors (geldanomycins)
Sunitinib,Imatinib
Tipifarnib
R115777
gamma-secretase inhibitor
Antibody to IGFR-1
NOTCH-1
Sorafenib, PLX4032, ARQ523,GSK compound, RAF-265,
Merck compound
BEZ235 Novartis
Bayer Pfizer Wyeth Novartis/Chiron AstraZeneca Mil
lenium Bristol-Myer-Squibb Schering Amgen/imclone
Plexikkon Exilexus
PD901 AZ6244 LBW624 CI-1040
mTOR
CCI-779 RAD001
BMS-345541 MLN0415 AS602868
Translation
Velcade NPI-0052
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V Foundation Clinical Translational Grant

• Preclinical phase
• Obtain tumor from advanced melanoma patients (20)
  at time of surgery
• Transplant tumor into immunosuppressed mice
  either under renal capsule or subcutaneous sites
• Characterize molecular phenotype of tumors-
  B-Raf, N-Ras, PTEN, Mitf, c-kit, NFkB state,
  Aurora kinase expression
• Treat each human tumor in a set of mice with
  either B-Raf inhibitor, IKK inhibitor, or Aurora
  kinase inhibitor and determine clinical efficacy-
  tumor size and apoptosis index
• Define molecular phenotype correlation with tumor
  response- define model
• Study the in vivo effects- MAP kinase, Akt
  phosphorylation, NFkB, gene expression array to
  better understand mechanism
• Clinical Phase
• Characterize patients tumor molecularly
• Based on molecular phenotype and preclinical
  model treat on one of three cohorts- B-Raf
  inhibitor, Aurora kinase inhibitor, or IKK
  inhibitor
• Assay effect on tumor when feasible with biopsy-
  signal inhibition of specific and other pathways
  and expression array
• Measure tumor responses

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Polymorphic receptor determines hair color,
ability to tan and freckle and fair skin