Download molecular pathology in melanoma

MOLECULAR PATHOLOGY
IN MELANOMA
Dr José Luis Rodríguez Peralto
Hospital Universitario 12 de Octubre, Madrid, Spain
AN IMPORTANT HEALTH
PROBLEM
• Melanoma is one of the most aggressive tumours (79% of skin cancer
death)
• In Europe, 2.5% of all cancers. 1-2% death by cancer (5000 deaths by
melanoma a year in Europe)
• Incidence has increased dramatically in last decades (15 times in last 50
years)
• Incidence in Spain: 15:100.000 inhabitants (depend on regions)
• The rate of mortality has not increased in the same average as
incidence (early diagnostic)
• Extremely bad prognosis, especially in advanced stages. Resistance to
chemotherapy
BIOLOGY OF MELANOMA
RADIAL
VERTICAL
METASTASIS
PROGRESSION IN THREE CLINICO-PATHOLOGIC STAGES
TISSUE-ARRAY
• An excellent tool to simultaneously study numerous
specimens using same conditions and criteria
•Preservation of the original paraffin block
40 proteins, by IHC
Cell Cycle: activators: cyclins,
inhibitors: Rb, p53, p16, p21, p27
Apoptosis: BCL-2, BCL-XL,Survivin
Nuclear Markers:
brown (LSAB- DAB)
Transcription Fact : MUM-1, PKCB,
Membrane Receptors:
Caveolin, C-KIT
Adhesion Molecules: E-cadherin,
B catenin, p120
Others: S-100, HMB-45, Melan A, Ki-67,
Cytoplasmic Markers:
red (APAAP, neo-fucsin)
BCL6, PTEN, IIa Topoisomerase, BMI-1
RESULTS
“Each progression stage of melanoma is characterized
by a specific protein expression profile”
RADIAL
Cell cycle
 Cyc A
 Cyc D1
 CDK1
 CDK2
 P21
 RB
 pRB
 HDM-2
Adhesion
Apoptosis
 Survivin
Transcription factors
 STAT-1
Membrane receptors
 C-KIT
 Caveolin
Others
 Topoisomerase II
 BMI-1
 Caderina E
 B Catenina
 p120
VERTICAL
Cell cycle
 Cyc D1
 CDK2
 p27
 MIB-1
Apoptosis
 Survivin
Transcription factors
 MUM-1
 PKCB
Membrane receptors
 C-KIT
DNA repair
 MLH-1
Adhesion
 Caderina E
 B Catenina
 p120
METASTASIS
Cell cycle
 Cyc D1
 Cyc D3
 CDK6
 p16
 p21
Apoptosis
 BCL-2
Transcription factors
 STAT-1
 PKCB
 MUM-1
DNA repair
 MLH-1
 MSH-2
Others
 Topoisomerase II
 RING 1B
Cell Cycle Activators
NEVUS
0%
RADIAL GP
p 0.007
48%
VERTICAL GP
p 0.002
14%
METASTASIS
p 0.021
32%
CYC- D1
0%
CYC- D3
p 0.303
22%
p 0.073
8%
p 0.009
32%
Cell Cycle Inhibitors
NEVUS
100 %
p16 INK4
RADIAL GP
p 0.553
88%
VERTICAL GP
p 1.000
89%
METASTASIS
p 0.009
71%
Cell Cycle Inhibitors
NEVUS
70%
p27 KIP
RADIAL GP
p 0.694
76%
VERTICAL GP
p 0.010
45%
METASTASIS
p 0.380
37%
Apoptosis
NEVUS
0%
RADIAL GP
p 0.038
36%
VERTICAL GP
p 0.002
71%
METASTASIS
p 0.356
62%
SURVIVIN
100%
BCL-2
p 0.157
77%
p 0.336
87%
p 0.000
45%
Adhesion Molecules
 E Cadherin
 B Catenin
 N Cadherin
 E Cadherin
 B Catenin
 Nuclear B Catenin
 N Cadherin
No differences
_
G0
M
CELL DIVISION
Cyclin B
Degradation
p14
_
p16
_
p53
CycD
CDK4/6
Cyclin A
Degradation
p27
+
_
CDK1 G2
CycA/B
INCREASE
CELL CYCLE
ACTIVATORS
Hdm2
P
p21
Rb
_
CDK2
CycE
P
Rb
G1
_
G2
CDK2
DNA REPAIR
CycA
S DNA
DNA
REPLICATION
REPLICATION
LOSS CELL
CYCLE
INHIBITOR
TNF
DEATH LIGAND
TNFR
DEATH RECEPTOR
APOPTOSIS
FADD
CASP
3,6,7
CASP 8
SURVIVIN
NF-kB
APOPTOSOMA
BID
SMAC
C-IAP
Bcl-2
CASP9
Surv
NF-kB
FLIP
Bcl-XL
APAF-1
BCL-2
Bcl-XL
Bcl-6
CELL SURVIVAL
p53
BAX
BAD
Cyt C
Melanoma Tissue Microarrays
Alonso et al AJPathol 2004, 164 p193
Results.- clinical follow up: predictive model
Statistical Analysis:
-Univariate. Relevant variables
were included
Three groups of risk: Ki67, p21, p16, BCL6
Supervivencia Global
1.00
-Multivariate. final model taking
the predicted HR*
BAJO
p16++y el
LOWRIESGO:
RISK p16
resto negativos
and rest negatives
0.75
INTERMEDIUM
RISK
RIESGO
INTERMEDIO:
y BCL-6
- -and
p16p16+
+ and
BCL6
con cualquiera Ki 67>20%
Ki 67y>20%
oand/or
/o p21+
0.50
p21 +
MULTIVARIATE ANALYSIS
0.25
Proteina RR
95 %IC
p-val
HIGH
RISK p16
p16- y/o
ALTO
RIESGO:
+ +
and/orBCL6
BCL6
0.00
1
BCL6 +
Ki67 
P16 +
P21 +
8.101
2.41
0.13
2.36
2.56-25.26
0.94-6.17
0.04-0.41
0.95-5.86
<0.001
0.068
0.001
0.065
2
3
4
5
6
Años desde el diagnóstico hasta el fallecimiento
Years from the diagnosis to the death
- Shorter
overall survival:
of p16
and/or
Fig
3B. Curva representando
el efecto de loss
la expresión
de las
proteínas p16, p21, Ki-67
y BCL-6
en la supervivencia global
BCL6
expression
de pacientes con Melanoma Cutáneo.
- No changes by including the Breslow’s index
*Performed by backwards elimination, starting with markers with a p < 0.10
OS PREDICTIVE MODEL
• Time from diagnostic to death by this condition
• 60 melanomas on vertical grow phase without metastasis at diagnostic time
1.00
1. Low risK: p16+ and rest negatives
0.75
2. Intermedium risk:
p16+ y BCL-6- and Ki 67>20%
and /or p21+
0.50
0.25
3. High risk P16 - and/or BCL6 +
0.00
1
2
3
4
5
6
Years
-Shorter overall survival: Loss of p16 and/or BCL6 expression
- No changes by including the Breslow’s index
cDNA Arrays in Cutaneous Melanoma
Aim: Identify the metastatic signature in a series of primary aggresive CMM
Training Set: Primary Melanomas, vertical growth phase, ≥ 1mm Breslow
METASTATIC DISEASE (22)
FOLLOW UP
Molecular Differences ???
36 months
NON METASTATIC DISEASE (13)
34 Primary Melanomas
Validation: To confirm the results. Independent series of 131 cases, 6 TMAs
(73 with metastasis /56 without metastasis)
Results
SIGNATURE OF METASTASES
233 highly differentially
expressed genes (median
differences > 2 fold ratio
threshold)
Up and down regulated genes
were classified according to
functional categories
91- GENES UP
REGULATED
2%
1%
18%
19%
3%
12%
3%
34%
2%
6%
EMT relacionado
Crecimiento y Diferenciación
Respuesta Inmune
Transducción de señales
Metabolismo
Funciones celulares básicas
Muerte celular
Ciclo Celular
Angiogénesis
Miscelánea
Gene Name
Dif S vs N
EDNRB
PDE5A
SPP1
SPARC
PRKCA
TUBB3
H2-ALPHA
TUBA2
TUBA3
CDH2
DSG2
NID2
EMP1
LUM
COL3A1
GPC3
SDCBP
HMMR
SMARCA1
CA9
CGI-141
LAPTM4A
IGFBP1
MYB
KIT
WEE1
PMP22
YWHAQ
DUSP12
NME2
ILF2
SERPINA3
CASP5
FAIM
GAS1
SKP1
1.1845
Function
Description
1.0360
Angiogenesis, negative regulation of EDNEndothelin receptor type B
Phosphodiesterase 5A, cGMP-specific
Angiogenesis, regulation of vessel size,dilatation
1.6610
EMT related, adhesion, migration, cell survival,
andphosphoprotein
tumorigenesis. 1 (osteopontin, bone
secreted
1.2270
1.5890
EMT related, bone remodeling
EMT related, cell adhesion
1.3450
EMT related, cell adhesion. link with cadherins
Tubulin, beta 3
1.1920
1.0825
EMT related, cell adhesion, link with cadherins
Tubulin, alpha 2
EMT related, cell adhesion, link with cadherins
Tubulin, alpha 2
1.0155
EMT related, cell adhesion, link with cadherins
Tubulin, alpha 3
1.4130
EMT related, cell adhesion
Cadherin 2, type 1, N-cadherin (neuronal)
1.1210
EMT related, cell adhesion
Desmoglein 2
1.0865
EMT related, cell adhesion
Nidogen 2 (osteonidogen)
1.1905
EMT related, cell adhesion regulator
Epithelial membrane protein 1
3.1030
1.1915
EMT related, organismal movement, matrix
Lumican
organization
2.0965
EMT related; cell growth and maintenanceGlypican 3
1.5895
1.3425
EMT related; cell motility
EMT related; cell motility
Syndecan binding protein (syntenin)
Hyaluronan-mediated motility receptor (RHAM
1.0150
EMT related; matrix associated
SWI/SNF related, matrix associated, actin dep
1.1375
Cell growth
Carbonic anhydrase IX
1.1700
Cell growth and differentiation
CGI-141 protein
1.4960
Cell growth and maintenance
Lysosomal-associated protein transmembrane
1.2980
Cell growth and maintenance
Insulin-like growth factor binding protein 1
1.2595
Cell growth and maintenance
V-myb myeloblastosis viral oncogene homolo
1.1910
Cell growth and maintenance
V-kit Hardy-Zuckerman 4 feline sarcoma viral
1.1395
Cell growth and maintenance
WEE1 homolog (S. pombe)
1.0755
Cell growth and maintenance
Peripheral myelin protein 22
1.0455
1.0185
Cell growth and maintenance
Cell growth and maintenance
Tyrosine 3-monooxygenase/tryptophan 5-mon
Dual specificity phosphatase 12
1.0105
Cell growth and maintenance
Non-metastatic cells 2, protein (NM23B)
1.3090
Immune response
Interleukin enhancer binding factor 2, 45kDa
1.0540
Immune response
Serpina 3 (alpha 1 antichymotrypsin y antitryp
1.3060
Cell death
Caspase 5, apoptosis-related cysteine protea
1.0845
Cell death
Fas apoptotic inhibitory molecule
1.0540
Cell death
Growth arrest-specific 1
1.1515
Cell cycle
SKP1(S-phase kinase-associated protein 1A)
EMT related, organogenesis
Secreted protein, acidic, cysteine-rich (osteon
Protein kinase C, alpha
Collagen, type III, alpha 1 (Ehlers-Danlos syn
EMT: role in melanoma mtx
Epithelial Mesenchymal Transition: EMT
-
Related with cell adhesion, cell motility and Extracellular
matrix interaction
- Operates during organogenesis
- First step in tumour invasion and metastases
- Melanocytes adquires mesenchymal phenotype with
migratory and invasive properties
EMT related genes
Switch of cadherin expression during melanoma progression : hypothesis
Cliff Perlis. The Oncologist 2004,9:182-187
NORMAL SKIN
• Expression of E-cadherin in
Keratinocytes and Melanocytes
•Trough E-cadherin keratinocytes
dictate melanocytes behaviour
EARLY
MELANOMAS (RGP)
ADVANCED
MELANOMAS (VGP)
• Maintenance of E-cadherin
• More advanced melanomas
expression by keratinocytes
begin to express N-cadherin and
interact with other cells that
express N-cadherin like
fibroblasts and endothelial cells
•Early melanomas begin to lose
expresssion of E-cadherin and
escape keratinocyte control
EMT: role in melanoma mtx
Supervised hierarchical clustering
Immunohistochemical Validation
Kaplan Meier Univariate Analysis, Free Time of disease:
PROTEÍNA
Caderina N
Osteonectina
Osteopontina
Glypican 3
PKC alfa
1.00
EXPRESIÓN
Número
de casos
Casos con
metástasis
Hazard
ratio
Negativa
Positiva
Negativa
Positiva
Negativa
Positiva
Negativo
Positivo
Negativo
Positivo
79
29
59
53
29
73
25
82
58
61
38
22
28
38
12
49
12
52
32
37
1.00
1.95
1.00
1.99
1.00
1.88
1.00
1.51
1.00
1.21
95% CI
p
Total
1.15-3.31
0.013
108
1.21-3.25
0.006
112
1.00-3.55
0.05
102
0.80-2.83
0.199
107
0.75-1.95
0.429
119
Kaplan-Meier de supervivencia libre enf. (Cadherina N)
0.75
0.50
0.25
1.00
Kaplan-Meier supervivencia libre enf. (Osteopontina)
0.00
0
5
10
Tiempo (años)
Negativa
15
20
Positiva
0.75
0.50
N-Cadherina
0.25
0.00
1.00
Kaplan-Meier supervivencia libre enf. (Osteonectina)
0
5
10
Tiempo (años)
Negativa
0.75
15
Positiva
Osteopontin
0.50
0.25
0.00
0
5
10
Tiempo (años)
Negativa
15
Positiva
Osteonectin
20
20
A high-throughput study in melanoma identifies
Epithelial-Mesenchymal Transition as a major
determinant of metastasis
Soledad R. Alonso1, Lorraine Tracey1, Pablo Ortiz4, Beatriz Pérez-Gómez5, José Palacios1,
Marina Pollán5, Juan Linares6, Salvio Serrano7, Ana I. Sáez-Castillo6, Lydia Sánchez2,
Raquel Pajares2, Abel Sánchez-Aguilera1 , Miguel A. Piris1 and José L. Rodríguez-Peralto3.
From the Molecular Pathology Programme1 and Histology and Immunohistochemistry Unit2, Centro
Nacional de Investigaciones Oncológicas (CNIO), Madrid; Departments of Pathology 3 and
Dermatology4, Hospital Universitario 12 de Octubre, Madrid; Centro Nacional de Epidemiología,
Instituto de Salud Carlos III5, Madrid; Departments of Pathology6 and Dermatology7, Hospital
Universitario San Cecilio, Granada, Spain.
Cancer Research 2007 67:3450-3460
MOLECULAR PATHOLOGY IN MELANOMA
TREATMENT
TREATMENT
• Surgery: Surgical excision with free
margin.
• Elective lymph node dissection
• Surgical excision of isolated metastasis
• Regional Radiotherapy
• Pharmacological Immunomodulator
• Chemotherapy…
 IFN a2b : In Medium risk melanomas (Breslow 1.54mm) or high risk of systemic recurrence (Breslow >4
mm and/or systemic lymph node involvement).
Limited efficacy. 10-20% improvement of free survival.
No decreasment of mortality rate.
 Chemotherapy: In advance stages.
Dacarbacin/Temozolomide.
Useful in less 20% of patients. Transitory responses.
 IL-2: approved for IV stage.
Useful in an small % of patient. Long Hospitality. High
toxicity.
… None systemic treatment has
demonstrated a significant survival
in mestastatic melanoma…
...“During 30 years, there was no significant
advances in treatment of melanoma”…
… ¿A new era? …
- Anti B-RAF  PLX4032
Vemurafenib
- Anti CTLA-4  Ipilimumab
MOLECULAR PATHOLOGY IN
MELANOMA
• A change in the role of pathologist
• Participation in multidisciplinary teams:
– Her2 neu and breast cancer
– K-Ras and colon cancer
– EGFR and pulmonary carcinoma
– EML4-ALK and pulmonary carcinoma
– Braf and metastatic melanoma
Vemurafenib inhibe kinasa BRAF
mutada V600
RTK
RAS
50%* of melanomas
BRAF
RAFV600mut
VEMURAFENIB
ATP
MEK
(PLX4032, RG7204, RO51
ATP
ERK
Cellular
Proliferation
Cellular
Survival
*Total V600 mutation rate for BRIM-3 (cobas® 4800 BRAF V600 Mutation Test); 9.9% of the cobas-positive cases subjected
to retrospective Sanger sequencing had V600K mutations
Cell growth and survival
The role of BRAF
Growth factor
Receptor tyrosine kinase
Cell membrane
RAS-RAF
pathway
RAS
BRAF is a protein
involved in sending
signals in cells for cell
growth
BRAF
MEK
ERK
Normal cell
proliferation and
survival
Normal cell growth
Nucleus
Mutated BRAF
The role of “V600”
Growth factor
Receptor tyrosine kinase
Cell membrane
RAS
A single codon mutation (V600) in the
gene for the BRAF protein leaves it
“switched on”
BRAF
mutation
MEK
Mutated BRAF is present in
many cancers:
>50% melanomas
~10% colorectal
~8% all solid tumors
ERK
Excessive cell
proliferation and survival
Abnormal
cell growth
Nucleus
CONCLUSIONS
 The conventional H&E criteria are still the most useful in
melanoma diagnostic and prognostic factors (Brelow I,
ulceration, Growth phase). Brelow’s index is the most
important prognostic factor
Some tools (tissue arrays, cDNA arrays, RT-PCR) may be
useful in order to discover new molecules that can help us to
predict melanoma aggressiveness
Melanomas with Bcl6 expression or loss p16 have much more
metastatic capability and kill the patient faster
Epithelium-mesenchymal transition molecular changes are
directly involved in melanoma progression
FUTURE
 B-Rafomas: Those melanomas of Non chronic sun damage
skin (trunk) susceptible to be treated with anti-BRAF
drugs
 C-Kitomas: Lentiginous Melanomas especially, lentigo maligna
and acral lentiginous melanomas with c-Kit
mutations
 Gnaqomas: Ocular Melanomas (Gnaq-11 mutations)