MOLECULAR PATHOLOGY IN MELANOMA

Dr José Luis Rodríguez PeraltoHospital 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

PROGRESSION IN THREE CLINICO-PATHOLOGIC STAGES

RADIAL VERTICAL METASTASIS

TISSUE-ARRAY

•Preservation of the original paraffin block

• An excellent tool to simultaneously study numerous specimens using same conditions and criteria

Cell Cycle: activators: cyclins,

inhibitors: Rb, p53, p16, p21, p27

Apoptosis: BCL-2, BCL-XL,Survivin

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,

BCL6, PTEN, IIa Topoisomerase, BMI-1

40 proteins, by IHC

Cytoplasmic Markers:

red (APAAP, neo-fucsin)

Nuclear Markers:

brown (LSAB- DAB)

“Each progression stage of melanoma is characterized by a specific protein expression profile”

Cell cycle Cyc A Cyc D1

CDK1 CDK2

P21

RB pRB HDM-2

Apoptosis SurvivinTranscription factors STAT-1

Membrane receptors C-KIT CaveolinOthers Topoisomerase II BMI-1

Apoptosis SurvivinTranscription factors MUM-1 PKCB

Cell cycle Cyc D1 CDK2 p27 MIB-1

Membrane receptors C-KITDNA repair MLH-1

Cell cycle Cyc D1 Cyc D3 CDK6

p16 p21

DNA repair MLH-1 MSH-2Others Topoisomerase II RING 1B

Apoptosis BCL-2 Transcription factors STAT-1 PKCB MUM-1

RESULTS

VERTICAL METASTASISRADIAL

Adhesion Caderina E B Catenina p120

Adhesion Caderina E B Catenina p120

0 % p 0.303 22% p 0.073 8% p 0.009 32%

CYC-

D3

CYC-

D1

0 % p 0.007 48% p 0.002 14% p 0.021 32%

Cell Cycle Activators NEVUS RADIAL GP VERTICAL GP METASTASIS

p16 INK4

100 % p 0.553 88% p 1.000 89% p 0.009 71%

Cell Cycle Inhibitors NEVUS RADIAL GP VERTICAL GP METASTASIS

p27 KIP

70% p 0.694 76% p 0.010 45% p 0.380 37%

Cell Cycle Inhibitors NEVUS RADIAL GP VERTICAL GP METASTASIS

SURVIVIN

0% p 0.038 36% p 0.002 71% p 0.356 62%

Apoptosis NEVUS RADIAL GP VERTICAL GP METASTASIS

100% p 0.157 77% p 0.336 87% p 0.000 45%

BCL-2

Adhesion Molecules

No differences E Cadherin

B Catenin

N Cadherin

E Cadherin

B Catenin

N Cadherin

Nuclear B Catenin

Cyclin ADegradation

Cyclin BDegradation

G2 DNA REPAIR

S DNA REPLICATION

G1

G0

M CELL DIVISION

p53

Hdm2p14

_

_

P

Rb

G2 +

RbP

CycA

CycDCDK4/6

CycECDK2

CDK2

CycA/B

CDK1

INCREASE CELL CYCLE ACTIVATORS

p21

p16

p27

_

_

_

_

LOSS CELL CYCLE

INHIBITORS DNA

REPLICATION

G1

Cyt C

SURVIVIN

Surv

p53

NF-kB

NF-kB

C-IAP

CELL SURVIVAL

DEATH LIGAND

DEATH RECEPTOR

TNF

TNFR

Bcl-6

FLIP

BCL-2

Bcl-XL

Bcl-2

APOPTOSIS

CASP 3,6,7

CASP9

FADD

Bcl-XL

BAD

CASP 8

BAX

BID

SMAC

APAF-1

APOPTOSOMA

Melanoma Tissue Microarrays

Results.- clinical follow up: predictive model

Fig 3B. Curva representando el efecto de la expresión de las proteínas p16, p21, Ki-67 y BCL-6 en la supervivencia global de pacientes con Melanoma Cutáneo.

1 2 3 4 5 6

0.00

0.25

0.50

0.75

1.00

Años desde el diagnóstico hasta el fallecimiento

BAJO RIESGO: p16+ y el

resto negativos

RIESGO INTERMEDIO:

p16+ y BCL-6 - con cualquiera Ki 67>20%

y /o p21+

ALTO RIESGO: p16 - y/o

BCL6 +

Supervivencia Global

Statistical Analysis:

-Univariate. Relevant variables were included

-Multivariate. final model taking the predicted HR*

Proteina RR 95 %IC p-val

BCL6 + 8.101 2.56-25.26 <0.001 Ki67 2.41 0.94-6.17 0.068P16 + 0.13 0.04-0.41 0.001P21 + 2.36 0.95-5.86 0.065

MULTIVARIATE ANALYSIS

*Performed by backwards elimination, starting with markers with a p < 0.10

Three groups of risk: Ki67, p21, p16, BCL6

- Shorter overall survival: loss of p16 and/or BCL6 expression

- No changes by including the Breslow’s index

Alonso et al AJPathol 2004, 164 p193

LOW RISK p16 + and rest negatives

INTERMEDIUM RISK p16 + and BCL6 -and Ki 67 >20% oand/or p21 +

HIGH RISK p16 - and/or BCL6 +

Years from the diagnosis to the death

OS PREDICTIVE MODEL

1. Low risK: p16+ and rest negatives

2. Intermedium risk: p16+ y BCL-6- and Ki 67>20%

and /or p21+

3. High risk P16 - and/or BCL6 +0.00

0.25

0.50

0.75

1.00

Years

1 2 3 4 5 6

-Shorter overall survival: Loss of p16 and/or BCL6 expression

- No changes by including the Breslow’s index

• Time from diagnostic to death by this condition• 60 melanomas on vertical grow phase without metastasis at diagnostic time

34 Primary Melanomas

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

FOLLOW UP

NON METASTATIC DISEASE (13)

METASTATIC DISEASE (22)

Molecular Differences ???

Validation: To confirm the results. Independent series of 131 cases, 6 TMAs (73 with metastasis /56 without metastasis)

36 months

19%

12%

2%

6%34%

3%

3%

1%

2%18%

EMT relacionado Crecimiento y DiferenciaciónRespuesta Inmune Transducción de señalesMetabolismo Funciones celulares básicas Muerte celularCiclo CelularAngiogénesis Miscelánea

Results

91- GENES UP REGULATED

Gene Name Dif S vs N Function Description

EDNRB 1.1845 Angiogenesis, negative regulation of EDNEndothelin receptor type B

PDE5A 1.0360 Angiogenesis, regulation of vessel size,dilatationPhosphodiesterase 5A, cGMP-specific

SPP1 1.6610 EMT related, adhesion, migration, cell survival, and tumorigenesis.secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1)

SPARC 1.2270 EMT related, bone remodeling Secreted protein, acidic, cysteine-rich (osteonectin)

PRKCA 1.5890 EMT related, cell adhesion Protein kinase C, alpha

TUBB3 1.3450 EMT related, cell adhesion. link with cadherinsTubulin, beta 3

H2-ALPHA 1.1920 EMT related, cell adhesion, link with cadherinsTubulin, alpha 2

TUBA2 1.0825 EMT related, cell adhesion, link with cadherinsTubulin, alpha 2

TUBA3 1.0155 EMT related, cell adhesion, link with cadherinsTubulin, alpha 3

CDH2 1.4130 EMT related, cell adhesion Cadherin 2, type 1, N-cadherin (neuronal)

DSG2 1.1210 EMT related, cell adhesion Desmoglein 2

NID2 1.0865 EMT related, cell adhesion Nidogen 2 (osteonidogen)

EMP1 1.1905 EMT related, cell adhesion regulator Epithelial membrane protein 1

LUM 3.1030 EMT related, organismal movement, matrix organizationLumican

COL3A1 1.1915 EMT related, organogenesis Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant)

GPC3 2.0965 EMT related; cell growth and maintenanceGlypican 3

SDCBP 1.5895 EMT related; cell motility Syndecan binding protein (syntenin)

HMMR 1.3425 EMT related; cell motility Hyaluronan-mediated motility receptor (RHAMM)

SMARCA1 1.0150 EMT related; matrix associated SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1

CA9 1.1375 Cell growth Carbonic anhydrase IX

CGI-141 1.1700 Cell growth and differentiation CGI-141 protein

LAPTM4A 1.4960 Cell growth and maintenance Lysosomal-associated protein transmembrane 4 alpha

IGFBP1 1.2980 Cell growth and maintenance Insulin-like growth factor binding protein 1

MYB 1.2595 Cell growth and maintenance V-myb myeloblastosis viral oncogene homolog (avian)

KIT 1.1910 Cell growth and maintenance V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog

WEE1 1.1395 Cell growth and maintenance WEE1 homolog (S. pombe)

PMP22 1.0755 Cell growth and maintenance Peripheral myelin protein 22

YWHAQ 1.0455 Cell growth and maintenance Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide

DUSP12 1.0185 Cell growth and maintenance Dual specificity phosphatase 12

NME2 1.0105 Cell growth and maintenance Non-metastatic cells 2, protein (NM23B)

ILF2 1.3090 Immune response Interleukin enhancer binding factor 2, 45kDa

SERPINA3 1.0540 Immune response Serpina 3 (alpha 1 antichymotrypsin y antitrypsin) inhibitor of N Killer cells

CASP5 1.3060 Cell death Caspase 5, apoptosis-related cysteine protease

FAIM 1.0845 Cell death Fas apoptotic inhibitory molecule

GAS1 1.0540 Cell death Growth arrest-specific 1

SKP1 1.1515 Cell cycle SKP1(S-phase kinase-associated protein 1A)_HUMAN Cyclin A/CDK2-associated protein p19

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

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: role in melanoma mtx

EARLY MELANOMAS (RGP)

NORMAL SKIN ADVANCED MELANOMAS (VGP)

EMT related genes

Cliff Perlis. The Oncologist 2004,9:182-187

• Expression of E-cadherin in Keratinocytes and Melanocytes

•Trough E-cadherin keratinocytes dictate melanocytes behaviour

• Maintenance of E-cadherin expression by keratinocytes

•Early melanomas begin to lose expresssion of E-cadherin and escape keratinocyte control

• More advanced melanomas begin to express N-cadherin and interact with other cells that express N-cadherin like fibroblasts and endothelial cells

Switch of cadherin expression during melanoma progression : hypothesis

EMT: role in melanoma mtx Supervised hierarchical clustering

Immunohistochemical Validation

PROTEÍNA EXPRESIÓN Número de casos

Casos con metástasis

Hazard ratio 95% CI p Total

Negativa 79 38 1.00 Caderina N

Positiva 29 22 1.95 1.15-3.31 0.013 108

Negativa 59 28 1.00 Osteonectina

Positiva 53 38 1.99 1.21-3.25 0.006 112

Negativa 29 12 1.00 Osteopontina

Positiva 73 49 1.88 1.00-3.55 0.05 102

Negativo 25 12 1.00 Glypican 3

Positivo 82 52 1.51 0.80-2.83 0.199 107

Negativo 58 32 1.00 PKC alfa

Positivo 61 37 1.21 0.75-1.95 0.429 119

Kaplan Meier Univariate Analysis, Free Time of disease:

0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 Tiempo (años)

Negativa Positiva

Kaplan-Meier supervivencia libre enf. (Osteonectina)

Osteonectin

0.00

0.25

0.50 0.75

1.00

0 5 10 15 20 Tiempo (años)

Negativa Positiva

Kaplan-Meier de supervivencia libre enf. (Cadherina N)

N-Cadherina

0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 Tiempo (años)

Negativa Positiva

Kaplan-Meier supervivencia libre enf. (Osteopontina)

Osteopontin

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 1.5-4mm) 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

- Anti CTLA-4 Ipilimumab

Vemurafenib

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

CellularProliferation

RTKRTK

RAFVEMURAFENIB (PLX4032, RG7204, RO5185426)

ATP

ATP

ERK

MEK

BRAFV600mut

RAS

50%* of melanomas

CellularSurvival

*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

Growth factor

RASRAS

MEKMEK

ERKERK

Normal cell growth

BRAFBRAF

Cell membrane

Nucleus

BRAF is a protein involved in sending signals in cells for cell growth

Cell growth and survivalThe role of BRAF

Receptor tyrosine kinase

RAS-RAFpathway

Normal cellproliferation and

survival

Nucleus

MEKMEK

ERKERK

Abnormalcell growth

BRAFmutation

BRAFmutation

A single codon mutation (V600) in the gene for the BRAF protein leaves it “switched on”

Growth factor

RASRAS

Cell membrane

Mutated BRAF is present in many cancers:

>50% melanomas~10% colorectal ~8% all solid tumors

Mutated BRAF is present in many cancers:

>50% melanomas~10% colorectal ~8% all solid tumors

Mutated BRAFThe role of “V600”

Receptor tyrosine kinase

Excessive cellproliferation and survival

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)