GENETICS OF MELANOMA

Ivana AntigoniGenetica Medica

“Sapienza – Università di Roma”Ospedale S. Camillo

Kidney cancer and melanoma:

progresses in clinical and translational research

Rome, November 23-24, 2007

Genetic susceptibility to Melanoma

5-10% of individuals who develop melanoma show an affected close

relative

Susceptibility to melanoma is inherited as an autosomal dominant trait, with a incomplete penetrance

Clinical risk factors for Melanoma

Genetics of melanoma

Several genes are involved in the progression of cutaneous melanoma

Two major groups of genes:

1. PROTO-ONCOGENES

2. TUMOR SUPPRESSOR GENES

1. Proto-oncogenes

GENE LOCUS FUNCTION/PATHOLOGYASSOCIATED

CDK4 12q14 Cell cycle

CTNNB1 3p22-p21.3 Cell cycle

* BRAF 7q34 Ser/Thr Kinasi

* NRAS 1p13.2 Melanoma

* KRAS2 12p12.1 Melanoma

* HRAS 11p15.5 Spitz Nevus

* MAPK PATHWAY (Mitogen-Activated Protein Kinase)

CDK4 (12q13)

Cyclin Dependent Kinases (promotes cell cycle progression from G1 to S phase)

Action: phosphorylation of Rb1

Same pathway of p16 (functionally antagonist)

Proto-oncogene

BRAF (7q34)MAPK PATHWAY: BRAF/NRAS/HRAS2/HRAS

Growth factor receptors interaction with its ligands induces Ras activation

Ras activation stimulates phosphorylation of RAF proteins (including BRAF)

The final step of this cascade (MEK/ERK/CCND1/Cdk4-6) is the

activation of transcription factors (G1/S)

Frequency of BRAF mutations in melanocytic nevi is similar to that in melanomas

suggesting that BRAF function inactivation could be an “early event” from healty skin

to nevi

BRAF mutations have been identified in more than 60% of melanomas

and 80% of these occur at a single site (hot spot) in position 600 (V600E)

MAPK PATHWAY

In contrast to BRAF mutations, NRAS alterations are more common in sun-exposed areas

HRAS mutations are less commonly observed and occurs in no more than 1.5-3% of melanomas

Activating changes in KRAS2 have been observed in rare cases and often associated with other

Ras genes mutations

2.Tumor Suppressor Genes

GENE LOCUS FUNCTION/PATHOLOGYASSOCIATED

CDKN2A 9p21 Cell cycle

ARF 9p21 Cell cycle

PTEN 10q23.31 Cowden Desease

RB1 13q14.2 Retinoblastoma

TP53 17p13.1 Li-Fraumeni Syndrome (LFS)

CDKN2A/p14ARF (9p21)

Encodes two distinct proteins: INK4A (p16), ARF (p14)

(alternative promoters and first exons: 1α, 1ß)

Coding exons: 3 (ex 1, 125 bp; ex 2, 307 bp; ex 3, 12 bp)

CDKN2A/p14ARF (1)

INK4A (p16): cell cycle G1/S

progression specific inhibitor.

Inhibits complex CDKs/cyclin D through

competitive binding to Cdks 4/6

p14ARF: inactivation of HDM2 which inhibits p53, by binding and degradation (apoptosis

inhibition)

PTEN (10q23.31)• The chromosome region in which this gene

maps is deleted in about 30-50% of melanomas

• Somatic PTEN mutations have been identified in approximately 3% of primary melanomas and

8% of metastatic melanomas

TP53 (17p13.1)• Mutations in melanomas are rare (7%)

• However, UVR is very likely the cause of these mutations, as well as in other non-melanocytic skin

tumors

• Identified in 15-20% of melanoma-prone families (at least two affected first or second degree

relatives)

• Present in 9-15 % of

sporadic multiple primary melanomas (MPM)

• Penetrance of 53% by age 80

• Interaction with sunlight exposure

• High frequency of multiple primary melanomas (MPM), pancreatic and larynx cancer

CDKN2A germline mutations in familial melanoma

MC1R (melanocortin-1 receptor) has been identified as a low-risk melanoma-susceptibility gene

Variants of the MC1R are major determinants of high-risk

phenotypes (red hair and pale skin) and of the ability to tan in response to UV exposure

(RR: 1 variant = 2-5; 2 variant = 7)

Some MCR1 polymorphisms could act as modifier alleles, able to

increase CDKN2A mutations penetrance

MC1R polymorphisms (16q24.3)MC1R polymorphisms (16q24.3)

FAMILIAL MELANOMA (FM)

MULTIPLE PRIMARY MELANOMAS (MPM)

FAMILIAL MULTIPLE MELANOMA (FAMMM)

156 ITALIAN MELANOMA PEDIGREES

97 FM (22 FAMMM)

59 Sporadic Multiple Primary Melanomas (MPM)

MOLECULAR ANALYSIS

CDKN2A (sequencing analysis of exon 1 α, 1 ß, 2, 3 and exon-intron boundaries)

CDKN2A (Multiple Ligation Probe Amplification)

CDK4 (sequencing analysis of exon 2)

FAMILY Nr of affected individuals

MPM CDKN2A mutation

FM008 3 SI gly101trp

FM009 3 SI asn71ser

FM012 3 NO pro48thr

FM013 2 SI 201delC

FM014 2 SI arg24pro

FM020 2 SI gly101trp

FM021 2 SI gly101trp

FM056 3 NO arg24pro

FM060 1 SI asp105glu/asn71ile *

FM068 1 SI IVS2-105A>G

FM108 3 NO asn71ile

FM132 4 SI arg24pro

FM136 1 SI asn71ser

FM144 1 SI gly101trp

FM148 2 NO gly101trp

FM152 2 NO 132delC

FM165 2 SI gly101trp* new mutation

17/156 (10,8%)

CDKN2A MUTATION

-------------------

12,3% FM/FAMMM

6,8% MPM

9 DIFFERENT MUTATIONS

6 Gly101Trp PEDIGREES

1 PZ: COMPOUND HETEROZYGOTE

1 PZ: INTRONIC MUTATION

FM/MPM/FAMMM

Absence of family history for melanoma

Father deceased for lung carcinoma

CDKN2A Compound Heterozygote

Asp105Glu/Asn71Ile

Female, 38 y.o.,

familiar origin: Avezzano (Central Italy)

March 1988: removal of 2 melanomas from deltoid region

March 1998: removal of 1 melanoma from right haunch

May 1998: removal of 5 melanomas

December 2001: removal of relapsing melanoma (right haunch)

Majore et al., Journal of Investigative Dermatology 122 (2), 450-451, 2004

Man, 49 y.o., familiar origin: Velletri (Central Italy), type II skin, blu eyes, more than 50 melanocytic nevi,

8 primary melanomas (at 37), absence of family history for melanoma

CDKN2A: IVS2-105 A>G

1 son with IVS2-105 A>G/+

(follow-up)

6 English families (same haplotype)

Our patient: “de novo” variant

Different haplotype from English pedigrees

IVS2-105 mutation “hot-spot”

IVS2-105A>G

Multiple Ligation Probe Amplification (MLPA)

90 PEDIGREES (CDKN2A negative) analysed:

absence of deletions/duplications

CDKN2A 1β (p14ARF): worldwide mutations

Locus Base Nr of geneal. Origin

g.192 A>T 1 UKg.193 G>C 1 UKg.193+1 G>A 1 Netherlandg.193+2 T>C 1 USAg.193+3 A>G 1 UKg.193+56 T>C 2 UK

Harland et al., Oncogene 24: 4604-4608, 2005

Mutation Nr of geneal. Origin

del ex 1ß 1 UKins 16 bp 1 Spain

Rizos et al., 2001; Randerson-Moor et al., 2001

g.193+1 G>A

(2 families)

CDKN2A/p14ARF: exon 1β

66/136 pedigrees CDKN2A negative

1992: removal of 1 melanoma from dorsum

1995: removal of 1 melanoma from trunk

1995: removal of 1 melanoma from dorsum

2002: removal of 1 melanoma from arm

1990: right haunch dysplastic nevi

1990: trunk dysplastic nevi

1991: trunk dysplastic nevi

LAST FOLLOW-UP 27-03-2007:

Disease free

Female patient, 20 y.o., red hair,

green eyes, type II skin, UV burns

Pz. S. Gallicano Rome/Univ. Catanzaro:Familial origin: Rome/S. Vito Romano

2 2 4

g.193+1 G>A

MELANOMA

45/62

30/34 2035

6472

> 70 > 70

MPM

MPM: MULTIPLE PRIMARY MELANOMAS

g.193+1 G>A

MELANOMA

61

40/54

g.193+1 G>A

g.193+1 G>A

66

WT

WTWTWT 5964

Pz S. Gallicano Rome – familial origin: Rome

CDKN2A/p14Arf NEGATIVE PEDIGREES (134)

(sequencing analysis of exon 2 CDK4 gene)

7 FM pedigrees with mutations in CDK4 (codon 24) described worldwide

2 French (Arg24His)2 USA (Arg24Cys)

1 Norvegian (Arg24His)1 Australian (Arg24His)

1 English (Arg24His)

1 Italian (Arg24His)

CDK4

MPM

38 41

4559

CDK4: Arg24His

Arg24His

Arg24His

Arg24His

Melanoma Gastric carcinoma

41

trunk melanoma

melanoma

melanoma

epatic carcinoma

Arg24His19

(follow-up)

Majore et al. Pigm Cell α Mel Res, in press.

CDK4 HAPLOTYPE (microsatellite)

Mutations are rare (8 cases)

High expressivity

100% penetrance (?)

No founder effect

CDK4

17/156 (10,8%)

CDKN2A MUTATIONS

12,3% FM, FMMM

6,8% MPM

9 DIFFERENT MUTATIONS

1 PZ: COMPOUND

HETEROZYGOTE

3 NEW MUTATIONS

FAMILIAL MELANOMA (FM, FAMMM),MULTIPLE PRIMARY MELANOMA (MPM)

CDKN2A EXON 1β:

2 FAMILIES: g.193+1 G>A

CDK4

1 FAMILY: Arg24His

20/156: 12.8%

UOC LABORATORIO DI GENETICA MEDICA,

“SAPIENZA” UNIVERSITA’ DI ROMA,

A.O. S. CAMILLO-FORLANINI, ROMA

Francesco Binni, Carmelilia De Bernardo, Silvia Majore, Alessandra Crisi, Ivana Antigoni, Paola Grammatico

UOC DERMATOLOGIA ONCOLOGICAS. GALLICANO, IFO, ROMA

Paola De Simone, Laura Eibenschutz, Caterina Catricalà

DERMATOLOGIA UNIVERSITA’ DI

CATANZARO

Ugo Bottoni

POLICLINICO MILITARE CELIO DI

ROMA

Tiziana Sbezzi

UOSD DERMATOLOGIA

OSP. S. CAMILLO, ROMA

Giovanni Cruciani

Genetics of Melanoma