genetics of melanoma ivana antigoni genetica medica “sapienza – università di roma” ospedale...
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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