Studi sull’eziologia dei
Tumori Cutanei:
risultati, problemi prospettive
CPO – Registro Tumori Piemonte
Stefano Rosso, Roberto Zanetti
Riunione del Comitato Scientifico CPO,
Torino, 3 ottobre 2008
Correva l’anno 1982…
• Studio promosso da IARC e IACR:
– Descriptive epidemiology of malignant
melanoma
In quegli anni i primi studi caso-controllo
sull’eziologia del melanoma
•
Klepp O, Magnus K. Some environmental and bodily characteristics of
melanoma patients. A case-control study. Int J Cancer. 1979 15; 23: 482-6.
• Adam SA, Sheaves JK, Wright NH, Mosser G, Harris RW, Vessey MP. A casecontrol study of the possible association between oral contraceptives and malignant
melanoma. Br J Cancer. 1981; 44: 45-50.
• MacKie RM, Aitchison T. Severe sunburn and subsequent risk of primary
cutaneous malignant melanoma in Scotland. Br J Cancer. 1982; 46: 955-60.
• Lew RA, Sober AJ, Cook N, Marvell R, Fitzpatrick TB. Sun exposure habits in
patients with cutaneous melanoma: a case control study. J Dermatol Surg Oncol.
1983; 9: 981-6.
• Acquavella JF, Wilkinson GS, Tietjen GL, Key CR, Stebbings JH, Voelz GL. A
melanoma case-control study at the Los Alamos National Laboratory. Health
Phys. 1983; 45: 587-92.
• Beral V, Evans S, Shaw H, Milton G. Cutaneous factors related to the risk of
malignant melanoma. Br J Dermatol. 1983; 109: 165-72.
… e nel 1984 parte il nostro primo studio
caso-controllo sull’eziologia del melanoma
Gli studi di quegli anni
• Holman CD, Armstrong BK. Cutaneous malignant melanoma and
indicators of total accumulated exposure to the sun: an analysis
separating histogenetic types. JNCI 1984; 73: 75-82.
• Holman CD, Armstrong BK. Pigmentary traits, ethnic origin,
benign nevi, and family history as risk factors for cutaneous
malignant melanoma. JNCI 1984; 72: 257-66.
• Elwood JM, Gallagher RP, Hill GB, Spinelli JJ, Pearson JC,
Threlfall W. Pigmentation and skin reaction to sun as risk
factors for cutaneous melanoma: Western Canada Melanoma
Study. Br Med J (Clin Res Ed). 1984; 288: 99-102.
• Beral V, Evans S, Shaw H, Milton G. Oral contraceptive use
and malignant melanoma in Australia. Br J Cancer. 1984; 50:
681-5.
I nostri risultati
•
•
•
•
Zanetti R, Rosso S, Colonna S, Martina G, Paudice A. Casecontrol study on malignant skin melanoma in the Turin
province. G Ital Dermatol Venereol. 1988; 123: 461-8.
Zanetti R, Rosso S, Faggiano F, Roffino R, Colonna S, Martina
G. A case-control study of melanoma of the skin in the province
of Torino, Italy. Rev Epidemiol Sante Publique. 1988; 36: 30917.
Zanetti R, Franceschi S, Rosso S, Bidoli E, Colonna S.
Cutaneous malignant melanoma in females: the role of
hormonal and reproductive factors. Int J Epidemiol. 1990;
19: 522-6.
Zanetti R, Franceschi S, Rosso S, Colonna S, Bidoli E.
Cutaneous melanoma and sunburns in childhood in a southern
European population. Eur J Cancer. 1992; 28A: 1172-6.
… the role of hormonal and
reproductive factors …
• Karagas MR, Stukel TA, Dykes J, Miglionico J, Greene
MA, Carey M, Armstrong B, Elwood JM, Gallagher RP,
Green A, Holly EA, Kirkpatrick CS, Mack T, Osterlind A,
Rosso S, Swerdlow AJ. A pooled analysis of 10 casecontrol studies of melanoma and oral contraceptive use. Br
J Cancer 2002; 86: 1085-92
• Karagas MR, Zens MS, Stukel TA, Swerdlow AJ, Rosso
S, Osterlind A, Mack T, Kirkpatrick C, Holly EA, Green
A, Gallagher R, Elwood JM, Armstrong BK. Pregnancy
history and incidence of melanoma in women: a pooled
analysis. Cancer Causes Control. 2006; 17: 11-9.
… Cutaneous melanoma and sunburns in
childhood …
• Zanetti R. et al. 1988 Rev Epidemiol Sante
Publique: …total number of weeks spent at
beach during holidays in childhood is
associated with an increased risk in men:
– OR=2.4(1.59-5.9) 3.7(1.4-9.7) …
• Østerlind et al. 1988 Int J Cancer:
– OR=3.7 (2.3-6.1)
Ipotesi dell’esposizione
“intermittente” al sole
• Armstrong BK. Epidemiology of malignant
melanoma: intermittent or total accumulated
exposure to the sun? J Dermatol Surg
Oncol. 1988; 14: 835-49.
• Zanetti R. et al. 1988 Rev Epidemiol Sante
Publique: … The increased risk of intense
and occasional sun exposure … has been
confirmed…
Gli studi sui carcinomi cutanei
• Kricker A, Armstrong BK, English DR, Heenan PJ (1991)
Pigmentary and cutaneous risk factors for non-melanocytic
skin cancer - a case-control study. Int J Cancer, 48: 650662.
• Gallagher RP, Hill GB, Bajdic CD, Fincham S, Coldman
AJ, McLean DI, Threlfall WJ (1995a) Sunlight exposure,
pigmentary factors, and risk of nonmelanocytic skin
cancer. I. Basal cell carcinoma. Arch Dermatol 131: 157163.
• Gallagher RP, Hill GB, Bajdic CD, Fincham S, Coldman
AJ, McLean DI, Threfall WJ (1995b) Sunlight exposure,
pigmentary factors, and risk of nonmelanocytic skin
cancer. II. Squamous cell carcinoma. Arch Dermatol 131:
164-169.
Lo studio Helios I sui carcinomi
cutanei
• Studio multicentrico (Italia, Francia,
Spagna, Svizzera) caso-controllo sui
carcinomi cutanei sqamocellulari e
basocellulari
• Reclutamento: 1989-1993
• 1549 BCC, 228 SCC, 1795 controlli
I nostri risultati sui carcinomi cutanei
•
•
•
•
•
•
•
Zanetti R, Rosso S, Martinez C, Navarro C, Schraub S, Sancho-Garnier H, Franceschi S, Gafa L, Perea
E, Tormo MJ, Laurent R, Schrameck C, Cristofolini M, Tumino R, Wechsler J. The multicentre south
European study 'Helios'. I: Skin characteristics and sunburns in basal cell and squamous cell
carcinomas of the skin. Br J Cancer. 1996; 73: 1440-6.
Rosso S, Zanetti R, Martinez C, Tormo MJ, Schraub S, Sancho-Garnier H, Franceschi S, Gafa L, Perea
E, Navarro C, Laurent R, Schrameck C, Talamini R, Tumino R, Wechsler J. The multicentre south
European study 'Helios'. II: Different sun exposure patterns in the aetiology of basal cell and squamous
cell carcinomas of the skin. Br J Cancer. 1996; 73: 1447-54.
Rosso S, Zanetti R, Pippione M, Sancho-Garnier H. Parallel risk assessment of melanoma and basal
cell carcinoma: skin characteristics and sun exposure. Melanoma Res. 1998; 8: 573-83.
Zanetti R, Gafa L, Franceschi S, Pippione M, Rosso S. Estimate of the proportion of skin tumors
attributable to sun exposure in 3 Italian populations. Epidemiol Prev. 1999; 23: 416-22.
Rosso S, Joris F, Zanetti R. Risk of basal and squamous cell carcinomas of the skin in Sion, Switzerland:
a case-control study. Tumori. 1999; 85: 435-42
Suarez B, Lopez-Abente G, Martinez C, Navarro C, Tormo MJ, Rosso S, Schraub S, Gafa L, SanchoGarnier H, Wechsler J, Zanetti R. Occupation and skin cancer: the results of the HELIOS-I multicenter
case-control study. BMC Public Health. 2007; 7: 180
Zanetti R, Rosso S, Martinez C, Nieto A, Miranda A, Mercier M, Loria DI, Osterlind A, Greiner R,
Navarro C, Fabbrocini G, Barbera C, Rancho-Garnier H, Gafa L, Chiarugi A, Mossoti R. Comparison of
risk patterns in carcinoma and melanoma of the skin in men: a multi-centre case-case-control study.
Br J Cancer. 2006; 94: 743-51.
Meta-analisi sul melanoma
•
•
•
•
•
•
Bliss JM, Ford D, Swerdlow AJ, et al. Risk of cutaneous melanoma associated
with pigmentation characteristics and freckling: systematic overview of 10
case-control studies. The International Melanoma Analysis Group (IMAGE).
Int J Cancer, 1995; 62: 367-376.
Elwood JM, Jopson J. Melanoma and sun exposure: an overview of published
results. Int J Cancer. 1997; 73: 198-203.
Whiteman DC, Whiteman CA, Green AC. Childhood sun exposure as a risk
factor for melanoma: a systematic review of epidemiologic studies. Cancer
Causes Control, 2001; 12: 69-82.
Gandini S, Sera F, Cattaruzza MS, Pasquini P, Abeni D, Boyle P, Melchi CF.
Meta-analysis of risk factors for cutaneous melanoma: I. Common and
atypical naevi. Eur J Cancer, 2005; 41: 28-44.
Gandini S, Sera F, Cattaruzza MS, Pasquini P, Picconi O, Boyle P, Melchi
CF. Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure.
Eur J Cancer, 2005; 41: 45-60.
Gandini S, Sera F, Cattaruzza MS, Pasquini P, Zanetti R, Masini C, Boyle P,
Melchi CF. Meta-analysis of risk factors for cutaneous melanoma: III. Family
history, actinic damage and phenotypic factors. Eur J Cancer. 2005; 41: 204059
LA curva dose-risposta
Rosso, Zanetti, Pippione, Sancho-Garnier,
Melanoma Res 1998; 8: 573-583
… senza trascurare gli aspetti di
metodologia della misura …
• Wechsler J, Zanetti R, Schrameck C, Rosso S, Pippione M,
Linares J, Laurent R, Ortuno G, Boi S, Gafa L, Joris F,
Spatz A, Barneon G, Sacerdote C, Sancho-Garnier H and
the HELIOS Group. Reproducibility of histopathologic
diagnosis and classification of non-melanocytic skin
cancer: a panel exercise in the framework of the
multicenter southern European study HELIOS. Tumori.
2001; 87: 95-100.
• Rosso S, Minarro R, Schraub S, Tumino R, Franceschi S,
Zanetti R. Reproducibility of skin characteristic
measurements and reported sun exposure history. Int J
Epidemiol. 2002; 31: 439-46.
All’inizio del millennio nuovi studi
su markers biologici
• Studi su markers somatici del melanoma
(es. BRAF), proto-oncogeni (es CDKN2C)
o polimorfismi (MC1R)
• Studi su marker biochimici: melanine
T y ro s in a s e
HO
N H2
HO
C O2H
HO
COOH O
2
N H2
HO
T y r o sin e
O2
C O2H
O
N H2
O
DO PA
D o p a q u in o n e
COOH
HS
HO
O
-
COOH
COOH
+
D op ac h r om e
Z n 2+
OH
L e uc od op ac hr om e
H2N
N
H
HO
5 - S - C y st e in y ld o p a
O
N
H
H
N
C O2H
DHI
D H IC A
N H2
COOH
HO
COOH
COOH
S
-C O 2
HO
HO
OH
N
H
HO
N
H
O
N H2
c y s te in y ld o p a
c y s te in y ld o p a
d o p a q u in o n e
OH
S
Z n 2+
[O ]
H
N
HO
OH
OH
H
N
CO2H
OH
COOH
NH
HO
HO
N
H
H2N
H2N
HO
H2N
CO2H
N
S
)2
S
N
H
COOH
OH
N
H
HO
HO
COOH
S
COOH
H2N
CO2H
HO
H2N
S
HO
N
H HO
N
H
COOH
C O2H
OH
OH
H O2C
OH
N
H2N
COOH
O
N H2
CO2H
O
H
N
S
NH
S
H
N
S
OH
E U M E L A N IN S
H
N
S
OH
N
HO
NH
CO2H
N
C O2H
HO
HO
COOH
H2N
OH
S
H2N
C O2H
OH
H2N
COOH
P H E O M E L A N IN S and T R IC H O C H R O M E S
CO2H
IDENTIFICATION AND QUANTITATION OF
MELANINS
IN PIGMENTED TISSUES
DEGRADATIVE APPROACH
(degradation of pigmented
tissue)
Spectrophotometric
analysis
HPLC
analysis
COMPLEMENTARY APPROACH:
In vitro reconstruction
of the biosynthetic pathway
… un lungo periodo trascorso ad
affinare le metodiche d’analisi …
• Zanetti R, Prota G, Napolitano A, Martinez C, Sancho-Garnier H,
Osterlind A, Sacerdote C, Rosso S. Development of an integrated
method of skin phenotype measurement using the melanins.
Melanoma Res. 2001; 11: 551-7.
• Zoccola M, Mossoti R, Innocenti R, Loria DI, Rosso S, Zanetti R.
Near infrared spectroscopy as a tool for the determination of
eumelanin in human hair. Pigment Cell Res. 2004; 17: 379-385.
• Zoccola M, Mossotti R, Innocenti R, Loria DI, Rosso S, Zanetti R. A
new approach in the determination of eumelanin in human hair using
near infrared spectroscopy. NIR news 2005. 16: 10-12
• Zanetti R, Zoccola M, Mossotti R, Innocenti R, Loria DI, Rosso S;
Helios 2 Working Group. PTCA determination in human hair:
reliability and analytical aspects. Med Sci Monit. 2006; 12: 23-8.
Studio Helios 2
• Studio multi-centrico (Italia, Francia,
Spagna, Portogallo, Danimarca, Germania,
Argentina) su BCC (215), SCC (139),
CMM (214) e gruppo di controllo (349)
• Reclutamento: 2001-2002
• Determinazione delle melanine su campioni
di capelli
Determinazione di eumelanine su campioni di capelli
Materiali: 100 campioni di capelli di casi (melanoma)
100 campioni di capelli di controlli
¾Le eumelanine nei campioni di
capelli sono state ossidate con
H2O2 e il marker strutturale
quantitativo delle eumelanine, il
PTCA, è stato determinato
tramite HPLC.
¾I campioni di capelli (10 mg) sono stati
immersi in NaOH 1N contenente H2O2 e
lasciati reagire per 24 h, a 20 °C sotto
agitazione.
Colonna:
C18 Xterra (Waters) 30 °C
Eluente:
97% acido formico 0.4 M e sodio
ottanosulfonato
0.5mM
pH
2.5,
3%metanolo, (vol/vol)
Flusso : 0.7ml/min
Detector: UV 270 nm
¾La H2O2 residua è stata decomposta con
aggiunta di
Na2S2O5 la miscela è stata
acidificata a pH 4 e centrifugata. Il
supernatante è stato filtrato e iniettato per
analisi HPLC.
PTCA
(ng/mg)
Determinazione di PTCA su
capelli tramite HPLC
Capelli rossi
Capelli biondi
Capelli castani
4
Capelli neri
Profili cromatografici ottenuti dalla miscela
di degradazione di quattro campioni di
capelli di differente colore: capelli rossi,
biondi, bruni e neri.
Capelli rossi
30
Capelli biondi
50
Capelli castani
100
Capelli neri
200
¾La resa di PTCA è di circa il 6-7% a
partire da DHICA melanine
¾Il PTCA deriva soprattutto da DHICA
unità delle melanine, e in bassa quantita
da DHI unità: In capelli di differente
colore il rapporto DHI/DHICA non è
costante.
¾PTCA è formato in bassa quantità da
feomelanine ossidate con H2O2..
Table 2. Risk of CMM for different levels and measurement scale of
PTCA (eumelanin) in hair. In parenthesis 95% credible regions
PANEL A
Category of exposure for
level of PTCA in ng/mg
50 <
Case
Control5
+
CaseControl+
4
Crude OR
6.2
50-75
13
8
75-100
26
13
100-125
18
22
125-150
16
19
150 +
6
18
Case+
Control26
CaseControl+
7
Case <
Control *
63
Case >
Control #
35
(1.12-38.78)
5.6
Adj OR
2.6
(0.50-30.20)
3.4
AdjOR FS
2.5
(0.22-28.38)
3.2
(1.57-23-89
5.6
(1.97-18.63)
2.6
(0.94-7.82)
2.5
(0.91-7.73)
1
(1.09-19.71)
3.2
(1.44-13.20)
1.4
(0.58-5.25)
1.7
(0.57-6.07)
1
(0.54-18.77)
3.0
(0.77-11.92)
1.4
(0.41-4.78)
1.7
(0.52-5.37)
1
Crude OR
3.7
(1.61-8.56)
Adj OR
4.4
(1.52-14.54)
AdjOR FS
4.8
(1.41-16.05)
Crude OR
1.016
Adj OR
1.013
AdjOR FS
1.013
(1.006-1.027)
(1.001-1.027)
(0.999-1.028)
PANEL B
Cut-off at 85 ng/mg
85 < vs 85+
PANEL C
Continuous scale
range 20-220 ng/mg
* number of pairs where PTCA levels were lower in cases
# number of pairs where PTCA levels were lower in controls
Table 4. Risk of CMM for eumelanin in hair measured with NIR (cut-off at 85
ng/mg) and corrected with calibration study. In parenthesis 95% credible
regions
Number of discordant pairs
Case+ ControlCase- Control+
18
9
Crude ORs
Crude &
Corrected OR
Crude OR
2.0
3.4
(0.93-4.76)
(1.73-7.83)
Adjusted by risk group strata*
Adjusted &
Corrected OR
Adjusted OR
0.9
2.3
(0.31-2.43)
(1.36-4.22)
* Four risk group strata from observed combinations of eye colour, number of naevi and hair colour
Human pigmentation-related genes
Locus
Protein
Function
Melanosome proteins
TYR
Tyrosinase
Oxidation of tyrosine, dopa
TYRP1
gp75, TRYP1
DHICA-oxidase, TYR stabilisation
DCT
DCT, TRYP2
Dopachrome tautomerase
SILV
gp100, pMel17, Silver
DHICA-polymerisation and melanosome striations
OCA2
P-protein
pH of melanosome and melanosome maturation
MATP
MATP, AIM-1
Melanosome maturation
Signal proteins
ASIP
Agouti signal protein
MC1R antagonist
MC1R
MSH receptor
G-protein coupled receptor
POMC
POMC, MSH, ACTH
MC1R agonist
OA1
OA1 protein
G-protein coupled receptor
MITF
MITF
Transcription factor
Proteins involved in melanosome transport or uptake by keratinocytes
MYO5A
MyosinVa
Motor protein
RAB27A
Rab27a
RAS family protein
HPS1
HPS1
Organelle biogenesis and size
HPS6
HPS6
Organelle biogenesis
Estimates of population attributable
fraction in different countries
Germany/Denmark
43.9
France
26.4
Italy
17.8
Spain
13.6
Summary of results from the
GEM study
• Nucleotide excision repair genes
– XPD 312 (OR 1.5) XPD 751 (OR 1.4) Millikan et al
2006
•
•
•
•
•
CDKN2A Berwick et al 2006 (OR 4.3 p<0.05)
MC1R Dwyer et al 2003 (OR 1.4 ns)
MC1R Kanetsky et al 2006 (OR 1.6 p<0.05)
EGF G/G vs A/A (OR 1.09 ns) Amend et al 2004
INK4A (OR 7.2 ns) Berwick et al 2004
Sun Exposure Analysis
Erythemal UV dose (age 10)
SPM
CDKN2A (-)
Low exposure
High
exposure
MPM
ORunadj
ORadj1
ORadj2
(controls) (cases)
994
422
1.00
(reference)
1.00
(reference)
1.00
(reference)
830
593
1.68
(1.44-1.97)
1.62
(1.38-1.91)
1.39
(1.03-1.86)
12
14
1.00
(reference)
1.00
(reference)
1.00
(reference)
10
17
1.46
(0.49-4.37)
1.25
(0.40-3.93)
3.44
(0.31-38.51)
CDKN2A (+)
Low exposure
High
exposure
ORunadj: unadjusted ORs
ORadj1 : Adjusted ORs - age, sex and age*sex interaction
ORadj2 : Adjusted ORs - centre (7 centres), tan (4 levels), European ancestry, age, sex and age*sex interaction
… un nuovo filone di studi su
melanoma, Parkinson e LevoDopa
• Zanetti R, Loria DI, Rosso S. Melanoma,
Parkinson's disease and levodopa: causal or
spurious link? A review of the literature.
Melanoma Res. 2006; 16: 201-6.
• Zanetti R, Rosso S Levodopa and risk of
melanoma. Lancet. 2007; 369: 257-8.
• Zanetti R, Rosso S, Loria DI. Parkinson’s disease
and cancer. Cancer Epidemiol Biomarkers Prev
2007; 16: 1081.
… le nuove sfide …
• Estensione delle analisi con metodica NIR
su melanine e carcinomi cutanei
• Studi congiunti sui marker biochimici e
genetici
• Ruolo dell’esposizione solare come fattore
prognostico
• Studio di coorte su pazienti con Parkinson
• Dosimetria UV
Estensione delle analisi con metodica NIR su
melanine e carcinomi cutanei
• Progetto Ricerca Sanitaria Finalizzata
bando 2007: “Individuazione di gruppi ad
alto rischio di tumore cutaneo:
misurazione con metodo spettrofotometrico
(nir) della eumelanina”.
Studi congiunti sui marker biochimici e genetici
Human pigmentation-related genes
Locus
Protein
Function
Melanosome proteins
TYR
Tyrosinase
Oxidation of tyrosine, dopa
TYRP1
gp75, TRYP1
DHICA-oxidase, TYR stabilisation
DCT
DCT, TRYP2
Dopachrome tautomerase
SILV
gp100, pMel17, Silver
DHICA-polymerisation and melanosome striations
OCA2
P-protein
pH of melanosome and melanosome maturation
MATP
MATP, AIM-1
Melanosome maturation
Signal proteins
ASIP
Agouti signal protein
MC1R antagonist
MC1R
MSH receptor
G-protein coupled receptor
POMC
POMC, MSH, ACTH
MC1R agonist
OA1
OA1 protein
G-protein coupled receptor
MITF
MITF
Transcription factor
Proteins involved in melanosome transport or uptake by keratinocytes
MYO5A
MyosinVa
Motor protein
RAB27A
Rab27a
RAS family protein
HPS1
HPS1
Organelle biogenesis and size
HPS6
HPS6
Organelle biogenesis
Ruolo dell’esposizione solare come
fattore prognostico
• Rosso S, Sera F, Segnan N, Zanetti R. Sun
exposure prior to diagnosis is associated with
improved survival in melanoma patients: Results
from a long-term follow-up study of Italian
patients. Eur J Cancer. 2008; 44: 1275-81.
• … e di altri fattori che possono influenzare la
prognosi dei melanomi:
– Studio GEM2: studio di follow-up di casi (MPM) e
controlli (SPM)
Studio di coorte su pazienti con
Parkinson
• Identificazione dei pazienti con Parkinson
attraverso i sistemi informativi correnti
(SDO e farmaceutica)
Dosimetria UV
• Progetto di Ricerca Sanitaria Finalizzata,
bando 1999. Prevenzione dei Tumori
Cutanei: Sviluppo di uno Strumento e di
un Metodo per la Dosimetria individuale
dell’Esposizione a Radiazione Solare.