Melanoma: aspetti biomolecolari. Elena Tamborini

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1 Melanoma: aspetti biomolecolari Elena Tamborini

2 - Malattia estremamente eterogenea - Ad oggi non esiste un correlazione tra fenotipo e genotipo Tuttavia il melanoma è tra i tumori con il piu alto tasso di mutazioni

3 Nature. 2013;499(7457):214-8 *

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5 Charles Lu, advance online publication 30 October doi: /jid Figure 1

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7 Proliferativo Fattori di trascrizione legati alla melanogenesi, MITF, SOX10 e PAX3 Al contrario, bassi livelli di MITF e alti livelli di EMT Fattori relati

8 Riprogrammazione dell apparato regolatorio del trascrittoma

9 Braf NRAS KIT

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11 Activating Signals NRAS ~20% *RAS Pl3K PTEN* 10-30% BRAF ~45% *RAF P AKT/PKB MEK P IKK P FKHR P BAD P P mtor P GSK ERK P IKB NFKB IKB NFKB P Summary (1) High Prevalence of ERK Pathway mutations (2) Concurrent Activation of Multiple Pathways (Subset)

12 ERK Pathway Mutations: Prevalence by Site Cutaneous w/o Chronic Sun Damage (CSD) Acral Melanoma Mucosal Melanoma Cutaneous with CSD Uveal 45% BRAF Mutations 20% NRAS Mutations Acral: 20% BRAF 10% NRAS Mucosal: 3% BRAF 5% NRAS Skin C.S.D. 5% BRAF 10% NRAS Virtually No BRAF/NRAS BRAF/NRAS 20-40% KIT ~1% KIT Acral: 11% Mut 25% Amp No KIT Mucosal: 21% Mut 29% Amp Skin C.S.D. 1-18% Mut 6% Amp C-KIT No GNAQ No GNA11 No GNAQ No GNA11 GNAQ 45% GNA11 35% GPCR

13 The most common somatic mutation in cutaneous melanomas 45% Non-CSD Cutaneous 20% Acral lentiginous 5% Mucosal 0% Uveal Pattern of mutations 95% Affect V600 (Exon 15) ~70% V600E ~20% V600K 10% other 5% Non-V600 Exon 15 Exon 11 Other Alterations in Melanoma Amplification Translocation/Gene Fusions (V. rare)

14 Molecular Effects of BRAF Mutations V600 Mutations: All markedly increase catalytic activity of BRAF Non-V600 Mutations: Variable effects on catalytic activity High activity, Intermediate activity, Low/Impaired activity Mutations that do not increase catalytic activity of BRAF promote dimerization of BRAF with CRAF still activate MEK & ERK Fold Increase in Catalytic Activit Versus Wild-Type BRAF WT R462I I463S G464V G464E G466A G466E G466V G469A G469E N581S E586K D594V F595L G596R L597V T599I V600 Mutants: X V600D V600E V600R V600K K601E A728V NRAS G12V Adapted from Garnett, et al Molec Cell, 2005

15 NRAS 2 nd Most Common Somatic Mutation Cutaneous 20% Acral 10% Mucosal 5% Uveal <1% Molecular Biology Hotspots: Q61 (~80%) & G12/13 (~20%) V600 BRAF mutations: Mutually exclusive Non-V600 BRAF mutations: Overlap Activate MAPK + other pathways PI3K-AKT, RAC, RAL, PLC, etc Prognosis Initial diagnosis (Devitt, PCMR, 2011) Stage IV (Jakob, Cancer, 2012) NRAS Devitt, PCMR, 2011 Treatment No NRAS-specific approved therapies Investigational: MEK162: n=28, PR 21%, median PFS 3.6 mos Ascierto, Lancet Oncol, 2013 Combinatorial approaches Jakob, Cancer, 2012

16 Mutations & Amplifications Cutaneous (-)CSD 1-2% MT <1% Amp Cutaneous (+)CSD 1-18% Mt 6% Amp Acral 11% Mt 25% Amp Mucosal 21% Mt 29% Amp Molecular Biology Mutations Same exons (11, 13, 17) as in GIST Mostly point mutations GIST: Deletions/Instertions Some mutated + amplified RTK can activate multiple pathways MAPK, PI3K, JAK-STAT KIT Treatment: KIT inhibitors FDA-Approved in GIST (~80% KIT Mutant) Unselected melanoma patients, ORR ~1% KIT-Mutant Patients Multiple case reports of responses 2 Phase II Clinical trials, ORR ~20% JAK STATs Pre-Treatment C-Kit RAS PI3K RAF AKT + Imatinib Hodi, J Clin Oncol, 2008

17 Extracellular Domain Juxtamembrane Domain Kinase Domain I Kinase Domain II Exon 11 Exon 13 Exon 17 Exon 18 Frequency of Mutation (%) K550N 2 Y553N 2 Del W557R 5 K558N 2 V559A/D/G 11 V560D 2 N566D 2 V569G 2 Del N566K 2 L576P 34 Del579 2 Insert/Dup583 2 K642E 15 R634W 2 D816V/H 6 Y823D 2 D820Y 2 N822I 2 A829P 2 I841V 2 Woodman SE & Davies MA. Biochem Pharmacol 2010; 80(5):

18 Techniques for Sequencing Sanger sequencing: Detects all variants (KIT) Slower, more time consuming Less sensitive; poorly quantitative Pyrosequencing: Focused on hot spot codons (BRAF, NRAS, GNAQ) Faster More sensitive; semiquantitive Sequenom (mass spectrometry): Very focused on very specific hot-spots (BRAF, NRAS, GNAQ) Multiplexed Sensitive; quantitative Specificity is complex (confirmation by second CLIA method)

19 To assure a correct molecular analysis to all the patients is fundamental! 1. Type of material 2. DNA amount 3. Sensitivity and specificity

20 Type of material: We can work: FFPE Cytologic material Frozen material IHC section It is better to have the best section for quality and abundancy of tumoral material The most recent lesion! We chose not to analyze materil present in eppendorf (no histological check)

21 Highest % of tumoral cells Avoid necrosis, or very pigmented areas if possible

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23 BRAF Intron 14 Exon 15 Intron 15 Fw 2 **** Amplification product: 224 bp 1 2b 3 4 C+ B NRAS Exons 2, 3 and 4 KIT Exons 9, 11, 13 and 17 DNA sequencing 224 pb Sanger methodology

24 GTG-GAG V600E BRAF: mutated, V600E NRAS: wt

25 Sensitivity ng DNA BRAF V600E ng DNA BRAF WT Sequencing. Cell line with BRAF V600E Cell line with wild type BRAF V600E V600E V600E V600E V600E V600E V600E V600E wild type The mutation is detected in samples with 90% of healty tissue.

26 Mut. DNA: 100% Mut. DNA : 33% Mut. DNA : 9% Mut. DNA: 3% Mut. DNA: 1.5% Mut. DNA: 0.7% 200 ng 50 ng ng 12.5 ng ng ng ng 1.5 ng ng 0.78 ng ng

27 Hot spot cancer panel: PGM Ion Torrent Step 1 Step 2 Step 3 Step 4

28 Tumor heterogenity: NGS experiments

29 La scelta del metodo di indagine è importante e altrettanto importante e conoscere i suoi limiti. L approccio NGS è attualmente il piu preciso e il piu informativo.

30 Grazie per l attenzione!

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