Melanoma: aspetti biomolecolari Elena Tamborini
- 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
Nature. 2013;499(7457):214-8 *
Charles Lu, advance online publication 30 October 2014. doi:10.1038/jid.2014.425 Figure 1
Proliferativo Fattori di trascrizione legati alla melanogenesi, MITF, SOX10 e PAX3 Al contrario, bassi livelli di MITF e alti livelli di EMT Fattori relati
Riprogrammazione dell apparato regolatorio del trascrittoma
Braf NRAS KIT
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)
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
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)
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 100 90 80 70 60 50 40 30 20 10 0 WT R462I I463S G464V G464E G466A G466E G466V G469A G469E N581S E586K D594V F595L G596R L597V T599I V600 Mutants: 150 700X V600D V600E V600R V600K K601E A728V NRAS G12V Adapted from Garnett, et al Molec Cell, 2005
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
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
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 Del554-559 2 W557R 5 K558N 2 V559A/D/G 11 V560D 2 N566D 2 V569G 2 Del566-572 2 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):568-74.
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)
To assure a correct molecular analysis to all the patients is fundamental! 1. Type of material 2. DNA amount 3. Sensitivity and specificity
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)
Highest % of tumoral cells Avoid necrosis, or very pigmented areas if possible
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
GTG-GAG V600E BRAF: mutated, V600E NRAS: wt
Sensitivity ng DNA BRAF V600E ng DNA BRAF WT Sequencing. Cell line with BRAF V600E Cell line with wild type BRAF 100 0 V600E 70 30 V600E 60 40 V600E 50 50 V600E 40 60 V600E 30 70 V600E 20 80 V600E 10 90 V600E 0 100 wild type The mutation is detected in samples with 90% of healty tissue.
Mut. DNA: 100% Mut. DNA : 33% Mut. DNA : 9% Mut. DNA: 3% Mut. DNA: 1.5% Mut. DNA: 0.7% 200 ng 50 ng + 100 ng 12.5 ng + 100 ng 3.125 ng + 100 ng 1.5 ng + 100 ng 0.78 ng + 100 ng
Hot spot cancer panel: PGM Ion Torrent Step 1 Step 2 Step 3 Step 4
Tumor heterogenity: NGS experiments
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.
Grazie per l attenzione!