Bersagli molecolari nel melanoma Giuseppe Palmieri - Unit of Cancer Genetics Institute of Biomolecular Chemistry, CNR, Sassari
Essential alterations in malignant cells Hanahan & Weinberg, Cell 2000
Essential alterations in malignant cells Hanahan & Weinberg, Cell 2011
Main pathways in melanomagenesis Fecher, JCO 2007; Palmieri, JCMM 2007; Palmieri, JTM 2009; Palmieri, Skin Cancer Book 2012
Role of BRAF gene in melanoma Nevi harbour BRAF mutations (mostly ) at quite same prevalence of melanoma BRAF mutations are necessary but not sufficient for MM development Pollock, Nature Genet 33, 2003; Casula, J.Clin.Oncol 22, 2004; Sharma, Cancer Res 65, 2005
The main road to melanoma Melanocytic proliferation Melanoma development Melanoma progression BRAF activation impairment of: 9p21 allelic deletions p16-rb pathway p14-p53 pathway AKT overexpression CyclinD1 amplification PTEN loss/inactivation Miller and Mihm, NEJM 2006; Palmieri, JCO 2007; Palmieri, JCMM 2007 loss of p16 CDKN2A MITF amplification Low expression of melanoma markers regulated by MITF
The Melanomas non CSD CSD Acral Mucosal Curtin, NEJM 2005
The Melanomas 50.0% KIT RAS BRAF 0.0% nocsd CSD acral mucosal Curtin, NEJM 2005
Incidence of key driver oncogenes in melanoma BRAF ~ 50% NRAS ~ 15% ckit ~ 1% Primarily mucosal and acral lentiginous GNAQ/GNA11 ~ 1% Almost exclusively uveal Nikolaou, J Invest Dermatol. 2012. Smalley, Semin Oncol. 2012
germline somatic BRAF/NRAS mutations in MM: our experience BRAF NRAS cell lines 18/32 (56%) 4/32 (12%) metastases 153/298 (51%) 34/216 (16%) primary MM 223/451 (49%) 46/312 (15%) peripheral blood 3*/897 (0.3%) not tested *No exon 15 mutation Casula, JCO 2004 - EJC 2007; Colombino, JCO 2012 - JTM 2013
metastases BRAF/NRAS mutations in MM metastasis BRAF NRAS Lymph node 78/151 (52%) 15/102 (15%) Visceral 25/47 (53%) 4/30 (13%) Liver 16/30 (53%) 3/22 (14%) Lung 9/17 (53%) 1/8 (12%) Skin 29/54 (54%) 5/38 (13%) Brain 21/46 (46%) 10/46 (22%) TOTAL 153/298 (51%) 34/216 (16%) Colombino JTM 2013
BRAF/NRAS mutations in primary MM NRAS mutations: 46/312 (15%) BRAF mutations: 223/451 (49%) Sardinian patients NRAS mutations: 2/105 (2%) BRAF mutations: 109/178 (61%) Non-Sardinian patients NRAS mutations: 44/207 (21%) BRAF mutations: 114/273 (42%) Colombino JTM 2013
Patients' geographical origin may account for different mutation rates in pathogenetic cancer genes
Pathogenetic mutations in MM No overlap between KIT mutations NRAS mutations BRAF mutations Beadling 2008; Ascida 2009; Colombino 2012 and 2013
Advanced melanoma: different strategies according to mutational status BRAF mut NRAS mut KIT mut BRAF NRAS KIT Modified from Ascierto
Targeting BRAF
BRAF inhibitors only inhibit in the context of B-RAF mutation NRAS BRAF CRAF MEK ERK Heidorn, Cell. 2010. Poulikakos, Nature 2010. Hatzivassiliou, Nature 2010
BRAF inhibitors only inhibit in the context of B-RAF mutation NRAS RAS-GTP BRAF CRAF BRAF mut CRAF CRAF MEK ERK MEK MEK ERK ERK Heidorn, Cell. 2010. Poulikakos, Nature 2010. Hatzivassiliou, Nature 2010
Objective tumor responses with vemurafenib Intrinsic resistance N Engl J Med 2012
Progression-free survival (%) Progression-free survival 100 90 80 70 60 50 40 30 20 10 No. of patients in follow up Dacarbazine Vemurafenib 0 Hazard Ratio 0.26 (95% CI; 0.20-0.33) Log-rank P<0.0001 0 1 2 3 4 5 6 7 8 9 10 11 12 274 275 Median 1.6 mos 213 268 Dacarbazine (N=274) 85 211 48 122 Vemurafenib (N=275) 28 105 Median 5.3 mos Months 16 10 6 3 0 Acquired resistance 50 35 16 4 3 N Engl J Med 2012
Intrinsic resistance Alterations predicted to cause resistance in pre-treatment sample Acquired resistance Alterations in the resistant sample at undetectable or subclonal level prior to therapy
Main mechanisms of resistance
Intrinsic BRAF resistance
Mutation patterns by NGS approaches Krauthammer, Nat Genet 2012
Acquired BRAF resistance
Acquired BRAF resistance
Resistance to target therapies Second biopsy?
Additional levels of complexity
BRAF multiclonality G/T Lin et al., J Natl Cancer Inst. 2009
Intratumor heterogeneity of BRAF Yancovitz, PLoS ONE 2012
Do prevalence of main mutations vary during the disease progression phases and among different types of metastasis?
BRAF/NRAS mutations among primary tumors and metastases in MM patients Tissue types Lymph node metastasis Visceral metastasis Consistency secondary/primary melanomas 91/101 (90%) 29/32 (91%) Primary tumor Metastasis BRAF NRAS BRAF NRAS V600K not tested not tested L597R not tested not tested
BRAF/NRAS mutations among primary tumors and metastases in MM patients Tissue types Brain metastasis Skin metastasis Consistency secondary/primary melanomas 18/22 (82%) 34/48 (71%) Primary tumor Metastasis BRAF NRAS BRAF NRAS Q61R Q61R Q61R not tested not tested Q61L Q61L Q61R Q61L not tested not tested
Example of expression plots for lower extremity in-transit melanomas in the same patient Augustine, Mol Cancer Ther 2010
Molecular classification of all tumor lesions in MM patients?
Molecular analysis in MM patient flow DISTINCT SETS OF GENETIC ALTERATIONS ~5% Uveal GNAQ mut + GNA11 mut + ckit mut-ampl + BAP1 mut ~5% Mucosal ckit mut + ckit ampl + CCND1 ampl + CDK4 ampl ~10% ~15% ~60% Acral CSD Non-CSD NRAS mut + BRAF mut + ckit mut-ampl + BAP1 mut + CCND1 ampl + CDK4 ampl NRAS mut + BRAF mut + ckit mut + ckit amplification + CCND1 ampl + CDK4 ampl NRAS mut + BRAF mut + BAP1 mut ~5% Conjuntival ckit mut + ckit ampl
new genes by next-generation sequencing? Whole genome sequencing of melanomas and matched germline DNA samples
Sardinian CNR SS University SS ASL1 SS Businco CA Non Sardinian Casula, Colombino, Manca, Palomba, Pisano, Rozzo, Sini Cossu, Lissia, Satta, Tanda Budroni, Contu, Scotto Bruder, Muggiano INT Pascale NA Ascierto, Ayala, Botti, Caracò, Mozzillo University SA University GE University FI University NA AOU Siena IDI Roma IRCCS Meldola Acknowledgements Rubino Bianchi-Scarrà, Ghiorzo De Giorgi, Massi Staibano Maio D Atri, Pagani Stanganelli