Milena Casula

BRAF/NRAS Mutation Frequencies Among Primary Tumors and Metastases in Patients With Melanoma

PURPOSE The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. PATIENTS AND METHODS In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. RESULTS BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. CONCLUSION In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

Main roads to melanoma

The characterization of the molecular mechanisms involved in development and progression of melanoma could be helpful to identify the molecular profiles underlying aggressiveness, clinical behavior, and response to therapy as well as to better classify the subsets of melanoma patients with different prognosis and/or clinical outcome. Actually, some aspects regarding the main molecular changes responsible for the onset as well as the progression of melanoma toward a more aggressive phenotype have been described. Genes and molecules which control either cell proliferation, apoptosis, or cell senescence have been implicated. Here we provided an overview of the main molecular changes underlying the pathogenesis of melanoma. All evidence clearly indicates the existence of a complex molecular machinery that provides checks and balances in normal melanocytes. Progression from normal melanocytes to malignant metastatic cells in melanoma patients is the result of a combination of down- or up-regulation of various effectors acting on different molecular pathways.

BRAF Gene Is Somatically Mutated but Does Not Make a Major Contribution to Malignant Melanoma Susceptibility: The Italian Melanoma Intergroup Study

PURPOSE Ocogenic activation of the BRAF gene has been demonstrated to be involved in the pathogenesis of malignant melanoma (MM). In this study, we investigated the contribution of BRAF to melanoma susceptibility, also making a comparison with frequency of CDKN2A germline mutations in MM patients from different areas in Italy. PATIENTS AND METHODS sing a combination of denaturing high-performance liquid chromatography analysis and automated sequencing on genomic DNA from peripheral blood or tumor tissue samples, 569 MM patients (211 from northern Italy and 358 from southern Italy) were screened for BRAF mutations. RESULTS Three BRAF germline sequence variants (M116R, V599E, and G608H) were identified in four (0.7%) of 569 MM patients. The most common BRAF mutation, V599E, was detected in one germline DNA sample only; M116R and G608H were newly described mutations. A high frequency (59%) of BRAF mutations was instead observed in tumor samples from patients also undergoing germline DNA analysis; at the somatic level, substitution of valine 599 was found to account for the majority (88%) of BRAF mutations. We then estimated the germline mutation rates in BRAF and CDKN2A among 358 consecutively collected patient samples originating in southern Italy; a low (2.5%) or very low (0.29%) prevalence of CDKN2A and BRAF mutations, respectively, was detected. CONCLUSION utation analysis of either blood DNA from a large collection of MM patients or matched MM tissues from a subset of such patients revealed that BRAF is somatically mutated and does not play a major role in melanoma susceptibility. The present study further suggests that patient origin may account for different mutation rates in candidate genes.

Molecular alterations at chromosome 9p21 in melanocytic naevi and melanoma

Background  The chromosome 9p21 and its CDKN locus, with the p16 tumour suppressor gene (CDKN2A), are recognized as the genomic regions involved in the pathogenesis of melanoma.

Microsatellite instability and mutation analysis of candidate genes in unselected sardinian patients with endometrial carcinoma

Microsatellite instability (MSI) is due mostly to a defective DNA mismatch repair (MMR). Inactivation of the two principal MMR genes, hMLH1 and hMSH2, and the PTEN tumor suppressor gene seems to be involved in endometrial tumorigenesis. In this study, Sardinian patients with endometrial carcinoma (EC) were analyzed to assess the prevalence of both the mutator phenotype (as defined by the presence of MSI and abnormal MMR gene expression at the somatic level) and the hMLH1, hMSH2, and PTEN germline mutations among patients with MSI positive EC.

Issues affecting molecular staging in the management of patients with melanoma.

•  Introduction •  Role of the RT‐PCR in prognostic prediction ‐  Melanoma micrometastases in peripheral blood ‐  Melanoma micrometastases in lymph nodes •  Molecular complexity of melanomagenesis ‐  Molecular alterations and disease phenotype •  Conclusions

Role of key-regulator genes in melanoma susceptibility and pathogenesis among patients from South Italy

BackgroundSeveral genetic alterations have been demonstrated to contribute to the development and progression of melanoma. In this study, we further investigated the impact of key-regulator genes in susceptibility and pathogenesis of such a disease.MethodsA large series (N = 846) of sporadic and familial cases originating from South Italy was screened for germline mutations in p16CDKN2A, BRCA2, and MC1R genes by DHPLC analysis and automated DNA sequencing. Paired primary melanomas and lymph node metastases from same patients (N = 35) as well as melanoma cell lines (N = 18) were analyzed for somatic mutations in NRAS, BRAF, and p16CDKN2Agenes.ResultsFor melanoma susceptibility, investigations at germline level indicated that p16CDKN2Awas exclusively mutated in 16/545 (2.9%) non-Sardinian patients, whereas BRCA2 germline mutations were observed in 4/91 (4.4%) patients from North Sardinia only. Two MC1R germline variants, Arg151Cys and Asp294His, were significantly associated with melanoma in Sardinia. Regarding genetic events involved in melanoma pathogenesis at somatic level, mutually-exclusive mutations of NRAS and BRAF genes were observed at quite same rate (about two thirds) in cultured and in vivo melanomas (either primary or metastatic lesions). Conversely, p16CDKN2Agene alterations were observed at increased rates moving from primary to metastatic melanomas and melanoma cell lines. Activation of the ERK gene product was demonstrated to be consistently induced by a combination of molecular alterations (NRAS/BRAF mutations and p16CDKN2Asilencing).ConclusionOur findings further clarified that: a) mutation prevalence in melanoma susceptibility genes may vary within each specific geographical area; b) multiple molecular events are accumulating during melanomagenesis.

Assessment of genetic instability in melanocytic skin lesions through microsatellite analysis of benign naevi, dysplastic naevi, and primary melanomas and their metastases

&NA; Microsatellite instability (MSI) is caused by replication errors due to deficient DNA mismatch repair and has been associated with tumour progression in various types of cancer. Controversial results have been reported concerning the frequency and significance of MSI in malignant melanoma. In this study, the time of onset and relative incidence of MSI were determined during the progression of melanocytic tumours, starting with benign melanocytic naevi. MSI was studied at 13 loci containing single, di‐ or trinucleotide repeat sequences and mapping to five different chromosomal locations. Tumours were classified as being low frequency MSI (L‐MSI+) or high frequency MSI (H‐MSI+) when either one or at least two marker loci, respectively, displayed mutant alleles in tumour DNA compared with the corresponding normal tissue DNA. None of the eight melanocytic naevi studied showed MSI, whereas a moderate frequency of H‐MSI was detected in dysplastic naevi (one out of 11; 9%) and primary melanomas (six out of 56; 11%). The incidence of H‐MSI was increased in melanoma metastases from the same patients (nine out of 42; 21%). In contrast to previously reported data showing higher rates of MSI in melanoma, genetic instability seems to be present in a minority of malignant melanoma lesions. However, our findings are consistent with the hypothesis that MSI may be sequentially induced during malignant evolution, contributing to the progression of a subset of melanocytic tumours.

Multiple Molecular Pathways in Melanomagenesis: Characterization of Therapeutic Targets

Molecular mechanisms involved in pathogenesis of malignant melanoma have been widely studied and novel therapeutic treatments developed in recent past years. Molecular targets for therapy have mostly been recognized in the RAS–RAF–MEK–ERK and PI3K–AKT signaling pathways; small-molecule inhibitors were drawn to specifically target key kinases. Unfortunately, these targeted drugs may display intrinsic or acquired resistance and various evidences suggest that inhibition of a single effector of the signal transduction cascades involved in melanoma pathogenesis may be ineffective in blocking the tumor growth. In this sense, a wider comprehension of the multiple molecular alterations accounting for either response or resistance to treatments with targeted inhibitors may be helpful in assessing, which is the most effective combination of such therapies. In the present review, we summarize the known molecular mechanisms underlying either intrinsic and acquired drug resistance either alternative roads to melanoma pathogenesis, which may become targets for innovative anticancer approaches.

Role of the EGF +61A>G polymorphism in melanoma pathogenesis: an experience on a large series of Italian cases and controls

BackgroundA single nucleotide polymorphism (61A>G) in the epidermal growth factor (EGF) gene has been implicated in both melanoma pathogenesis and increased melanoma risk. To further evaluate this association, we conducted a case-control study in a clinic-based Italian population.MethodsIndividuals with less than 10 (N = 127) or more than 100 (N = 128) benign nevi, and patients with cutaneous melanoma (N = 418) were investigated for the EGF +61A>G polymorphism, using an automated sequencing approach.ResultsOverall, no difference in EGF genotype frequencies was observed among subjects with different number of nevi as well as when non-melanoma healthy controls were compared with the melanoma patients. However, a heterogeneous distribution of the frequencies of the G/G genotype was detected among cases and controls originating from North Italy (21.1 and 18.3%, respectively) vs. those from South Italy (12.6 and 17.1%, respectively).ConclusionOur findings further suggest that EGF +61A>G polymorphism may have a limited impact on predisposition and/or pathogenesis of melanoma and its prevalence may vary in different populations.