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.

The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. However, only partial effects have been reported for candidate genes experimentally tested so far. Here we demonstrate that inactivation of two ovarian somatic factors, Wnt4 and Foxl2, produces testis differentiation in XX mice, resulting in the formation of testis tubules and spermatogonia. These genes are thus required to initiate or maintain all major aspects of female sex determination in mammals. The two genes are independently expressed and show complementary roles in ovary morphogenesis. In addition, forced expression of Foxl2 impairs testis tubule differentiation in XY transgenic mice, and germ cell-depleted XX mice lacking Foxl2 and harboring a Kit mutation undergo partial female-to-male sex reversal. The results are all consistent with an anti-testis role for Foxl2. The data suggest that the relative autonomy of the action of Foxl2, Wnt4 and additional ovarian factor(s) in the mouse should facilitate the dissection of their respective contributions to female sex determination.

The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. BRAFV600E/V600K and NRASQ61R/Q61L were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines. COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations.

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 inhibitors may display intrinsic or acquired resistance and various evidences suggest that targeting a single component in pathogenetic signaling pathways could not yield significant anti-tumor responses. Therefore, knowledge of the multiple altered signaling events involved in response and resistance to targeted treatments will allow to speed up the development of more effective combination therapies. In this 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.

Poor data have been previously reported about the mutation rates in K-RAS, BRAF, and PIK3CA genes among patients with hepatocellular carcinoma (HCC). Here we further elucidated the role of these genes in pathogenesis of primary hepatic malignancies. Archival tumour tissue from 65 HCC patients originating from South Italy were screened for mutations in these candidate genes by direct sequencing. Overall, oncogenic mutations were detected in 15 (23%) patients for BRAF gene, 18 (28%) for PIK3CA gene, and 1 (2%) for K-RAS gene. Using statistical analysis, BRAF mutations were significantly correlated with the presence of either multiple HCC nodules (P=0.021) or higher proliferation rates (P=0.034). Although further extensive screenings are awaited in HCC patients among different populations, our findings clearly indicated that mutational activation of both BRAF and PIK3CA genes does contribute to hepatocellular tumorigenesis at somatic level in Southern Italian population.

Purpose of Review Conventional clinico-pathological features in melanoma patients should be integrated with new molecular diagnostic, predictive, and prognostic factors coming from the expanding genomic profiles. Cutaneous melanoma (CM), even differing in biological behavior according to sun-exposure levels on the skin areas where it arises, is molecularly heterogeneous. The next-generation sequencing (NGS) approaches are providing data on mutation landscapes in driver genes that may account for distinct pathogenetic mechanisms and pathways. The purpose was to group and classify all somatic driver mutations observed in the main NGS-based studies. Recent Findings Whole exome and whole genome sequencing approaches have provided data on spectrum and distribution of genetic and genomic alterations as well as allowed to discover new cancer genes underlying CM pathogenesis. Summary After evaluating the mutational status in a cohort of 686 CM cases from the most representative NGS studies, three molecular CM subtypes were proposed: BRAF^mut, RAS^mut, and non-BRAF^mut/non-RAS^mut.