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 FMR1 gene, associated with fragile X syndrome, has recently been cloned and the sequence of partial cDNA clones is known. We have determined additional cDNA sequences both at the 5' and 3' end. We have characterized the expressed gene by means of RT-PCR in various tissues and have found that alternative splicing takes place in the FMR1 gene, which does not seem to be tissue specific. When the different alternative splicing events are combined, 12 distinct mRNA products could result from FMR1 expression in each tested tissue. In all these transcripts the open reading frame is maintained until the same stop codon. At the 3' end alternative use of polyadenylation signals is found. The alternative splicing allows functional diversity of the FMR-1 gene. Whether all the possible proteins will be synthesized and whether they will be functionally active has to be determined.

As an essential step towards an exhaustive analysis of the coding potential of large regions of the genome, we have developed a protocol allowing the rapid isolation of transcripts defmed by overlapping clone libraries. The method is based on the hybridisation of cDNA inserts, which had been amplified by PCR from cDNA libraries, to biotiiiylated DNA from cosmids or cosmid pools. Nonspecific hybrids are then removed, the selected cDNAs are eluted and reamplifled by PCR. Using a cosmid containing part of the FMR-1 gene as test, we were able to demonstrate an eighty thousand fold enrichment of cDNAs for this gene after two rounds of selection-amplification. The technique was applied to the analysis of transcripts from two cosmid contigs, together encompassing a region of 900 kb in Xq28. These experiments have thus far resulted in the identification of 81 cDNA clones, of which 54 clones were mapped back to the cosmid contigs. Of the 54 clones placed on the contig maps, 12 cDNA clones can be shown to belong to two genes which have been previously reported (L1CAM and QM).

To elucidate the function of the FMR1 gene, we applied RNA in situ hybridization to cryosectlons of mice from different developmental stages. The murlne Fmr-1 was found transcribed in a ubiquitous manner with an expression pattern similar to glyceraldehyd phosphate dehydrogenase, Gapdh, which was used as a control gene. A significant difference in the Fmr-1 expression pattern, however, was markedly enhanced expression specifically confined to the testis and the fetal ovary. In the immature and mature testis an elevated level of Fmr-1 expression is found in type A₁ spermatogonla. Expression in the testis is observed in fetal life, reaches the highest level in the immature testis, and declines early in adult life. In the mature ovary no specific Fmr-1 expression signal was found but enhanced levels were seen in the fetal ovary. At this developmental stage proliferation of oogonia takes place. It is suggested that FMR1 serves a special function during germ cell proliferation in males and females. These findings are discussed in the light of the current observation that fraglie X patients produce only sperm with a premutation sized allele. Two hypotheses are put forward: (1) In males lack of FMR1 function results in a premeiotic defect preventing spermatogonia with a full mutation to reach meiosis. A fragile X mutation can be passed on to offsprings only as a premutation (selection hypothesis). (2) Transition of a premutation alleie to full mutation occurs in a postzygotic stage after separation of the germ line and is restricted to soma cells (restriction hypothesis). Expression of FMR1 in proliferating germ cells is in line with both hypothesis.

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.