Low prevalence of RAS-RAF-activating mutations in Spitz melanocytic nevi compared with other melanocytic lesions.

Melanocytic lesions, including Spitz nevi (SN), common benign nevi (CBN) and cutaneous metastatic melanoma (CMM), were analyzed for activating mutations in NRAS, HRAS and BRAF oncogenes, which induce cellular proliferation via the MAP kinase pathway. One of 22 (4.5%) SN tested showed an HRAS G61L mutation. Another lesion, a 'halo' SN, showed a BRAF V600E (T1796A) mutation. BRAF V600E mutations were found in two thirds (20/31) of CBN, while a further 19% (6/31) showed NRAS codon 61 mutations. One third of CMM (10/30) had various BRAF mutations of codon 600, and a further 6% (2/31) showed NRAS codon 61 mutations. Seventeen SN tested for loss of heterozygosity (LOH) at 9p and 10q regions, known to be frequently deleted in melanoma, showed LOH at the 9p loci D9S942 and IFNA. A further lesion was found with low-level microsatellite instability at one locus, D10S214. The low rate of RAS-RAF mutations (2/22, 9.1%) observed in SN suggests that these lesions harbor as yet undetected activating mutations in other components of the RAS-RAF-MEK-ERK-MAPK pathway. Germline DNA from members of 111 multiple-case melanoma families, representing a range of known (CDKN2A) and unknown predisposing gene defects, was analyzed for germline BRAF mutations, but none was found.

Deletion mapping suggests that the 1p22 melanoma susceptibility gene is a tumor suppressor localized to a 9-Mb interval.

Loss of the short arm of chromosome 1 is frequently observed in many tumor types, including melanoma. We recently localized a third melanoma susceptibility locus to chromosome band 1p22. Critical recombinants in linked families localized the gene to a 15-Mb region between D1S430 and D1S2664. To map the locus more finely we have performed studies to assess allelic loss across the region in a panel of melanomas from 1p22-linked families, sporadic melanomas, and melanoma cell lines. Eighty percent of familial melanomas exhibited loss of heterozygosity (LOH) within the region, with a smallest region of overlapping deletions (SRO) of 9 Mb between D1S207 and D1S435. This high frequency of LOH makes it very likely that the susceptibility locus is a tumor suppressor. In sporadic tumors, four SROs were defined. SRO1 and SRO2 map within the critical recombinant and familial tumor region, indicating that one or the other is likely to harbor the susceptibility gene. However, SRO3 may also be significant because it overlaps with the markers with the highest 2-point LOD score (D1S2776), part of the linkage recombinant region, and the critical region defined in mesothelioma. The candidate genes PRKCL2 and GTF2B, within SRO2, and TGFBR3, CDC7, and EVI5, in a broad region encompassing SRO3, were screened in 1p22-linked melanoma kindreds, but no coding mutations were detected. Allelic loss in melanoma cell lines was significantly less frequent than in fresh tumors, indicating that this gene may not be involved late in progression, such as in overriding cellular senescence, necessary for the propagation of melanoma cells in culture.

Frequent loss of heterozygosity targeting the inactive X chromosome in melanoma.

After previous preliminary observations of paradoxical deletion events affecting the inactive X chromosome in melanoma, we have surveyed the X chromosome for deletions using 23 polymorphic microsatellite markers in 28 informative (female XX) metastatic melanomas. Ten tumors (36%) showed at least one loss of heterozygosity (LOH) event, and in two cases an entire chromosome showed LOH at all informative loci. Four distinct X chromosome smallest regions of overlap can be resolved. An 18.6-Mb region on the p arm involving 9 of 28 (32%) samples lies between the markers DXS1061 and DXS1068. An equally frequently deleted smallest region of overlap straddled the centromere, bounded by DX1204 on the p arm and DXS983 14.6 Mb away in Xq11-12. One tumor potentially defines this region more tightly to a 10.6-Mb smallest region of overlap bounded by DXS1190 and DXS981 that contains the androgen receptor (AR) gene. A 6.2-Mb deleted region can be defined between the markers DXS8051 and DXS9902 in 8 of 28 (28%) tumors. An additional, less frequently deleted region of 25.7 Mb was found on distal Xq between the markers DXS1212 and DXS1193 in 5 of 28 (18%) tumors. X inactivation analysis of five tumors with LOH, using the AR exon 1 CAG repeat, showed that in each case, the inactive, hypermethylated allele was the one deleted. Analysis of copy number in this region by quantitative PCR showed restoration to disomy and, in one case, trisomy at AR.