Molecular genetic investigation of Japanese cutaneous melanoma

在46例日本散发性原发性皮肤黑色素瘤的系统分析中，我们检测到11例（24%）肿瘤的染色体区域9 p21（p16所在区域）的杂合性丢失（洛）。然而，直接测序显示p16基因没有体细胞突变。在另外19个没有9 p21洛证据的肿瘤中进一步的测序分析仅在密码子81处鉴定出一个杂合的C->T突变。甲基化特异性PCR检测显示，在这12例携带9 p21洛LOH或p16杂合突变的肿瘤中，未发现p16基因启动子5 'CpG岛的从头甲基化，这可能导致转录沉默。尽管如此，完全丢失的p16蛋白，最有可能是由于纯合性缺失的p16基因，观察到6（15%）的39个可评估的情况下，免疫组化分析冷冻切片。结果表明，失活的p16是不经常在原发性黑色素瘤已在细胞系中报道，并保证进一步寻找另一种肿瘤抑制因子9 p21这可能是更重要的黑色素瘤的启动。同时调查检查相应的转移14例显示完全丢失的p16表达在转移进展过程中的4例，这表明p16的失活在散发性黑色素瘤的进展（而不是启动）中起着重要作用。该分析还比较了染色体臂6 q、9 p、9 q、10 q、11 q和18 q的洛，并提供了明确的证据，即在3个病例中，在转移部位发现的细胞克隆不是来自原发肿瘤内的优势亚克隆，表明黑色素瘤进展的线性模型过于简单，因为在肿瘤发生的最早阶段可能存在相当大的遗传异质性，并且来自同一肿瘤的转移可能具有不同的遗传变化。

In a systematical analysis in 46 Japnese sporadic primary cutaneous melanomas, we detected loss of heterozygosity (LOH) of chromosome region 9p21 (where the p16 resides) in 11 (24%) tumors. Direct sequencing, however, revealed no somatic mutation of the p16 gene. Further sequencing analyses in 19 additional tumours with no evidence of LOH of 9p21 identified only one heterozygous C->T mutation at codon 81. De novo methylation of the promoter 5'CpG island of the p16 gene, which would lead to transcriptional silencing, was not demonstrated in any of these 12 tumours harboring 9p21 LOH or heterozygous p16 mutation by methylation-specific PCR assay. Nonetheless, complete loss of p16 protein, most likely due to homozygous deletion of the p16 gene, was observed in 6 (15%) out of 39 evaluable cases by immunohistochemical analyses on frozen sections. The results show that inactivation of p16 is not as frequent in primary melanoma as has been reported in cell lines, and warrant further search for another tumour suppressor on 9p21 which is likely to be more important in the initiation of melanoma. Simultaneous investigation examining corresponding metastases in 14 cases showed complete loss of p16 expression during matastatic progression in 4 cases, suggesting that inactivation of p16 plays an important role in progression (rather than initiation) of sporadic melanoma. This analysis also compared LOH of chromosome arms 6q, 9p, 9q, 10q, 11q and 18q, and provided clear evidence that in 3 cases clones of cells found in the sites of metastasis did not derive from the dominant subclone within the primary tumor, indicating that a linear model of melanoma progression is too simplistic, as there is likely to be considerable genetic heterogeneity at the earliest stages of tumourigenesis and that metastases from the same tumor may harbor different genetic change.