Molecular genetic investigation of Japanese cutaneous melanoma

在对46例日本散发性原发性皮肤黑色素瘤的系统分析中，我们在11例（24%）肿瘤中检测到染色体9p21区（p16所在）的杂合性缺失。然而，直接测序显示p16基因没有体细胞突变。进一步的测序分析在另外19个没有9p21缺失证据的肿瘤中发现，在密码子81处只有一个杂合的C->T突变。通过甲基化特异性PCR检测，在这12个携带9p21 LOH或杂合p16突变的肿瘤中未发现p16基因启动子5'CpG岛的重新甲基化，这将导致转录沉默。尽管如此，通过冷冻切片的免疫组织化学分析，39例可评估病例中有6例（15%）观察到p16蛋白完全丢失，这很可能是由于p16基因的纯合缺失。结果表明，p16的失活在原发性黑色素瘤中并不像在细胞系中报道的那样频繁，并且需要进一步寻找9p21上的另一个肿瘤抑制因子，这可能在黑色素瘤的发生中更重要。同时对14例相应转移灶的研究显示，4例在转移进展过程中p16表达完全丧失，提示p16失活在散发性黑色素瘤的进展（而不是起始）中起重要作用。染色体臂的分析还比较LOH 6问,9 p, q, 9 10 q, 11日和18,并提供明确的证据表明,在3例克隆的细胞中发现的网站转移没有来自主导subclone在原发肿瘤,黑色素瘤进展表明线性模型过于简单,有可能是相当大的遗传异质性的最早阶段tumourigenesis和转移相同的肿瘤可能港口不同的基因变化。

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.