Abstract Introduction: Despite advances in the understanding of the molecular pathogenesis of cutaneous melanoma, relatively little is known about the genetic changes that occur in the progression of conjunctival melanocytic from intraepithelial lesions to invasive conjunctival melanoma. Methods: We exposed four exenteration specimens that each contained varying grades of intraepithelial conjunctival melanocytic neoplasia and invasive neoplasia to a combination of various techniques, including array comparative genomic hybridisation (aCGH), RNA seq, fluorescence in-situ hybridisation (FISH) and immunohistochemistry. Results: Three out of four of the invasive melanomas showed gains in 11q13 (CCND1 locus) by aCGH. FISH demonstrated CCND1 gain in invasive melanoma and in conjunctival melanocytic intraepithelial lesions of all grades (Low grade CMIL and in-situ melanoma) and this was paralleled by increased expression of Cyclin D1 protein within the atypical melanocytes by immunohistochemistry, using a double staining method with a red end point for Melan A cytoplasmic staining and a brown end-point for nuclear Cyclin D1 expression. Higher grades of melanocytic intraepithelial lesions showed more cells expressing Cyclin D1 compared to lower grade melanocytic intraepithelial lesions. The Cyclin D1 protein expression was in the same location as the amplified CCND1 signal by FISH. One out of three of these cases also showed amplification of the 12q13-15 locus corresponding to MDM2 and FISH confirmed gains in the conjunctival melanocytic intraepithelial neoplasia and invasive melanoma. The remaining fourth case showed a homozygous deletion of 9p21 (CDKN2A) by aCGH only, with immunohistochemistry showing clonal loss of p16 protein expression in the invasive and conjunctival melanocytic intraepithelial lesion. Two out of four of the invasive melanomas harboured classical driver mutations in NRAS and NF-1respectively. None of the cases showed mutations in BRAF, KIT and TERT mutations. RNAseq data showed secondary mutations in ARAF, PLCB4, MET, EZH2, MAP2K2, CTNNB1, CIITA, NF2, TP53 and MEN1, some of which are implicated in the MAPK pathway. Conclusion: Conjunctival melanocytic intraepithelial lesions harbour amplifications of CCND1 (3 cases), MDM2 (1 case) and loss of CDKN2A (1 case), which are also present when the lesion progresses to invasive melanoma, implicating these amplifications in the early pathogenesis of conjunctival melanocytic intraepithelial lesions. This study represents the first attempt to capture the mutational landscape of at all stages of conjunctival melanoma in a single tissue excision.


中文翻译：

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结膜黑色素瘤从上皮内疾病到侵袭性黑色素瘤的遗传变化的单一时间框架概述。一项对 4 个排液样本的研究，说明了 Cyclin D1 的潜在作用。

摘要 简介：尽管对皮肤黑色素瘤的分子发病机制的理解取得了进展，但对于结膜黑色素细胞从上皮内病变发展为侵袭性结膜黑色素瘤的过程中发生的遗传变化知之甚少。方法：我们将四份含有不同级别的上皮内结膜黑色素细胞瘤形成和侵袭性瘤形成的肠切除标本暴露于各种技术的组合中，包括阵列比较基因组杂交 (aCGH)、RNA seq、荧光原位杂交 (FISH) 和免疫组织化学。结果：四分之三的侵袭性黑色素瘤通过 aCGH 在 11q13（CCND1 基因座）中显示出增益。FISH 显示 CCND1 在侵袭性黑色素瘤和所有级别的结膜黑色素细胞上皮内病变（低级别 CMIL 和原位黑色素瘤）中增加，这与免疫组织化学在非典型黑色素细胞中 Cyclin D1 蛋白的表达增加平行，使用双染色方法Melan A 细胞质染色的红色终点和核 Cyclin D1 表达的棕色终点。与较低级别的黑色素细胞上皮内病变相比，较高级别的黑色素细胞上皮内病变显示更多细胞表达 Cyclin D1。Cyclin D1 蛋白表达与 FISH 放大的 CCND1 信号位于同一位置。这些病例中有三分之一也显示出对应于 MDM2 的 12q13-15 基因座的扩增，并且 FISH 证实了结膜黑色素细胞上皮内瘤变和侵袭性黑色素瘤的增加。剩下的第四个病例仅显示 aCGH 纯合缺失 9p21 (CDKN2A)，免疫组织化学显示侵袭性和结膜黑色素细胞上皮内病变中 p16 蛋白表达的克隆性丧失。四分之二的侵袭性黑色素瘤分别在 NRAS 和 NF-1 中具有经典驱动突变。所有病例均未显示 BRAF、KIT 和 TERT 突变。RNAseq 数据显示 ARAF、PLCB4、MET、EZH2、MAP2K2、CTNNB1、CIITA、NF2、TP53 和 MEN1 的二次突变，其中一些与 MAPK 通路有关。结论：结膜黑色素细胞上皮内病变包含 CCND1（3 例）、MDM2（1 例）和 CDKN2A 缺失（1 例）的扩增，当病变进展为侵袭性黑色素瘤时也会出现，提示这些扩增与结膜黑色素细胞上皮内的早期发病机制有关病变。这项研究首次尝试在单次组织切除中捕获结膜黑色素瘤各个阶段的突变情况。