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.
Immunohistochemical Analysis of Cyclin D1 Protein in Hematopoietic Neoplasms with Special Reference to Mantle Cell Lymphoma
