The Landscape Of Somatic Mutations And The Functions Of Major Mutant Genes In Acral Melanoma

Background:Melanoma is the cancer derived from melanocytes.The main clinical classifications are skin,acral,mucosal and ocular subtypes.Cutaneous melanoma is the most prevalent subtype in the world,mainly occurring on the trunk or the face and neck in European and American populations.Cutaneous melanoma has a strong correlation with UV exposure and has high frequencies of mutations on BRAF and NRAS gene,which are therapy targets of melanoma.In addition,immune checkpoint inhibitors achieve clinical benefits among advanced melanoma patients.The studies of cutaneous melanoma have clarified its landscape of somatic mutations.According to the characteristics of somatic mutant genes,cutaneous melanoma could be classified into four molecular subtypes,which is helpful for the understanding of the pathogenesis,prognosis and treatment on cutaneous melanoma.Acral melanoma is the most common subtype of melanoma among Asian populations,but the pathogenic factors or the molecular mechanism are still not clear.The deficiency of the research blocks efficient clinical treatments on this subtype.Therefore,it is urgent to carry out a study among large scale samples of acral melanoma to identify the landscape of somatic mutations,explore potential pathogenic factors,and provide novel molecular targets for clinical treatments.Methods:1.Extract DNA samples from acral melanoma tumors and adjacent normal tissues.Also collect the clinical and pathological information of these patients.2.Using the technology of exome sequencing,target region sequencing and m RNA sequencing to analysis the landscape of somatic mutations among 108 cases of acral melanoma.3.For the recurrent amplified EP300 gene,real-time PCR with probes was used to verify its mutation rates.And the effect of P300 inhibitor on the cell cycle and proliferation in melanoma cells were detected by cell counting experiments and flow cytometry.4.We constructed the plasmids of wild type and mutant PARP4 and transfected these plasmids into melanoma cells to identify the effects of PARP4 mutation on apoptosis by flow cytometry.Results:1.We successfully extracted DNA samples from 108 acral melanoma tissue and 19 RNA samples among them and complete the collection of clinical and pathological information of the samples.The analysis of clinical information revealed that subungual melanoma has longer course of disease than melanomas on foot.2.The main features of the landscape of somatic mutation in acral melanoma are as follows: 1)The mutation burden of acral melanoma is significantly lower than that of cutaneous melanoma;2)The mutations of acral melanoma display non-UV associated characteristics;3)The mutation frequency of BRAF and NRAS genes is significantly lower than that in cutaneous melanomas,while KIT is the gene with the highest mutation frequency in acral melanomas;4)We identifies novel significantly mutant genes such as PARP4 and MED16;5)Acral melanoma has high-frequency of copy number variations,and the major involved genes are CCND1,KIT,TERT,EP300,CDK4 and CDKN2A;6)We find a novel fusion gene,DPH7-PAPD3 A,with high-frequency of mutation in acral melanoma;7)RTKs,MAPK pathway and cell cycle pathways are the main pathways involved with somatic mutations in acral melanoma.8)The burden of neoantigens is positively correlated with the mutation burden and the level of intratumoral lymphocyte infiltration.3.The features of mutation landscape,including the mutation burden,the mutation characteristics,the frequency of significantly mutant genes,the major mutant pathways,potential drug targets and the burden of neoantigens,are significantly different among subungual and foot melanomas.4.High frequency copy number amplification of EP300 was verified among another cohort of acral melanoma.The inhibitor of P300 suppresses the process of cell cycle and the proliferation of melanoma cells.5.PARP4 inhibits cisplatin-induced apoptosis of melanoma cells,while the recurrent T1170 I mutation of PARP4 blocks its inhibitory effect.Conclusion:In this study,we used the technology of second-generation sequencing for the first time to identify the landscape of somatic mutations in a large scale of acral melanoma samples.According to the results,we not only confirmed the genomic features of acral melanoma in previous studies,such as the relatively lower mutation burden,the non-UV related mutation characteristics,high-frequency of copy number variations,but also identified novel significantly mutant genes DPH7-PAPD3 A,PARP4,MED16,EP300 and so on.We innovatively elucidated the differences of somatic mutations between acral melanomas derived from different primary sites and provided a molecular basis for further classification of acral melanomas.Further explorations of the mechanism of EP300 and PARP4 involved in melanoma indicated the potential of these mutant genes as targets for clinical therapy.