Exome and transcriptome sequencing for the identification of immunogenic mutations in a human melanom model

Somatic point mutations in tumors can lead to the generation of tumor-specific neoantigens which can be recognized by the patients’ own T cells. Such immunogenic mutations were searched for in the human melanoma model Ma-Mel-86 by whole exome and transcriptome sequencing of four tumor cell lines and an autologous EBV-transformed B cell line as germline control. The melanoma cell lines had been established from distinct lymph node metastases occurring over six years. Tumor cell lines derived from the later occurring metastases partially or completely lacked cell surface expression of HLA class I molecules. Considering biological variance, DNA duplicates and RNA triplicates isolated from separate cultures and two different time points each were sequenced. Paired-end exome sequencing of 65.8 million ± 11.8 million (standard deviation, SD) reads per sample achieved on avarage a 49.3-fold coverage of target exons. Per RNA sample, 69.4 million ± 25.5 million (SD) paired-end reads were generated. Single nucleotide substitutions (SNS) accounted for 85-89% of the somatic variants covering SNS and small insertions and deletions detected in the tumor exomes. C>T/G>A substitutions indicating a UV-induced disease dominated with 46-56% the somatic SNS. Per melanoma cell line, 265 non-synonymous somatic SNS were reported on average. Altogether 181 expressed non-synonymous somatic SNS could be identified in the four melanoma cell lines by combining the data from both sequencing procedures. About one third of these mutations were common to all four metastases-derived cell lines. Synthetic peptides carrying somatic SNS that were selected based on their presence in at least one of the two exome replicates and two of the three transcriptome replicates of the HLA class I-expressing melanoma cell lines were subjected to immunogenicity testing. By this means, autologous MHC I-restricted T cell responses against four neoantigens encoded by the mutated genes HERPUD1, INSIG1, MMS22L and PRDM10 were identified.