Pathway Enrichment Analysis of Whole-Exome Sequencing Data from Formalin-Fixed, Paraffin-Embedded Enucleated Eyes with Retinoblastoma and Choroidal Malignant Melanoma

Objectives: Intraocular tumors are extremely rare, small in size, and difficult to approach by biopsy. In the era of cancer genome analysis, we designed a pilot study to perform whole-exome sequencing of formalin-fixed paraffin-embedded enucleated eyes of retinoblastoma and choroidal malignant melanoma as two major intraocular malignancies.
Methodology: Genomic DNA was isolated from intraocular tumor areas of 105 paraffin sections with a 5 μm thickness of seven enucleated eyes with retinoblastoma and seven eyes with choroidal malignant melanoma. One of 7 samples of retinoblastoma and another of seven samples of choroidal malignant melanoma were excluded from the study since the sequencing output and depth of reads were lower compared with the other samples. The sequencing data after quality control were aligned to the reference genome sequence (hg38, GRCh38 Assembly, Genome Reference Consortium Human Build 38), and the mapped reads were processed to improve data quality. Somatic mutations (single nucleotide variants, insertions and deletions, and multiple nucleotide variants) in each sample were extracted after excluding variants reported in a Panel of Normals (PON) from the 1000 Genomes Project. Additional selection criteria included a mutation depth of ≥5 reads and either no registration in or an allele frequency of less than 5% in the Tohoku Medical Megabank of Japan (ToMMo 60KJPN-SNV/INDEL Allele Frequency Panel).
Results: Candidate genes with somatic mutations were selected by three criteria: genes with the same mutation shared by two samples or more, recurrently mutated genes three times or over, and genes of driver candidates identified in combining several different driver mutation-detecting programs by Integrative OncoGenomics (IntOGen). Using candidate genes detected by any of the three criteria as input, enrichment analyses identified 28 pathways in Gene Ontology (GO) and 2 pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) for retinoblastoma, while 385 pathways in GO, 12 in KEGG, 2 in the Hallmark gene set of the Molecular Signatures Database (MSigDB), and 47 in Reactome were identified for choroidal malignant melanoma. The enrichment maps showed three major pathways differently in retinoblastoma and choroidal malignant melanoma: one with dynein in retinoblastoma and another with MET in choroidal malignant melanoma.
Conclusions: Although there were limitations related to the small amounts of DNA available from formalin-fixed, paraffin-embedded small-sized tissues and the absence of matched normal control tissue, whole-exome sequencing provided clues to somatic mutations that were enriched in specific pathways and differed between retinoblastoma and choroidal malignant melanoma.