Whole Exome Sequencing and Analysis Of Mutations In Sézary Syndrome

Abstract Introduction and Rationale Sézary Syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma CTCL), is characterized by generalized erythroderma and >1000 circulating central memory T-cells with a CD4+CD26- phenotype, Other clinical features include reactive adenopathy, keratoderma, ectropion, pruritus, and colonization with Staphylococcus aureus. SS can arise de novo or emerge in patients with Mycosis Fungoides (MF). Morphologically, SS cells were first recognized by their convoluted nuclei and karyotype analysis has shown the presence of multiple chromosomal abnormalities and copy number variants, none of which are disease defining; however, the mutational landscape of SS has not been investigated systematically. Here we present initial findings from profiling somatic mutation by whole exome sequencing. Methods In order to delineate the genomic mutation profile of Sézary cells we compared protein coding sequences from CD4+ and CD4- peripheral blood cell DNA to that from autologous cultured fibroblasts derived from ten newly diagnosed SS patients with high percentages of circulating SS cells. CD4+ T-cells were selected by a Miltenyi Biotec CD4+ T Cell Isolation Kit and genomic DNA was extracted from CD4+ T-cells, residual CD4- cells, and autologous fibroblasts from each patient using an All Prep DNA/RNA/Protein Mini Kit (Qiagen). Purity of CD4+ T-cell sorting confirmed by flow cytometry was > 90% purity. Skin biopsies from SS non-lesional skin were explanted, cultured for 5-6 weeks, and used to extract genomic control DNA for each patient. Whole exome DNA was enriched by capture using a Nimblegen VCRome version 2.1 array. Nine billion bases of sequence was generated for each sample on an Illumina HiSeq 2000 run for 101 cycles. This resulted in approximately 95% target covered to a minimum depth of 20 bases, with and overall average coverage of over 130 bases in all samples. Somatic mutations were called by comparing sequence from all tissues to the human reference sequence (GRC37) and then subtracting nucleotide variation found in the fibroblast lines from variation found in the CD4- and CD4+ cell populations. Results We found an average of 82 mutations per patient in the CD4+ cells. TP53 with 4 mutations was the most commonly mutated, three patients harbored MSH3 mutations, which may contribute to higher mutation frequencies in those patients. Singleton mutations in MYC, MLLT4 are among cancer-related transcription factors, and singleton NF1 and Notch4 suggest aberrant signaling. Surprisingly, the CD4- cells also harbored TP53 mutations in 3 of the 4 patients. Conclusions Whole exome sequencing revealed recurrent mutations in two genes: TP53 and MSH3 and hinted at involvement of RAS and Notch pathway signaling, although no RAS mutations were observed. The presence of TP53 mutation in CD4- cells was unanticipated, and may result from mutation of a common progenitor cell population in the CD4- cells. Studies are underway, including analysis of copy number gains and losses to further elucidate the mutational patterns in SS patients. Disclosures: No relevant conflicts of interest to declare.