40. Clonal evolution of Cutaneous T Cell Lymphoma (CTCL) revealed at single cell resolution

Patients with CTCL subtypes Mycosis Fungoides and Sézary Syndrome (MF/SS) frequently progress to advanced stage disease, which has a 20% 5-year survival. The molecular factors that drive MF/SS progression remain undefined. To address this, we subjected 41 longitudinal tumor samples (peripheral blood, skin and lymph node biopsies, 49-1050 days apart) and matched, purified non-malignant cells from 10 MF/SS patients to single-cell (sc)RNA-seq and exome+whole genome sequencing (eWGS). and patient-specific TCR clonotype assignment (scRepertoire). Clustering and cell type inference of scRNAseq (UMAP/Seurat/singleR) showed canonical CTCL marker genes and high clonal proportions (1-100%) of patient-specific TCR clonotypes. Somatic genome copy number variants (CNV) from scRNAseq (copykat) and eWGS (cnvkit), and deleterious/damaging single nucleotide variants (SNV) (eWGS, GATK/Mutect2) were identified in CTCL versus matched, non-malignant cells. To segregate early genomic events from progression-associated, we developed an integrative analysis pipeline to prioritize somatic CNV/SNV in genes with altered expression in >=30% of patients. This novel approach revealed alterations in NF-KB and T-cell receptor pathways (NFKB2, STAT3/5, TOX) as early events that were maintained in subsequent CTCL samples. We demonstrate gain-of-function activity in a novel-to-CTCL mutation in STAT3. Losses of some chromatin-modifying enzymes (DNMT3A, KDM6B) are early events, while gains of others (EZH2) are acquired with progression. Strikingly, progression-associated mutations were enriched in cell metabolism, growth, and motility genes, some of which are new to MF/SS: HIF1A, ITPR3, GTPase/RAS (IQGAP1/2, RASA1), and PI kinase (AKT1, MTOR, PIK3R1). These findings represent new insights into MF/SS progression and identify new precision medicine targets for currently available therapies.