Unraveling the Structural Variations of Early-Stage Mycosis Fungoides—CD3 Based Purification and Third Generation Sequencing as Novel Tools for the Genomic Landscape in CTCL

SIMPLE SUMMARY: Mycosis fungoides is the most common cutaneous T-cell lymphoma, but knowledge of the genetic alterations, particularly of the early stages, is limited. A major problem is that biopsies from early stages contain few tumor cells and many “healthy” skin cells, making accurate analysis of tumor cells difficult. Here, we demonstrate a workflow to enrich tumor cells and thereby obtain better results for mutation detection, especially for deletions and amplifications. For the same sample, we also demonstrate the advantages of long-read sequencing for a more comprehensive elucidation of genetic alterations in early stages of mycosis fungoides. ABSTRACT: Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). At present, knowledge of genetic changes in early-stage MF is insufficient. Additionally, low tumor cell fraction renders calling of copy-number variations as the predominant mutations in MF challenging, thereby impeding further investigations. We show that enrichment of T cells from a biopsy of a stage I MF patient greatly increases tumor fraction. This improvement enables accurate calling of recurrent MF copy-number variants such as ARID1A and CDKN2A deletion and STAT5 amplification, undetected in the unprocessed biopsy. Furthermore, we demonstrate that application of long-read nanopore sequencing is especially useful for the structural variant rich CTCL. We detect the structural variants underlying recurrent MF copy-number variants and show phasing of multiple breakpoints into complex structural variant haplotypes. Additionally, we record multiple occurrences of templated insertion structural variants in this sample. Taken together, this study suggests a workflow to make the early stages of MF accessible for genetic analysis, and indicates long-read sequencing as a major tool for genetic analysis for MF.