Genomic analysis of atypical fibroxanthoma

Abstract

Atypical fibroxanthoma (AFX), is a rare type of skin cancer affecting older individuals with sun damaged skin. Since there is limited genomic information about AFX, our study seeks to improve the understanding of AFX through whole-exome and RNA sequencing of 8 matched tumor-normal samples. AFX is a highly mutated malignancy with recurrent mutations in a number of genes, including COL11A1, ERBB4, CSMD3, and FAT1. The majority of mutations identified were UV signature (C>T in dipyrimidines). We observed deletion of chromosomal segments on chr9p and chr13q, including tumor suppressor genes such as KANK1 and CDKN2A, but no gene fusions were found. Gene expression profiling revealed several biological pathways that are upregulated in AFX, including tumor associated macrophage response, GPCR signaling, and epithelial to mesenchymal transition (EMT). To further investigate the presence of EMT in AFX, we conducted a gene expression meta-analysis that incorporated RNA-seq data from dermal fibroblasts and keratinocytes. Ours is the first study to employ high throughput sequencing for molecular profiling of AFX. These data provide valuable insights to inform models of carcinogenesis and additional research towards tumor-directed therapy.

Fig 1. Histopathology of AFX.

AFX samples were microdissected from snap-frozen, OCT-embedded surgical specimens. The spectrum of predominant cytomorphology included four pleomorphic/histocytic (AFX 1, 5, 6, and 8), two spindled (AFX 2 and 7), and two epithelioid (AFX 3 and 4). Scale bar indicates 300μm.

Fig 2. Whole exome sequencing identifies copy number variations, and frequent UV mutations in AFX.

(a) Histogram of somatic mutation rate (number of mutations/megabase of DNA) for each AFX tumor. (b) Stacked plot of the percentage of mutations of each type. (c) Matrix illustrates genes that are mutated in at least 75% of the AFX tumors and the type of mutations found. When more than one mutation is present for a single gene, only one type of mutation is shown delineated in order by the legend; insertion/deletion, nonsense/nonstop, missense, splice-site, 3’ or 5’UTR. (d) A heatmap of copy number variations in the 8 tumor samples relative to the normal. Red represents gains, blue represents deletions, and white represents no losses or gains in that location in the genome.

Fig 3. AFX gene expression clusters with other sarcomas.

T-SNE plot of 8 AFX tumors with publicly available tumors from TCGA. AFX are highlighted as black dots, clustering within other sarcomas (colored purple).

Fig 4. RNA sequencing reveals differentially expressed genes and pathways in AFX.

(a) Heatmap of DE genes extracted from literature reviews that are associated with TAMs (higher expression is red, while lower expression is shown as blue). (b) Heatmap of genes from the Hallmark Epithelial Mesenychmal Transition Pathway from GSEA.

Fig 5. M2 tumor-associated macrophages (TAM) are enriched in AFX.

A. Schematic of successive gating strategy of live myeloid cells expressing CD45 and HLADR. These were further gated on CD14+CD163+ to identify macrophages; CD206 expression was measured on this subset. A representative sample is shown. B. CD206 expression in four AFX (blue) and patient-matched normal skin (red). C. CD206+ TAM are enriched in AFX as a proportion of CD45+HLADR+CD14+CD163+ macrophages (left, p = 0.049); CD14+CD163+CD206+ macrophages are enriched as a proportion of CD45+HLADR+ myeloid cells (right, p = 0.01).