AKT1 expression is lost in a subset of VIN and the epidermis of the K14-HPV16 mouse.

<p>A. Histology and AKT1 expression in two representative VIN. Inset shows specific AKT1 expression associated with nucleated upper epidermal cells. Secondary alone control of upper epidermis is also shown. B. Immunohistochemical analysis of AKT1 in the dorsal epidermis of the K14-HPV8 complete early region (CER) and ear epidermis of the K14-HPV16 complete early region mouse and corresponding wildtype controls (wildtype). Arrowheads indicate AKT1 positivity in the granular layer of controls. Sec alone is a no primary antibody control for staining specificity. Bar (A–B) 50 µm.</p

Summary of nested PCR analysis and sequencing for HPV16/18, and Beta-PV types.

<p>-, sample is negative for this PV-type. AKT1 indicates the presence (pos) or absence (neg) of AKT1 expression in the epidermis of the VIN/vSCC. Copy no. indicates the HPV16 copy number per cell in each sample. Integrated? denotes the integration status of HPV16 based on the HPV16 genomic PCR. N.B. Several samples were PCR-positive for cutaneous alpha-PV species 4, however sequencing showed this was due to non-specific amplification of HPV16 in all cases; gamma and mu/nu types were not detected. E1-E4 and E7, summary of the immunohistochemistry for HPV16 EE4 and E7. n.d – not determined.</p

AKT1 expression and HPV16E7 expression in vSCC cohort.

<p>Representative data of immunohistochemistry of the archive vSCC cohort. AKT1 expression and HPV16E7 expression were analysed by immunohistochemistry. Loss of AKT1 associated with low HPV16E7 levels, while maintained AKT1 correlated with high HPV16E7 expression. Bar 50 µm.</p

Keratinocyte differentiation marker expression in epidermis, vulva and cervix.

<p>Immunohistochemical analysis of AKT1, loricrin and keratin 1 expression in extragenital cutaneous epidermis, vulval external epithelium and cervix. Bar 50 µm.</p

Early gene expression in VIN.

<p>Expression of the early genes 16E1∧E4 and 16E7 in a representative AKT1 negative (AKT1 −ve) and AKT1 positive (AKT1 +ve) VIN. Note the increased 16E7 expression in AKT1 positive VIN, and the change of 16E1∧E4 expression from perinuclear low expression in AKT1 negative VIN to high nuclear expression in AKT1 positive VIN (see insets). Bar 50µm.</p

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

<p><b>(a)</b> Histogram of somatic mutation rate (number of mutations/megabase of DNA) for each AFX tumor. <b>(b)</b> Stacked plot of the percentage of mutations of each type. <b>(c)</b> 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. <b>(d)</b> 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.</p

AFX gene expression clusters with other sarcomas.

<p>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).</p

RNA sequencing reveals differentially expressed genes and pathways in AFX.

<p>(<b>a</b>) 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>b</b>) Heatmap of genes from the Hallmark Epithelial Mesenychmal Transition Pathway from GSEA.</p

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

<p><b>(a)</b> Histogram of somatic mutation rate (number of mutations/megabase of DNA) for each AFX tumor. <b>(b)</b> Stacked plot of the percentage of mutations of each type. <b>(c)</b> 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. <b>(d)</b> 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.</p

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

<p>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).</p