Transcriptomic analysis identifies differences in gene expression in actinic keratoses after treatment with imiquimod and between responders and non responders

Abstract

The presence of actinic keratoses (AKs) increases a patient's risk of developing squamous cell carcinoma by greater than six-fold. We evaluated the effect of topical treatment with imiquimod on the tumor microenvironment by measuring transcriptomic differences in AKs before and after treatment with imiquimod 3.75%. Biopsies were collected prospectively from 21 patients and examined histologically. RNA was extracted and transcriptomic analyses of 788 genes were performed using the nanoString assay. Imiquimod decreased number of AKs by study endpoint at week 14 (p < 0.0001). Post-imiquimod therapy, levels of CDK1, CXCL13, IL1B, GADPH, TTK, ILF3, EWSR1, BIRC5, PLAUR, ISG20, and C1QBP were significantly lower (adjusted p < 0.05). Complete responders (CR) exhibited a distinct pattern of inflammatory gene expression pre-treatment relative to incomplete responders (IR), with alterations in 15 inflammatory pathways (p < 0.05) reflecting differential expression of 103 genes (p < 0.05). Presence of adverse effects was associated with improved treatment response. Differences in gene expression were found between pre-treatment samples in CR versus IR, suggesting that higher levels of inflammation pre-treament may play a part in regression of AKs. Further characterization of the immune micro-environment in AKs may help develop biomarkers predictive of response to topical immune modulators and may guide therapy.

Figure 1

Comparison of maximum number of AKs (Lmax), during treatment with imiquimod (black bars) with number of AKs at week 14, two months post-treatment with imiquimod (grey bars). Numbers above bars indicate number of AKs. Patients 15 and 17 had seborrheic keratoses on initial biopsy and are excluded from this figure. The samples with both pre- and post- treatment biopsies were: 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19.

Figure 2

(A) Volcano plot of gene expression changes in post versus baseline of pre-treatment samples shows significant downregulation of CXCL13, IL1β, GAPDH, CDK1, TTK, ILF3, EWSR1, PLAUR (p < 0.05). (B) H&E 10 × magnification of pre-treatment AK shows dense immune infiltration (C) IHC staining of CD3 cells at 10 × magnification in pre-treatment AK shows significant staining for CD3 cells (D) IHC staining of CD20 cells at 10 × magnification in pre-treatment AK shows few CD20 positive cells (E) IHC staining of CD68 cells at 10 × magnification in pre-treatment AK shows moderate staining for CD68 cells.

Figure 3

Heatmap of 103 genes differentially expressed between complete responders and incomplete responders. Data shown is pre-treatment gene expression for genes that are differentially expressed between CR and IR. Blue indicates a lower expression, red indicates a higher expression. Heatmap was generated using Morpheus v.1 Software. URL: https://software.broadinstitute.org/morpheus/.

Figure 4

(A) Violin plot examining the effects of potential confounders on clinical outcomes. (B) Variance explained by gene for the first ten genes in the analysis.