Distinctive Transcriptional and Microbial Signature in Cutaneous Acute Graft-vs-Host-Disease

Skin acute graft-vs-host disease (aGVHD) is often the first manifestation of GVHD, yet very few preclinical and clinical studies have focused on this target organ, leaving a critical information gap in the pathophysiology of GVHD. We hypothesized that analysis of host gene expression and microbiome profiling could yield novel insights into the molecular and immunologic mechanisms underlying skin GVHD. Our objectives were to determine the differential host gene expression and microbiome profile of human skin aGVHD samples compared to normal skin, and aGVHD corticosteroid responders to non-responders. We performed RNA-Sequencing on lower arm biopsies from 45 patients compared to 10 healthy controls. Our findings suggest a distinctive transcriptional signature of cutaneous aGVHD, that could identify potentially actionable targets for prevention or treatment of corticosteroid refractory disease. Our analysis suggests a key role of dendritic cells and macrophages, potentially mediated by differential expression of MIF, in the development of cutaneous aGVHD and corticosteroid responsiveness. Additionally, we describe a unique microbial signature in cutaneous aGVHD that includes skin microbes not previously described in this population. Performed RNA-Sequencing (RNA-seq) on 20 µm slices of formalin-fixed paraffin embedded (FFPE) diagnostic lower arm skin biopsies from 45 adult patients with histologic grade 2-2.5 acute GVHD (aGVHD). Compared these to 10 healthy controls from discarded surgical lower arm skin tissue. Assessed aGVHD treatment response at day 28 and identified 35 complete or partial responders (CR or PR) and 10 non-responders (NR) based upon their response to systemic immunosuppression with corticosteroids at day 28 of aGVHD therapy. extracted RNA from the FFPE biopsies using PureLink FFPE RNA Isolation Kit. FFPE samples yielded an adequate quantity of RNA for sequencing (median = 98717524.5 reads, range = 74316820 to 229178960). After extraction, we prepared the libraries with the SMARTer Stranded Total RNA Kit v2 and performed sequencing on a single lane of a NovaSeq S4 2x150-bp. Aligned and assembled using hisat2 and Cufflinks. Single gene analysis performed and pathway analysis via Enrichr. Unaligned reads alignment via Pathseq - microbiome analysis.