In situ targeted gene expression analysis of Marjolin ulcers

Marjolin's ulcer is a a rare and aggressive cutaneous malignancy that can arise on previously injured skin, established scars, and chronic non-healing wounds. It is most often found in burn scars, but it can also occur in other types of wounds, including venous stasis ulcers, pressure sores, and vaccination sites. The most common histological type of Marjolin’s ulcers is squamous cell carcinoma, however basal cell carcinomas, malignant melanomas, and sarcomas have also been reported. All parts of the body could potentially be affected yet the lower extremities are the anatomic sites most commonly involved. While SCCs commonly have a metastasis rate of 0.5 to 3.0 percent, those arising from burn scars metastasize at a rate in excess of 30 percent. The 5-year survival after a diagnosis of Marjolin’s ulcer was found to be 50 percent. The transformation to a malignancy can occur either chronically, over a period of more than 35 years, or occasionally within a year of the original injury. The exact mechanism of this transformation is not fully understood, but it is thought to be related to chronic keratinocyte dysfunction during the healing process of severe burn wounds. Surgical excision is the main treatment for Marjolin's ulcer and provides the best chance of survival. In a previous study, we investigated the bulk transcriptional changes that lead to Marjolin's ulcer by comparing global gene expression changes between squamous cells present in a squamous cell carcinoma versus those present within Marjolin's ulcer (MU) (Sinha et. al. JBCR 2017). This imparted novel insights into mechanisms underlying divergent clinical features of these cutaneous cancers. The goal of our current study is to characterize a new case of Marjolin's ulcer in a patient under our care by analyzing the cell types, their frequencies, and their individual transcriptional responses within a burn scar versus within Marjolin's ulcer. To achieve this, we conducted single-cell RNA sequencing on two excised tissues: 1. a sample from the center of the tumor (tumor core), and 2. another sample from the margin of tumor-free scar tissue. The Xenium In Situ platform employs a microscopy-based method for analysis. A formalin-fixed and paraffin-embedded (FFPE) MU tissue block was obtained. Adhering to the manufacturer's protocols, the tissue underwent processing, including sectioning, organizing the sections, and performing Hematoxylin and Eosin (H&E) staining to ensure quality. The tissue sections were precisely trimmed to match the dimensions of the Xenium slide, with careful attention to include areas of both the burn scar and the tumor core. Once positioned on Xenium slide, the section was stained with Hematoxylin and Eosin (H&E) for tissue morphology analysis. Subsequent steps involved the hybridization and ligation of specific DNA probes to target mRNA, followed by a process of rolling circle amplification. The slide underwent several rounds of fluorescent probe hybridization and imaging in the Xenium Analyzer. This process, leveraging each gene's unique optical signature, allowed for the decoding of target genes and the creation of a spatial transcriptomic map of the entire tissue section.