Inflammation-related gene biomarkers in human diabetic foot ulcer healing

Project summary Diabetic foot ulcers (DFUs) occur in 15% of diabetic patients, leading to over 82,000 lower limb amputations annually in the United States and a 5-year mortality rate of up to 74%. The reasons for impaired DFU healing are complex, but the downstream effects of chronic inflammation are major contributors. Our research has shown that while initial pro-inflammatory activation of immune cells is critical for the initiation of healing processes, prolonged activation directly impairs wound healing. Recognizing that transition from the early inflammatory phase to the late resolution phase is required for successful healing, we developed a composite biomarker that uses the change in the ratio of 4 early stage pro-inflammatory gene markers to 3 late stage inflammation-resolution gene markers over 4 weeks to predict responsiveness to treatment. By using a ratio of these early-stage to late-stage genes, referred to here as the Inflammation Index, higher values indicate wounds that are earlier in the healing process (and further from healing), while lower values indicate a wound that is later in the healing process (and closer to healing). Thus, a decrease in this index over time is linked to healing, while an increase is linked to exacerbating inflammation and non-healing. In our preliminary studies (3 cohorts of 21 subjects), the change in the Inflammation Index correctly predicted healing in 10 out of 10 healing wounds (responders), and it correctly predicted non-healing in 9 out of 11 non-healing wounds (non-responders), with an area under the ROC curve of 0.9. This biomarker utilizes debrided wound tissue so that it can be easily incorporated into standard wound care practice without adding any new techniques or time into the visit. Expression of the 7 genes that comprise the composite biomarker is measured using qRTPCR, a widely available, low-cost, and reliable technique. Finally, the use of a ratio self-normalizes the gene expression values to reduce patient-to-patient variability and increase reproducibility. In this project, we will develop and internally validate detection of this composite ratio-based biomarker; establish proof of concept of its prognostic utility; lay the foundation for a specific Context of Use (COU); and ultimately ensure that is ready for Phase II clinical trials. The proposed COU is a prognostic biomarker to identify individuals who are not likely to heal their ulcer when treated with the standard of care, for use in the personalization of treatment and/or the refinement of entry criteria for clinical trials of new treatments. In the R61 phase of this project, we will optimize biomarker measurement and standardization, and determine the quality control (QC) metrics that can be used to determine if the biomarker is accurately measured. We will also validate storage/shipping conditions and measure reliability and reproducibility. After meeting rigorous milestones the biomarker will progress to the R33 phase of the project, in which we will measure its ability to predict healing in response to the standard of care, in order to ultimately personalize treatment for patients with hard-to-heal ulcers and to refine entry criteria for clinical trials of new treatments.