Inflammation-related gene biomarkers in human diabetic foot ulcer healing

项目总结 糖尿病足溃疡(DFU)发生在15%的糖尿病患者中，导致超过82,000条肢体 在美国，每年的截肢和5年死亡率高达74%。致残的原因 DFU的愈合是复杂的，但慢性炎症的下游影响是主要因素。我们的 研究表明，虽然免疫细胞的初始促炎激活对于启动 愈合过程中，长时间的激活直接损害伤口的愈合。认识到从 早期炎症阶段到晚期消退阶段是成功愈合所必需的，我们开发了一种 使用4个早期促炎基因标志物与3个晚期促炎基因标志物比率的变化的复合生物标志物 在4周内分期炎症消解基因标记物以预测治疗反应。通过使用 这些早期和晚期基因的比率，在这里被称为炎症指数，值越高表明 愈合过程中较早(离愈合较远)的伤口，而较低的值表示伤口 这是在康复过程的后期(也更接近于康复)。因此，随着时间的推移，该指数的下降与 治愈，而增加与加剧炎症和无法愈合有关。在初步研究中(3 21名受试者)，炎症指数的变化正确地预测了十分之十的愈合情况 伤口(应答者)，它正确地预测了11个未愈合伤口(无应答者)中的9个无法愈合， ROC曲线下的面积为0.9。这种生物标记物利用清创的伤口组织，因此可以很容易地 纳入标准伤口护理实践，无需增加任何新的技术或时间的访问。 组成复合生物标记物的7个基因的表达使用qRTPCR来测量， 成本低，技术可靠。最后，使用比率使基因表达值自我归一化以减少 患者与患者之间的可变性，并增加重复性。 在这个项目中，我们将开发和内部验证这种基于比率的复合生物标志物的检测； 建立其预测效用的概念证据；为具体的使用背景(COU)奠定基础；以及 最终确保为第二阶段临床试验做好准备。建议的cou可作为判断预后的生物标志物。 在接受标准护理治疗时不太可能治愈溃疡的个人，用于 个性化治疗和/或完善新疗法临床试验的准入标准。 在这个项目的R61阶段，我们将优化生物标记物的测量和标准化，以及 确定可用于确定生物标记物是否被准确测量的质量控制(QC)指标。 我们还将验证存储/运输条件，并测量可靠性和重复性。会后 严格的里程碑生物标记物将进入项目的R33阶段，在此阶段我们将衡量其 能够根据护理标准预测康复情况，以便最终实现个性化治疗 为治疗难愈的溃疡患者提供技术援助，并完善新疗法临床试验的准入标准。

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.