Discovery and validation of urine markers of acute pediatric appendicitis using high-accuracy mass spectrometry

Abstract

Study objective: Molecular definition of disease has been changing all aspects of medical practice, from diagnosis and screening to understanding and treatment. Acute appendicitis is among many human conditions that are complicated by the heterogeneity of clinical presentation and shortage of diagnostic markers. Here, we sought to profile the urine of patients with appendicitis, with the goal of identifying new diagnostic markers.

Methods: Candidate markers were identified from the urine of children with histologically proven appendicitis by using high-accuracy mass spectrometry proteome profiling. These systemic and local markers were used to assess the probability of appendicitis in a blinded, prospective study of children being evaluated for acute abdominal pain in our emergency department. Tests of performance of the markers were evaluated against the pathologic diagnosis and histologic grade of appendicitis.

Results: Test performance of 57 identified candidate markers was studied in 67 patients, with median age of 11 years, 37% of whom had appendicitis. Several exhibited favorable diagnostic performance, including calgranulin A (S100-A8), alpha-1-acid glycoprotein 1 (orosomucoid), and leucine-rich alpha-2-glycoprotein (LRG), with the receiver operating characteristic area under the curve and values of 0.84 (95% confidence interval [CI] 0.72 to 0.95), 0.84 (95% CI 0.72 to 0.95), and 0.97 (95% CI 0.93 to 1.0), respectively. LRG was enriched in diseased appendices, and its abundance correlated with severity of appendicitis.

Conclusion: High-accuracy mass spectrometry urine proteome profiling allowed identification of diagnostic markers of acute appendicitis. Usage of LRG and other identified biomarkers may improve the diagnostic accuracy of clinical evaluations of appendicitis.

Figure 1

Experimental scheme, outlining methods used for protein capture and fractionation, identification for discovery urine proteomics, and validation of candidate diagnostic markers. The discovery phase of the study involved comparisons of 12 specimens obtained from 9 patients (6 patients without appendicitis, and 3 patients with appendicitis before and after they underwent appendectomies), whereas the validation phase of the study involved all 67 patients.

Figure 2

Boxplots of the relative urine protein abundance (logarithm normalized ion current units) of the validated candidate diagnostic markers for the non-appendicitis (open) and appendicitis (gray) patient groups for leucine-rich α-2-glycoprotein (LRG), calgranulin A (S100-A8), α-1-acid glycoprotein 1 (ORM1), plasminogen (PLG), mannan-binding lectin serine protease 2 (MASP2), zinc-α-2-glycoprotein (AZGP1), α-1-antichymotrypsin (serpin3A), and apolipoprotein D (ApoD). Normalized value of 1 corresponds to the apparent abundance of internal reference standard (Online Appendix). Boxes contain the 25-75 % interquartile range, with the dividing bars representing medians, and whiskers representing 10-90 % range. Square symbols represent means. Abundance of LRG in patients with pyelonephritis (solid dot, ●) and those who underwent appendectomies with findings of histologically normal appendices (open dot, ○)

Figure 3

A. Receiver operating characteristics of urine protein markers validated by targeted mass spectrometry, demonstrating the relative diagnostic performance of leucine-rich α-2-glycoprotein (LRG), calgranulin A (S100-A8), α-1-acid glycoprotein 1 (ORM1), and peripheral blood absolute neutrophil count (ANC). The listed confidence intervals were computed for single comparisons, and do not include possible correction for multiple testing, which is expected to broaden them in proportion to the correlation and number of simultaneous tests. B. Enrichment of LRG in a random sample of urine of patients with histologically proven appendicitis (+) as compared to those without (−) by using Western immunoblotting. LRG signal was observed in 5/5 patients with appendicitis and no signal was observed in 5/6 patients without appendicitis. Development of quantitative LRG urine immunoblotting and assessment of its diagnostic performance in interventional studies are important directions of future work.

Figure 4

Top panel. Boxplots of the relative urine protein abundance (normalized ion current units) of leucine-rich α-2-glycoprotein (LRG) and calgranulin A (S100-A8) as a function of appendicitis severity, as assessed using histologic classification. Crosses represent 1-99 % range. Note that the group with histologically normal appendices includes both patients who underwent appendectomies and patients without clinical diagnosis of appendicitis. Bottom panel. Representative micrographs of appendectomy specimens and immunohistochemistry staining against LRG.