Metabolomics Identifies and Validates Serum Novel Biomarker for Diagnosing Primary Angle Closure Glaucoma and Predicting the Visual Field Progression

Reviewer #1 (Public Review):

The prevalence of primary angle closure glaucoma (PACG) is high in Asia compared to all over the world. This study focused on characterizing the metabolite profile associated with PACG, identifying potential blood diagnostic markers, assessing their specificity for PACG, and verifying their applicability to predict the progression of visual field loss. To this end, Li et al. implemented a 5-phases multicenter prospective study to identify novel candidate biomarkers of PACG. A total of 616 individuals were recruited, identifying 1464 distinct metabolites in the serum by metabolomics and chemiluminescence immunoassays. By applying different machine learning algorithms the metabolite androstenedione showed good discrimination between PACG and control subjects, in both the discovery and validation phases. This metabolite also showed alterations in the aqueous humor and higher levels of androstenedione seemed to be associated with faster loss of visual field. Overall, the authors claimed that serum androstenedione levels may provide a new biomarker for early detection and monitoring/predicting PACG severity/progression.

Strengths:
• Omics research on glaucoma is constrained by inadequate sample sizes, a dearth of validation sets to corroborate findings, and an absence of specificity analyses. The 5-phases study was designed to try to overcome these limitations. The study design is very robust, with well well-described discovery set (1 and 2), validation phase (1 and 2), supplemental phase, and cohort phase. Large and well-characterized patients with adequate control subjects contributed to the robustness of the results.
• Combining untargeted and targeted metabolomics using mass spectrometry instruments (high resolution and low resolution) with an additional chemiluminiscence immunoassay determining androstenedione levels
• Androstenedione achieved better diagnostic accuracy across the discovery and validation sets, with AUC varying between 0.85 and 1.0. Interestingly, baseline androstenedione levels can predict glaucoma progression via visual field loss results.
• A positive correlation was observed between levels of androstenedione in serum and aqueous humor of PACG patients.
• A level higher of 1.66 ng/mL of the metabolite androstenedione seems to imply a high risk of visual field loss. Androstenedione may serve as a predictor of glaucomatous visual field progression.

Weakness:
• A single biomarker seems very unlikely to be of much help in the detection of glaucoma due to the etiological heterogeneity of the disease, the existence of different subtypes, and the genetic variability among patients. Rather, a panel of biomarkers may provide more useful information for clinical prediction, including better sensitivity and specificity. The inclusion of additional metabolites already identifying in the study, in combination, may provide more reliable and correct assignment results.
• The number of samples in the supplementary phase is low, larger sample sizes are mandatory to confirm the diagnostic accuracy.
• Cohorts from different populations are needed to verify the applicability of this candidate biomarker.
• Sex hormones seem to be associated also with other types of glaucoma, such as primary open-angle glaucoma (POAG), although the molecular mechanisms are unclear (see doi:10.1167/iovs.17-22708). The inclusion of patients diagnosed with other subtypes of glaucoma, like POAG, may contribute to determining the sensitivity and specificity of the proposed biomarker. Androstenedione levels should be determined in POAG, NTG, or PEXG patients.
• In addition, the levels of androstenedione were found significantly altered during other diseases as described by the authors or by conditions like polycystic ovary syndrome, limiting the utility of the proposed biomarker.
• Uncertainty of the androstenedione levels compromises its usefulness in clinical practice.

Reviewer #2 (Public Review):

Summary:
The objective of authors using metabolomics analysis of primary angle closure glaucoma (PACG) is to demonstrate that serum androstenedione is a novel biomarker that can be used to diagnose PACG and predict visual field progression.

Strengths:
Use of widely targeted and untargeted metabolite detection conditions. Use of liquid chromatography-tandem mass spectrometry and a chemiluminescence method for confirmation of androstenedione.

Weaknesses:
The "predict" part is on much less solid ground. The visual field progression and association with serum androstenedione within the current experimental design eludes to a correlation. It truly cannot be stated as predictive. To predict one needs to put the substance when nothing is there and demonstrate that the desired endpoint is reached. Conversely, the substance (androstenedione) can be removed, and show that the condition regresses. None of these are possible without model system experiments, which have not been done. The authors could put some additional details in the methods, such as: 1) how much sample was collected, 2) whether equal serum volume for analysis had equal serum proteins (or cells). They have used a LC-MS/MS and a Chemiluminescence method, but another independent method such as GC-MS/MS or NMR to detect androstenedione for a subset of patients with different stages of visual field defect would be desirable.