Scott Bringans

Background: Diabetes-related chronic kidney disease (DKD) is a leading cause of end-stage kidney disease (ESKD), requiring costly dialysis or kidney transplantation. Existing standard- of-care tests for DKD have several limitations, and an alternative is Promarker®D, a validated plasma biomarker test system that predicts DKD in adults with diabetes up to 4 years before symptoms develop. Methods: To enable high-throughput application of PromarkerD, a novel CaptSureTM immunoassay version of the test was developed targeting plasma biomarkers Apolipoprotein A4 (ApoA4) and CD5 antigen-like (CD5L). The analytical performance of the assay was assessed, and clinical samples from 2 independent clinical cohorts (>1700 adults with type 2 diabetes [T2D]) were used for the development and external validation of the DKD predictive test. Results: The PromarkerD test system combined ApoA4 and CD5L concentrations with clinical factors age and estimated glomerular filtration rate (eGFR) to calculate risk scores (0% to 100%) and classify study participants as either at low, moderate, or high risk for future kidney decline. PromarkerD demonstrated reliable analytical performance and provided a high discriminative capability in adults with T2D (receiver operating characteristic area under the curve [ROC-AUC]: 0.78 to 0.88) to predict 4-year kidney decline, defined as incident DKD (eGFR 92% at the high-risk cutoff across the two cohorts. Conclusion: The next-generation PromarkerD test system offers a convenient yet highly effective tool for DKD risk assessment. By introducing PromarkerD to standard diabetes care, preventative treatment strategies may be implemented early before permanent kidney function loss occurs.

Background: Esophageal adenocarcinoma (EAC) diagnosis involves invasive and expensive endoscopy with biopsy, but rising EAC incidence has not been reduced by increased surveillance. This study aimed to develop and clinically validate a novel glycoprotein biomarker blood test for EAC, named PromarkerEso. Methods: Serum glycoprotein relative concentrations were measured using a lectin-based magnetic bead array pulldown method, with multiple reaction monitoring mass spectrometry in 259 samples across three independent cohorts. A panel of glycoproteins: alpha-1-antitrypsin, alpha-1-antichymotrypsin, complement C9 and plasma kallikrein, were combined with clinical factors (age, sex and BMI) in an algorithm to categorize the samples by the risk of EAC. Results: PromarkerEso demonstrated a strong discrimination of EAC from the controls (area under the curve (AUC) of 0.91 in the development cohort and 0.82 and 0.98 in the validation cohorts). The test exhibited a high sensitivity for EAC (98% in the development cohort, and 99.9% and 91% in the validation cohorts) and a high specificity (88% in the development cohort, and 86% and 99% in the validation cohorts). PromarkerEso identified individuals with and without EAC (96% and 95% positive and negative predictive values). Conclusions: This less invasive approach for EAC detection with the novel combination of these glycoprotein biomarkers and clinical factors coalesces in a potential step toward improved diagnosis.

Background/Objectives: The current standard of care for assessing chronic kidney disease complicating diabetes (DKD) includes measurement of estimated glomerular filtration rate (eGFR) and urinary albumin:creatinine ratio (uACR) but both tests have limitations. The present study compared the biomarker-based Promarker^®D test with conventional biochemical measures for predicting future kidney function decline in adults with type 2 diabetes (T2D). Methods: Baseline concentrations of apolipoprotein A-IV, CD5 antigen-like protein and insulin-like growth factor binding protein 3 were combined with age, serum HDL cholesterol and eGFR to generate PromarkerD risk scores for incident DKD/eGFR decline ≥ 30% (the primary endpoint) in 857 adults with T2D (mean age 65.4 years, 54% males). Logistic regression modelling was used to compare the association of (i) PromarkerD, (ii) eGFR, (iii) uACR, and (iv) eGFR plus uACR with this outcome during 4 years of follow-up. Results: Study participants were classified by PromarkerD as low (63%), moderate (13%), or high risk (24%) for kidney function decline at baseline. Over a mean 4.2 years, 12.5% developed the primary endpoint. PromarkerD scores showed significantly higher predictive performance (area under the receiver operating characteristic curve (AUC) 0.88 (95% confidence interval (CI) 0.85–0.91)) compared to conventional biochemical measures (AUC = 0.63–0.82). There was a progressive increase in risk with moderate and high risk by PromarkerD exhibiting greater odds of the primary endpoint compared to those at low risk (odds ratios (OR) (95% CI) 8.11 (3.99–16.94) and 21.34 (12.03–40.54), respectively, both p < 0.001). Conclusions: PromarkerD more accurately identifies adults with T2D at risk of kidney function decline than current usual care biochemical tests.

PromarkerD is a biomarker-based blood test that predicts kidney function decline in people with type 2 diabetes (T2D) who may otherwise be missed by current standard of care tests. This study examined the association between canagliflozin and change in PromarkerD score (Δ score) over a three-year period in T2D participants in the CANagliflozin cardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline and Year 3 in 2008 participants with preserved kidney function (baseline eGFR ≥60 mL/min/1.73 m²). Generalized estimating equations were used to assess the effect of canagliflozin versus placebo on PromarkerD scores. At baseline, the participants (mean age 62 years, 32% females) had a median PromarkerD score of 3.9%, with 67% of participants categorized as low risk, 14% as moderate risk, and 19% as high risk for kidney function decline. After accounting for the known acute drop in eGFR following canagliflozin initiation, there was a significant treatment-by-time interaction (p < 0.001), whereby participants on canagliflozin had decreased mean PromarkerD scores from baseline to Year 3 (Δ score: −1.0% [95% CI: −1.9%, −0.1%]; p = 0.039), while the scores of those on placebo increased over the three-year period (Δ score: 6.4% [4.9%, 7.8%]; p < 0.001). When stratified into PromarkerD risk categories, participants with high risk scores at baseline who were randomized to canagliflozin had significantly lower scores at Year 3 (Δ score: −5.6% [−8.6%, −2.5%]; p < 0.001), while those on placebo retained high scores (Δ score: 4.5% [0.3%, 8.8%]; p = 0.035). This post hoc analysis of data from CANVAS showed that canagliflozin significantly lowered PromarkerD risk scores, with the effect greatest in those T2D participants who were classified at study entry as at high risk of a subsequent decline in kidney function.

Immunoaffinity mass spectrometry as an approach for diagnostic biomarker assays combines the advantages of antibody selectivity with the multiplexing and analytical performance of mass spectrometry. A method has been developed to detect and quantify three protein biomarkers for a diabetic kidney disease prognostic assay, PromarkerD. The methodology reflects an immunoaffinity approach compatible with higher throughput and robust clinical application. After preparation and purification of antibody-bead conjugates for the three target proteins, an immunoaffinity capture step provides a solution for reduction, alkylation, and digestion on-bead. Targeted mass spectrometry provides a quantitative measure of each biomarker in a rapid 8 min run using a microflow LCMS workflow. Key words Biomarkers Assay development Diabetic kidney disease Immunoaffinity Targeted mass spectrometry

PromarkerD is a proteomics derived test for predicting diabetic kidney disease that measures the concentrations of three plasma protein biomarkers, APOA4, CD5L and IBP3. Antibodies against these proteins were developed and applied to a multiplexed immunoaffinity capture mass spectrometry assay. In parallel, and facilitating current clinical laboratory workflows, a standard ELISA was also developed to measure each protein. The performance characteristics of the two technology platforms were compared using a cohort of 100 samples, with PromarkerD test scores demonstrating a high correlation (R = 0.97). These technologies illustrate the potential for large scale, high throughput clinical applications of proteomics now and into the future.

Background: PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application. Methods: A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories. Results: Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer. Conclusions: An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients.

The ability of current tests to predict chronic kidney disease (CKD) complicating diabetes is limited. This study investigated the prognostic utility of a novel blood test, PromarkerD, for predicting future renal function decline in individuals with type 2 diabetes from the CANagliflozin CardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline in 3568 CANVAS participants (n = 1195 placebo arm, n = 2373 canagliflozin arm) and used to predict incident CKD (estimated glomerular filtration rate (eGFR) 2 during follow-up in those above this threshold at baseline) and eGFR decline ≥30% during the 4 years from randomization. Biomarker concentrations (apolipoprotein A-IV (apoA4), CD5 antigen-like (CD5L/AIM) and insulin-like growth factor-binding protein 3 (IGFBP3) measured by mass spectrometry were combined with clinical data (age, serum high-density lipoprotein (HDL)-cholesterol, eGFR) using a previously defined algorithm to provide PromarkerD scores categorized as low-, moderate- or high-risk. The participants (mean age 63 years, 33% females) had a median PromarkerD score of 2.9%, with 70.5% categorized as low-risk, 13.6% as moderate-risk and 15.9% as high-risk for developing incident CKD. After adjusting for treatment, baseline PromarkerD moderate-risk and high-risk scores were increasingly prognostic for incident CKD (odds ratio 5.29 and 13.52 versus low-risk, respectively; both p < 0.001). Analysis of the PromarkerD test system in CANVAS shows the test can predict clinically significant incident CKD in this multi-center clinical study but had limited utility for predicting eGFR decline ≥30%.