Analytical validation of a novel multiplex test for detection of advanced adenoma and colorectal cancer in symptomatic patients.

Early detection of colorectal cancer (CRC) is key to reducing associated mortality. Despite the importance of early detection, approximately 40% of individuals in the United States between the ages of 50-75 have never been screened for CRC. The low compliance with colonoscopy and fecal-based screening may be addressed with a non-invasive alternative such as a blood-based test. We describe here the analytical validation of a multiplexed blood-based assay that measures the plasma concentrations of 15 proteins to assess advanced adenoma (AA) and CRC risk in symptomatic patients. The test was developed on an electrochemiluminescent immunoassay platform employing four multi-marker panels, to be implemented in the clinic as a laboratory developed test (LDT). Under the Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) regulations, a United States-based clinical laboratory utilizing an LDT must establish performance characteristics relating to analytical validity prior to releasing patient test results. This report describes a series of studies demonstrating the precision, accuracy, analytical sensitivity, and analytical specificity for each of the 15 assays, as required by CLIA/CAP. In addition, the report describes studies characterizing each of the assays' dynamic range, parallelism, tolerance to common interfering substances, spike recovery, and stability to sample freeze-thaw cycles. Upon completion of the analytical characterization, a clinical accuracy study was performed to evaluate concordance of AA and CRC classifier model calls using the analytical method intended for use in the clinic. Of 434 symptomatic patient samples tested, the percent agreement with original CRC and AA calls was 87% and 92% respectively. All studies followed CLSI guidelines and met the regulatory requirements for implementation of a new LDT. The results provide the analytical evidence to support the implementation of the novel multi-marker test as a clinical test for evaluating CRC and AA risk in symptomatic individuals.

Identification of carbamylated alpha 1 anti-trypsin (A1AT) as an antigenic target of anti-CarP antibodies in patients with rheumatoid arthritis.

In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA.

Anti-CarP antibodies as promising marker to measure joint damage and disease activity in patients with rheumatoid arthritis.

Anti-citrullinated protein antibodies (ACPA) are important serological markers in the diagnosis of rheumatoid arthritis (RA) and are part of the recent disease classification criteria. However, there is a strong need for reliable markers for measuring and predicting joint damage and disease activity. Recently, antibodies directed against carbamylated antigens (anti-CarP antibodies) were identified. A total of 120 RA patients were tested for anti-CCP antibodies using different methods and for anti-CarP antibodies using carbamylated fetal calf serum according to the method described by Shi et al. Additionally, ACPA fine specificities (to three citrullinated peptides) were measured. Disease activity was assessed at baseline using the disease activity score 28 (DAS28) in 80 patients. For 40 RA patients, joint erosion score (JES) was established. The median JES was 14.1 with a standard deviation of 11.5. Anti-CarP antibodies were correlated with joint erosion score (ρ = 0.34, 95% CI 0.03-0.59; p = 0.0332). No correlation between ACPA and joint erosion score was observed. No individual marker correlated with DAS28. When one ACPA peptide was combined with anti-CarP antibodies in a score (ACPA peptide 1 divided by anti-CarP), a statistically relevant correlation was found (p = 0.0264). In this small cohort, the presence of anti-CarP antibodies, but not ACPA correlate with joint erosion score. Anti-CarP antibodies combined with ACPA fine specificities correlated with DAS28. Therefore, anti-CarP antibodies might represent a promising marker to predict joint damage and disease activity in RA patients.