Liquid profiling of circulating tumor DNA in colorectal cancer: steps needed to achieve its full clinical value as standard care

The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid profiling (LP), its acceptance by clinicians, and its integration into clinical workflows in real‐world settings remain limited. Here, LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at < 0.1% in over 20% of cases, a high frequency of concomitant driver mutations (in up to 14% of cases), and ctDNA levels reflecting the clinical course of disease were revealed. However, certain limitations hampering successful translation of ctDNA into clinical practice were uncovered, including the lack of clinically relevant ctDNA thresholds, appropriate time points of LP requests, and integrative evaluation of ctDNA, imaging, and clinical findings. In conclusion, these results highlight the potential clinical value of LP for CRC patient management and demonstrate issues that need to be addressed for successful long‐term implementation in clinical workflows.

SARS-CoV-2 Antibody Testing - How to Increase the Positive Predictive Value.

BACKGROUND: Despite increasing COVID-19 infection rates, low overall prevalence resulting in a poor positive predictive value (PPV) of serological tests requires strategies to increase specificity. We therefore investigated a dual diagnostic strategy and evaluated the correlation between the severity of a SARS-CoV-2 infection and the detectable immune-response. METHODS: Participants were systematically categorized into positive and control cohorts and a probability score of COVID-19 was calculated based on clinical symptoms. Six hundred eighty-two serum samples were analyzed using a highly specific high-throughput system. Combining the serological test result and probability score was performed as a dual diagnostic strategy. RESULTS: Specificity of 99.61% and sensitivity of 86.0% were the basis of our approach. A dual diagnostic strategy led to increased pre-test probability and thus to a test specificity of 100%. In a flu-like symptomatic population, we estimated a COVID-prevalence of 4.79%. Moreover, we detected significantly higher antibody values in patients with fever than without fever. CONCLUSIONS: Based on sensitivity and specificity results of our study being in line with previous findings, we demonstrated a dual assessment strategy including a symptom-based probability score and serological testing to increase the PPV. Moreover, the presence of fever seems to trigger a stronger immune-response.

Assessing the Quality of Serological Testing in the COVID-19 Pandemic: Results of a European External Quality Assessment (EQA) Scheme for Anti-SARS-CoV-2 Antibody Detection.

External quality assessment (EQA) is a key instrument for achieving harmonization, and thus a high quality, of diagnostic procedures. As reliable test results are crucial for accurate assessment of SARS-CoV-2 infection prevalence, vaccine response, and immunity, and thus for successful management of the ongoing COVID-19 pandemic, the Reference Institute for Bioanalytics (RfB) was the first EQA provider to offer an open scheme for anti-SARS-CoV-2 antibody detection. The main objectives of this EQA were (i) to gain insights into the current diagnostic landscape and the performance of serological tests in Europe and (ii) to provide recommendations for diagnostic improvements. Within the EQA, a blinded panel of precharacterized human serum samples with variable anti-SARS-CoV-2 antibody titers was provided for detection of anti-SARS-CoV-2 IgG, IgA, and IgM antibodies. Across the three distribution rounds in 2020, 284 laboratories from 22 countries reported a total of 3,744 results for anti-SARS-CoV-2 antibody detection using more than 24 different assays for IgG. Overall, 97/3,004 results were false for anti-SARS-CoV-2 IgG, 88/248 for IgA, and 34/124 for IgM. Regarding diagnostic sensitivity and specificity, substantial differences were found between the different assays used, as well as between certified and noncertified tests. For cutoff samples, a drop in the diagnostic sensitivity to 46.3% and high interlaboratory variability were observed. In general, this EQA highlights the current variability of anti-SARS-CoV-2 antibody detection, technical limitations with respect to cutoff samples, and the lack of harmonization of testing procedures. Recommendations are provided to help laboratories and manufacturers further improve the quality of anti-SARS-CoV-2 serological diagnostics.

Results of the first pilot external quality assessment (EQA) scheme for anti-SARS-CoV2-antibody testing.

Objectives Assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevalence and immunity is cornerstones in the fight against COVID-19 pandemic. For pandemic control, reliable assays for the detection of anti-SARS-CoV-2 antibodies are required. This pilot external quality assessment (EQA) scheme aimed to independently assess the participants' clinical performance of anti-SARS-CoV-2 testing, to identify shortcomings in clinical practice and to evaluate the suitability of the scheme format. Methods The EQA scheme consisted of eight serum samples with variable reactivity against SARS-CoV-2 intended for the analysis of anti-SARS-CoV-2 immunoglobulin (Ig)G, IgA, and IgM antibodies. Laboratories reported: (1) results for each sample and the respective method, (2) raw data from replicate testing of each sample. Results The 16 selected pilot EQA participants reported 294 interpreted results and 796 raw data results from replicate testing. The overall error rate for the anti-SARS-CoV-2 IgG, IgA, and IgM tests was 2.7, 6.9, and 16.7%, respectively. While the overall diagnostic specificity was rated as very high, sensitivity rates between 67 and 98% indicate considerable quality differences between the manufacturers, especially for IgA and IgM. Conclusions Even the results reported by the small number of participants indicate a very heterogeneous landscape of anti-SARS-CoV-2 serological testing. Differences of available tests and the individual performance of laboratories result in a success rate of 57.1% with one laboratory succeeding for all three antibody-classes. These results are an incentive for laboratories to participate in upcoming open EQA schemes that are needed to achieve a harmonization of test results and to improve serological testing.

Comparison of test performance of commercial anti-SARS-CoV-2 immunoassays in serum and plasma samples.

BACKGROUND: For epidemiologic, social and economic reasons, assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevalence and immunity are important to adapt decisions to current demands. Hence, immunoassays for detection of anti-SARS-CoV-2 antibodies are introduced rapidly without requiring FDA emergency use authorization approval. Thus, evaluation of test performance predominantly relies on laboratories. This study aimed to evaluate the test performance of recently launched commercial immunoassays in serum and plasma samples. METHODS: 51 serum samples from 26 patients with confirmed SARS-CoV-2 infection after end of quarantine and 25 control patients were analyzed using anti-SARS-CoV-2 IgG immunoassays from Roche, Euroimmun and Epitope to assess diagnostic sensitivity and specificity. 20 matching pairs of serum and plasma samples were included to analyze comparability between different specimens. RESULTS: Overall, a diagnostic sensitivity of 92.3%, 96.2-100% and 100% with a respective diagnostic specificity of 100%, 100% and 84-86% for the immunoassays from Roche, Euroimmun and Epitope were determined. In total, 84-96% of samples were correctly classified as negative and 92.3-95.2% as positive. The level of concordance between plasma- and serum-based testing diverged between the assays (Epitope r2 = 0.97; Euroimmun r2 = 0.91; Roche r2 = 0.76). CONCLUSIONS: The immunoassays from Euroimmun and Roche revealed a higher specificity than the Epitope assay without a substantial drop of diagnostic sensitivity. Significant differences between plasma- and serum-based testing highlights the need for determination of appropriate cut-offs per specimen type. Hence, there is an urgent need for test harmonization and establishment of quality standards for an appropriate use of COVID-19 serological tests.