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Mass spectrometry-based assays have been increasingly implemented in various disciplines in clinical diagnostic laboratories for their combined advantages in multiplexing capacity and high analytical specificity and sensitivity. It is now routinely used in areas including reference methods development, therapeutic drug monitoring, toxicology, endocrinology, paediatrics, immunology and microbiology to identify and quantify biomolecules in a variety of biological specimens. As new ionisation methods, instrumentation and techniques are continuously being improved and developed, novel mass spectrometry-based clinical applications will emerge for areas such as proteomics, metabolomics, haematology and anatomical pathology. This review will summarise the general principles of mass spectrometry and specifically highlight current and future clinical applications in anatomical pathology.

Immune-checkpoint blockade (ICB) uses antibody targeting of specific inhibitory receptors and ligands. The major limitations of ICB, such as high cost, limited success rate, and immune-related adverse events (irAE), highlight the need for predictive biomarkers. We analyzed preimmunotherapy and post-immunotherapy serum samples of 24 patients treated with pembrolizumab for changes in PD-1 and over 1,000 additional protein markers using a multiplex proximity extension assay (PEA) to identify potential predictive biomarkers of response and/or toxicity. Candidates were selected based on the criteria that at least 2 patients within any of 3 patient groups (responders without irAEs, responders with irAEs, or nonresponders with irAEs) had either a !4-fold increase or 4-fold decrease in expression post-immunotherapy. Female and male control samples were used as technical duplicates. A patient group with no response and no irAEs was used to exclude candidates. Following treatment with pembrolizumab, there was a relative increase of PD-1 in the serum of all patients, compared with controls (average 4.4-fold). We identified 7 additional serum proteins that met our candidate selection criteria. These candidate markers did not have any significant association with response or toxicity to pembrolizumab. Overall, we show that serum PD-1 increases post-therapy with pembrolizumab treatment but has no predictive value for response or toxicity in this small set of patients.

Despite significant investment of funds and resources, few new cancer biomarkers have been introduced to the clinic in the last few decades. Even though many candidates produce promising results in the laboratory, deficiencies in sensitivity, specificity and predictive value make them less than desirable in a patient setting. This review will analyze these challenges in detail as well as discuss false discovery, problems with reproducibility and tumor heterogeneity. Circulating tumor DNA (ctDNA), an emerging cancer biomarker is also analyzed, particularly in the contexts of assay specificity, sensitivity, fragmentation, lead time, mutant allele fraction and clinical relevance. Emerging artificial intelligence technologies will likely be valuable tools in maximizing the clinical utility of ctDNA which is often found in very small quantities in patients with early stage tumors. Finally, the implications of challenging false discoveries are examined and some insights about improving cancer biomarker discovery are provided.

Serum MUC13 is frequently elevated not only in a variety of malignant cases but also in some benign pathologies, thus appearing to be a non-specific disease biomarker. Nonetheless, serum MUC13 is clearly highly elevated in some carcinoma patients, and its relationship with tumor progression in this context warrant further research. Future studies that examine the correlation between serum MUC13 levels to stage of cancer could elucidate prognostic potential.

Objectives Widespread SARS-CoV-2 testing is invaluable for identifying asymptomatic/pre-symptomatic individuals. There remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests suitable for frequent mass testing. Compared to nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling. Current saliva-based SARS-CoV-2 rapid antigen tests (RATs) are hindered by limited analytical sensitivity. Here, we report one of the first ultrasensitive, saliva-based SARS-CoV-2 antigen assays with an analytical sensitivity of <0.32 pg/mL, corresponding to four viral RNA copies/µL, which is comparable to that of PCR-based tests. Methods Using the novel electrochemiluminescence (ECL)-based immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 salivas, obtained from non-COVID-19 and COVID-19 patients. We then verified the results with a second, independent cohort of 689 patients (3.8% SARS-CoV-2 positivity rate). We also compared our method with a widely used point-of-care rapid test. Results In the first cohort, at 100% specificity, the sensitivity was 92%. Our assay correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were obtained for 86 cases. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (<0.32 pg/mL) and strongly positive (>2 pg/mL) N antigen concentrations. In the second cohort, at 100% specificity, sensitivity was also 92%. Our assay is about 700-fold more sensitive than the Abbott Panbio Rapid Test. Conclusions We demonstrated the ultrasensitivity and specificity assay and its concordance with PCR. This novel assay is especially valuable when compliance to frequent swabbing may be problematic.

Widespread SARS-CoV-2 testing is highly valuable for identifying asymptomatic/pre-symptomatic individuals to slow community disease transmission. However, there remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests that are suitable for frequent mass testing. Compared to the conventional nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling protocols. Despite its merits in rapid turn-around-time and high throughput compared to traditional PCR-based technologies, the widespread use of saliva-based SARS-CoV-2 rapid antigen tests is hindered by limited analytical sensitivity of current methods. Here, we report the first ultrasensitive, saliva-based SARS-CoV-2 antigen assay with an analytical sensitivity of < 0.32 pg/ml, corresponding to 4 viral RNA copies/ul, which is comparable to that of PCR-based tests. Using the novel electrochemiluminescence (ECL)-based S-PLEX immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 saliva samples obtained from non-COVID-19 and COVID-19 patients. Our assay displayed absolute specificity and high sensitivity (90.2%), where it correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were also obtained for 86 cases for comparison. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (< 0.32 pg/ml) and strongly positive (> 2 pg/ml) N antigen concentrations. Our study unveiled the ultrasensitivity and specificity of the saliva-based S-PLEX assay, demonstrating its clinical value as a high throughput, complementary alternative to PCR-based techniques. The novel technique is especially valuable in cases where compliance to frequent swabbing may be problematic (e.g. schools, nursing homes, etc.).

Background: To account for cancer heterogeneity, we previously introduced the concept of “personalized” tumor markers, which are biomarkers that are informative in subsets of patients or even a single patient. Recent developments in various multiplex protein technologies create excitement for the discovery of markers of tumor burden in individual patients, but the reliability of the technologies remains to be tested for this purpose. Here, we sought to explore the potential of a novel proteomics platform, which utilizes a multiplexed antibody microarray, to detect changes in serum protein concentration that may correlate to tumor burden in pancreatic cancer. Methods: We applied the Quantibody® Human Kiloplex Array to simultaneously measure 1,000 proteins in sera obtained pre- and post-surgically from five pancreatic cancer patients. We expected that proteins which decreased post-surgery may correlate to tumor burden. Sera from two healthy individuals, split into two aliquots each, were used as controls. To validate the multiplexed results, we used single-target ELISA assays to measure the proteins with the largest serum concentration changes after surgery in sera collected pre- and post-surgically from the previous five patients and 10 additional patients. Results: The multiplexed array revealed nine proteins with more than two-fold post-surgical decrease in at least two of five patients. However, validation using single ELISAs showed that only two proteins tested displayed more than two-fold post-surgical decrease in one of the five original patients. In the independent cohort, six of the proteins tested showed at least a two-fold decrease post-surgery in at least one patient. Conclusions: Our study found that the Quantibody® Human Kiloplex Array results could not be reliably replicated with individual ELISA assays and most hits would likely represent false positives if applied to biomarker discovery. These findings suggest that data from novel, high-throughput proteomic platforms need stringent validation to avoid false discoveries.