Advances in preeclampsia testing.

Preeclampsia is a multisystem hypertensive disorder and one of the leading causes of maternal and fetal morbidity and mortality. The clinical hallmarks such as hypertension and proteinuria, and additional laboratory tests currently available including liver enzyme testing, are neither specific nor sufficiently sensitive. Therefore, biomarkers for timely and accurate identification of patients at risk of developing preeclampsia are extremely valuable to improve patient outcomes and safety. In this chapter, we will first discuss the clinical characteristics of preeclampsia and current evidence of the role of angiogenic factors, such as placental growth factor (PlGF) and soluble FMS like tyrosine kinase 1 (sFlt-1) in the pathogenesis of preeclampsia. Second, we will review the clinical practice guidelines for preeclampsia diagnostic criteria and their recommendations on laboratory testing. Third, we will review the currently available PlGF and sFlt-1 assays in terms of their methodologies, analytical performance, and clinical diagnostic values. Finally, we will discuss the future research needs from both an analytical and clinical perspective.

Analytical evaluation and Sigma metrics of 6 next generation chemistry assays on the Abbott Architect system.

BACKGROUND: We evaluated analytical and Sigma performance for 6 next generation chemistry assays on the Abbott Architect c8000 system. METHODS: Albumin with bromocresol purple or green, amylase, cholesterol, total protein, and urea nitrogen were analyzed using photometric technology. Analytical performance goals were defined based on Accreditation Canada Diagnostics (ACD) and Clinical Laboratory Improvement Amendments (CLIA). Precision study consisted of testing 2 quality control concentrations and 3 patient serum sample pools, twice a day in quintuplicate over 5 days. Linearity testing consisted of 5-6 concentrations of commercial linearity materials. We tested a minimum of 120 serum/plasma specimens on the new and current Architect methods for comparison. We assessed accuracy with reference materials for 5 assays, and a calibration standard for cholesterol. Bias from the reference standard target value was used for Sigma metric analysis. RESULTS: Observed total imprecision of the assays ranged from 0.5 to 4%, meeting pre-defined goals. Linearity was acceptable over the tested range. Measurements on the new and current Architect methods were comparable. Accuracy ranged from 0 to 2.0% absolute mean difference from target value. All 6 next generation clinical chemistry assays demonstrated Six Sigma quality, using CLIA standards. CONCLUSION: Applying ACD recommendations, 5 assays showed Six Sigma, while cholesterol showed Five Sigma performance.

Multiplex proteomics using proximity extension assay for the identification of protein biomarkers predictive of acute graft-vs.-host disease in allogeneic hematopoietic cell transplantation.

OBJECTIVES: Allogeneic hematopoietic cell transplantation (HCT) is associated with acute graft-vs.-host disease (aGVHD). The presented study applied a novel multiplex antibody-based proximity extension assay (PEA) proteomic platform that can detect thousands of serum proteins simultaneously for the identification of potential biomarkers of aGVHD. METHODS: Serum samples from 28 patients who underwent allogeneic HCT for acute myeloid leukemia (AML) were analyzed; 17 were diagnosed with grade II-IV aGVHD while 11 patients were not. Samples collected on day -6, day 0, +14, +30, +60 and +90 post-HCT were analyzed for the relative concentrations of 552 proteins. The concentration of each protein from baseline to the closest time point before onset of aGVHD, or to the latest time point in control patients, was documented. RESULTS: Individualized analysis identified 26 proteins demonstrating ≥3-fold increase at aGVHD onset compared to baseline, eliminating proteins with a similar increase in controls. Another approach used paired t-testing and logistic regression that identified a four-marker panel, including SLAMF7, IL-1ra, BTN3A2 and DAB2, where individual log-likelihood ratios ranged from 3.99 to 8.15 (logistic regression, p=0.004-0.046). When combined, the four-marker panel demonstrated an area under the curve (AUC) of 0.90 (95% CI: 0.78-1.00; p=0.0006) with high negative predictive value of 81.8% and positive predictive value of 86.7%. All four markers play a physiological role in immune regulation. Among these, three were also present in the individualized analysis (SLAMF7, IL-1ra and BTN3A2). CONCLUSIONS: We conclude that serum proteins identified using multiplex proteomics, particularly SLAMF7, IL-1ra, BTN3A2 and DAB2, may potentially predict aGVHD.

Mucin 13 (MUC13) as a candidate biomarker for ovarian cancer detection: potential to complement CA125 in detecting non-serous subtypes.

OBJECTIVES: Ovarian cancer is the most lethal gynecological malignancy in developed countries. One of the key associations with the high mortality rate is diagnosis at late stages. This clinical limitation is primarily due to a lack of distinct symptoms and detection at the early stages. The ovarian cancer biomarker, CA125, is mainly effective for identifying serous ovarian carcinomas, leaving a gap in non-serous ovarian cancer detection. Mucin 13 (MUC13) is a transmembrane, glycosylated protein with aberrant expression in malignancies, including ovarian cancer. We explored the potential of MUC13 to complement CA125 as an ovarian cancer biomarker, by evaluating its ability to discriminate serous and non-serous subtypes of ovarian cancer at FIGO stages I-IV from benign conditions. METHODS: We used our newly developed, high sensitivity ELISA to measure MUC13 protein in a large, well-defined cohort of 389 serum samples from patients with ovarian cancer and benign conditions. RESULTS: MUC13 and CA125 serum levels were elevated in malignant compared to benign cases (p<0.0001). Receiver-operating characteristic (ROC) curve analysis showed similar area under the curve (AUC) of 0.74 (MUC13) and 0.76 (CA125). MUC13 concentrations were significantly higher in mucinous adenocarcinomas compared to benign controls (p=0.0005), with AUC of 0.80. MUC13 and CA125 showed significant elevation in early-stage cases (stage I-II) in relation to benign controls (p=0.0012 and p=0.014, respectively). CONCLUSIONS: We report the novel role of MUC13 as a serum ovarian cancer biomarker, where it could complement CA125 for detecting some subtypes of non-serous ovarian carcinoma and early-stage disease.

Sensitive Serology Measurements in the Saliva of Individuals with COVID-19 Symptoms Using a Multiplexed Immunoassay.

BACKGROUND: There are numerous benefits to performing salivary serology measurements for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen for coronavirus disease 2019 (COVID-19). Here, we used a sensitive multiplex serology assay to quantitate salivary IgG against 4 SARS-CoV-2 antigens: nucleocapsid, receptor-binding domain, spike, and N-terminal domain. METHODS: We used single samples from 90 individuals with COVID-19 diagnosis collected at 0 to 42 days postsymptom onset (PSO) and from 15 uninfected control subjects. The infected individuals were segmented in 4 groups (0-7 days, 8-14 days, 15-21 days, and >21 days) based on days PSO, and values were compared to controls. RESULTS: Compared to controls, infected individuals showed higher levels of antibodies against all antigens starting from 8 days PSO. When applying cut-offs with at least 93.3% specificity at every time interval segment, nucleocapsid protein serology had the best sensitivity at 0 to 7 days PSO (60% sensitivity [35.75% to 80.18%], ROC area under the curve [AUC] = 0.73, P = 0.034). Receptor-binding domain serology had the best sensitivity at 8 to 14 days PSO (83.33% sensitivity [66.44%-92.66%], ROC AUC = 0.90, P < 0.0001), and all assays except for N-terminal domain had 92% sensitivity (75.03%-98.58%) at >14 days PSO. CONCLUSIONS: This study shows that our multiplexed immunoassay can distinguish infected from uninfected individuals and reliably (93.3% specificity) detect seroconversion (in 60% of infected individuals) as early as the first week PSO, using easy-to-collect saliva samples.

Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection.

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.

Comparison of two multiplexed technologies for profiling >1,000 serum proteins that may associate with tumor burden.

Background: In this pilot study, we perform a preliminary comparison of two targeted multiplex proteomics technologies for discerning serum protein concentration changes that may correlate to tumor burden in ovarian cancer (OC) patients. Methods: Using the proximity extension assay (PEA) and Quantibody® Kiloplex Array (QKA), we measured >1,000 proteins in the pre-surgical and post-surgical serum from nine OC patients (N=18 samples). We expect that proteins that have decreased significantly in the post-surgical serum concentration may correlate to tumor burden in each patient. Duplicate sera from two healthy individuals were used as controls (N=4 samples). We employed in-house ELISAs to measure five proteins with large serum concentration changes in pre- and post-surgical sera, from four of the original nine patients and the two original controls. Results: Both platforms showed a weak correlation with clinical cancer antigen 125 (CA125) data. The two multiplexed platforms showed a significant correlation with each other for >400 overlapping proteins. PEA uncovered 15 proteins, while QKA revealed 11 proteins, with more than a two-fold post-surgical decrease in at least six of the nine patients. Validation using single enzyme-linked immunosorbent assays (ELISAs) showed at least a two-fold post-surgical decrease in serum concentration of the same patients, as indicated by the two multiplex assays. Conclusion: Both methods identified proteins that had significantly decreased in post-surgical serum concentration, as well as recognizing proteins that had been implicated in OC patients. Our findings from a limited sample size suggest that novel targeted proteomics platforms are promising tools for identifying candidate serological tumor-related proteins.  However further studies are essential for the improvement of accuracy and avoidance of false results.

Uncovering the Depths of the Human Proteome: Antibody-based Technologies for Ultrasensitive Multiplexed Protein Detection and Quantification.

Probing the human proteome in tissues and biofluids such as plasma is attractive for biomarker and drug target discovery. Recent breakthroughs in multiplex, antibody-based, proteomics technologies now enable the simultaneous quantification of thousands of proteins at as low as sub fg/ml concentrations with remarkable dynamic ranges of up to 10-log. We herein provide a comprehensive guide to the methodologies, performance, technical comparisons, advantages, and disadvantages of established and emerging technologies for the multiplexed ultrasensitive measurement of proteins. Gaining holistic knowledge on these innovations is crucial for choosing the right multiplexed proteomics tool for applications at hand to critically complement traditional proteomics methods. This can bring researchers closer than ever before to elucidating the intricate inner workings and cross talk that spans multitude of proteins in disease mechanisms.

Novel severe traumatic brain injury blood outcome biomarkers identified with proximity extension assay.

OBJECTIVES: Severe traumatic brain injury (sTBI) patients suffer high mortality. Accurate prognostic biomarkers have not been identified. In this exploratory study, we performed targeted proteomics on plasma obtained from sTBI patients to identify potential outcome biomarkers. METHODS: Blood sample was collected from patients admitted to the ICU suffering a sTBI, using standardized clinical and computerized tomography (CT) imaging criteria. Age- and sex-matched healthy control subjects and sTBI patients were enrolled. Targeted proteomics was performed on plasma with proximity extension assays (1,161 proteins). RESULTS: Cohorts were well-balanced for age and sex. The majority of sTBI patients were injured in motor vehicle collisions and the most frequent head CT finding was subarachnoid hemorrhage. Mortality rate for sTBI patients was 40%. Feature selection identified the top performing 15 proteins for identifying sTBI patients from healthy control subjects with a classification accuracy of 100%. The sTBI proteome was dominated by markers of vascular pathology, immunity/inflammation, cell survival and macrophage/microglia activation. Receiver operating characteristic (ROC) curve analyses demonstrated areas-under-the-curves (AUC) for identifying sTBI that ranged from 0.870-1.000 (p≤0.005). When mortality was used as outcome, ROC curve analyses identified the top 3 proteins as Willebrand factor (vWF), Wnt inhibitory factor-1 (WIF-1), and colony stimulating factor-1 (CSF-1). Combining vWF with either WIF-1 or CSF-1 resulted in excellent mortality prediction with AUC of 1.000 for both combinations (p=0.011). CONCLUSIONS: Targeted proteomics with feature classification and selection distinguished sTBI patients from matched healthy control subjects. Two protein combinations were identified that accurately predicted sTBI patient mortality. Our exploratory findings require confirmation in larger sTBI patient populations.

Plasma Protein Profiling by Proximity Extension Assay Technology Reveals Novel Biomarkers of Traumatic Brain Injury-A Pilot Study.

BACKGROUND: Traumatic brain injury (TBI) is a significant public health issue affecting nearly 69 million patients worldwide per year. Reliable diagnostic biomarkers are urgently needed to aid in disease diagnosis and prognosis and to guide patient aftercare. Blood biomarkers represent an attractive modality to quickly, cheaply, and objectively evaluate clinical status. We hypothesize that deep and quantitative plasma proteomic profiling with a novel technology, proximity extension assay, may lead to the discovery of diagnostic and/or prognostic biomarkers of TBI. METHODS: We used high-throughput proximity extension assays (PEA) to quantify the relative abundance of over 1000 unique proteins in plasma. PEA is a highly sensitive multiplex immunoassay capable of detecting very low-abundance proteins (down to fg/mL) in complex biological matrices. Our patient cohort consisted of severe TBI (sTBI) patients, matched healthy controls, and another non-TBI group that was included in the analysis to validate the specificity of the candidates during the selection process. The obtained protein quantification data was then filtered to identify candidate biomarkers through statistical analysis, literature searches, and comparison to our reference control groups. RESULTS: Overall, we identified 6 novel candidate TBI biomarkers. Candidates exhibit a significant increase in plasma protein abundance in sTBI when comparing between healthy controls and sTBI patients. Candidates generally had low expression in our reference groups compared with the sTBI group. CONCLUSIONS: Our preliminary findings represent a starting point for future validation. These biomarkers, either alone or in combination, may have significant clinical utility in aiding in TBI diagnosis, prognosis, and/or management.

Investigating a novel multiplex proteomics technology for detection of changes in serum protein concentrations that may correlate to tumor burden.

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.

Utility of a Fifth-Generation Ultrasensitive Prostate-Specific Antigen Assay for Monitoring Prostate Cancer Patients after Radical Prostatectomy with 3 Years of Follow-Up.

BACKGROUND: We investigated an ultrasensitive prostate-specific antigen (uPSA) immunoassay (MesoScale; lower limit of detection (LLD) of 0.0035 pg/mL) to monitor patients with prostate cancer (PCa) following radical prostatectomy (RP) and to examine whether changes in PSA in the conventionally undetectable range (<1 pg/mL) can predict biochemical relapse (BCR). METHODS: We measured uPSA in serial serum samples (N = 100) collected from 20 RP cases with a third-generation ELISA (LLD of 1 pg/mL) and the fifth-generation MesoScale assay. We analyzed the PSA nadir changes to classify patients into BCR or non-BCR groups, observed the trends in PSA kinetics, and associated BCR status with clinicohistopathological features. RESULTS: The ELISA could quantify PSA in only 38% of the RP samples, detecting BCR in 7 of 20 patients with PCa. The MesoScale assay quantified PSA in all samples, showing 8 of 20 patients with BCR. However, there was no significant difference between the median time to BCR detection based on ELISA (1016 days) compared with MesoScale data (949 days). Gleason scores were higher in the BCR groups compared with non-BCR. There was no significant difference for other clinicohistopathological parameters. CONCLUSIONS: The uPSA MesoScale technology could track miniscule changes in serum PSA in the range of 0.003-1 pg/mL in all RP cases. However, PSA kinetics and nadir at concentrations <2 pg/mL fluctuated, and increases below this range could not reliably suggest signs of BCR. Instead, ultrasensitive fifth-generation PSA assays may hold clinical potential for measuring the low concentrations of PSA in women for various medical contexts.

Pitfalls in Cancer Biomarker Discovery and Validation with Emphasis on Circulating Tumor DNA.

Despite significant investment of funds and resources, few new cancer biomarkers have been introduced to the clinic in the last few decades. Although 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.See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible."

Kallikrein-related peptidases protein expression in lymphoid tissues suggests potential implications in immune response.

OBJECTIVES: Kallikrein-related peptidases (KLKs) are a subgroup of 15 secreted chymotrypsin- and trypsin-like serine proteases that have been reported to possess novel functions in innate immunity and inflammation. Since the potential role of KLKs in immunity has not been studied in detail at the protein level, we examined the expression pattern of 12 members of the KLK family in immune-related tissues. DESIGN & METHODS: Protein expression in tissue extracts was evaluated using immunoassays (ELISA). Immunohistochemistry (IHC) was performed on representative sections of tonsil and lymph nodes to determine the cellular localization of the KLK family members. RESULTS: ELISA profiling of KLK3-KLK15 (except KLK12) revealed higher protein levels in the tonsil, compared to the lymph nodes and spleen. Relatively high protein levels in the tonsil were observed for KLK7, KLK9, KLK10 and KLK13. Expression of these KLKs was significantly lower in lymph nodes and spleen. IHC analysis in tonsil unveiled that KLK9 and KLK10 were differentially expressed in lymphoid cells. KLK9 was strongly expressed in the germinal center of lymphoid follicles where activated B-cells reside, whereas KLK10 was expressed in the follicular dendritic cells (FDCs) that are vital for maintaining the cycle of B cell maturation. CONCLUSION: Overall, our study revealed the possible implications of KLK expression and regulation in the immune cells of lymphoid tissues.

Emerging role of clinical mass spectrometry in pathology.

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.

Serum PD-1 Is Elevated after Pembrolizumab Treatment but Has No Predictive Value.

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 pre-immunotherapy 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.

Expression profile of human tissue kallikrein 15 provides preliminary insights into its roles in the prostate and testis.

BACKGROUND: Human tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer. METHODS: In the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC). RESULTS: Highest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3. CONCLUSIONS: Collectively, our data provide insights into the localization and possible role of KLK15 in human physiology.

Biochemical characterization of human tissue kallikrein 15 and examination of its potential role in cancer.

OBJECTIVE: Human tissue kallikrein 15 (KLK15) is the last cloned member of the KLK-related gene family. Despite being implicated in multiple cancers, its pathophysiological role remains unknown. We aimed to biochemically characterize KLK15 and preliminarily study its role in cancer. DESIGN & METHODS: Recombinant KLK15 protein was produced, purified to homogeneity and quantified by mass spectrometry (parallel reaction monitoring analysis). We profiled the enzymatic activity of KLK15 using fluorogenic peptide substrates, and performed kinetic analysis to discover the cleavage sites. As KLK15 has mainly been associated with prostate cancer, we used a degradomic approach and subsequent KEGG pathway analysis to identify a number of putative protein substrates in the KLK15-treated prostate cancer cell line PC3. RESULTS: We discovered trypsin-like activity in KLK15, finding that it cleaves preferentially after arginine (R). The enzymatic activity of KLK15 was regulated by different factors such as pH, cations and serine protease inhibitors. Notably, we revealed that KLK15 most likely interacts with the extracellular matrix (ECM) receptor group. CONCLUSION: To our knowledge, this is the first study that experimentally verifies the trypsin-like activity of KLK15. We show here for the first time that KLK15 may be able to cleave many ECM components, similar to several members of the KLK family. Thus the protease could potentially be linked to tumorigenesis by promoting metastasis via this mechanism.

Exploring the potential of mucin 13 (MUC13) as a biomarker for carcinomas and other diseases.

BACKGROUND: Mucin 13 (MUC13) is a cell surface glycoprotein aberrantly expressed in a variety of epithelial carcinomas. Thus far, the role of MUC13 in various diseases remains elusive. To the best of our knowledge, this is the first study to examine the potential of MUC13 as a serum biomarker in a variety of carcinomas and other conditions. METHODS: We developed a recombinant MUC13 protein, mouse monoclonal antibodies and enzyme immunoassay (ELISA) for MUC13. We used this assay to measure MUC13 levels in the supernatants of cancer cell lines and a large cohort of serum samples from healthy and diseased individuals. RESULTS: MUC13 is secreted from cancer cell lines, with highest levels found in ovarian cancer cell lines. MUC13 levels in human sera were significantly increased in patients with renal failure and 20%-30% of patients with ovarian, liver, lung and other cancers. MUC13 was also elevated in 70% of patients with active cutaneous melanoma, but not uveal melanoma. Furthermore, we identified significant MUC13 elevations in the serum of patients with vasculitis (ANCA-positive) autoantibodies, but not in those with inflammatory bowel disease. CONCLUSIONS: 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.