Prognostic urinary miRNAs for the assessment of small renal masses.

BACKGROUND: Renal cell carcinoma (RCC) is often detected incidentally as a small renal mass (SRM; pT1a, ≤4 cm). It is clinically challenging to predict progression in patients with SRMs. This is largely due to the recent recognition of clinically progressive and non-progressive RCC-SRMs. It is critical to accurately stratify SRM patients according to risk to avoid unnecessary treatment. This is especially significant for elderly and infirm patients, where the risk of surgery outweighs mortality from SRMs. METHODS: We employed a qRT-PCR array-based approach and targeted qRT-PCR to identify and validate early, non-invasive diagnostic and prognostic biomarkers of RCC-SRMs. In total, we evaluated eighty urine samples, including 30 renal oncocytoma (≤4 cm) cases, 26 progressive and 24 non-progressive clear cell RCC-SRM (ccRCC-SRM) cases. RESULTS: We identified nine urinary miRNAs which displayed significantly elevated expression in ccRCC-SRMs (pT1a; ≤4 cm) relative to renal oncocytoma (≤4 cm). Additionally, miR-328-3p displayed significantly down-regulated expression in progressive relative to non-progressive ccRCC-SRMs. Patients with elevated miR-328-3p expression had significantly longer overall survival (HR = 0.29, 95% CI = 0.08-1.03, p = 0.042) compared to patients with low miR-328-3p expression. We also found no significant association between miR-328-3p expression levels and gender, age, laterality, tumor size, or grade, suggesting that miR-328-3p is an independent prognostic biomarker. CONCLUSIONS: Our in-depth miRNA profiling approach identified novel biomarkers for early-stage ccRCC-SRMs. Pretreatment characterization of urinary miRNAs may provide insight into early RCC progression and could potentially aid clinical decision-making, improving patient management and reducing overtreatment.

Quantitative assessment and clinical relevance of kallikrein-related peptidase 5 mRNA expression in advanced high-grade serous ovarian cancer.

BACKGROUND: In ovarian cancer, dysregulation of mRNA expression of several components of the family of the kallikrein-related peptidases (KLKs) is observed. In this study, we have analyzed the KLK5 mRNA expression pattern in tumor tissue of patients suffering from high-grade serous ovarian cancer stage FIGO III/IV. Moreover, we have correlated the KLK5 mRNA levels with clinical outcome. METHODS: We assessed the mRNA expression levels of KLK5 in tumor tissue of 138 patients using quantitative PCR (qPCR). The mRNA levels were correlated with KLK5 antigen tumor tissue levels measured by ELISA (available for 41 of the 138 patients), established clinical features as well as patients' outcome, using Chi-square-tests, Mann-Whitney U-tests and Spearman rank calculations as well as Cox regression models, Kaplan-Meier survival analysis and the log-rank test. RESULTS: A highly significant correlation between the mRNA expression levels and protein levels of KLK5 in tumor tissues was observed (rs = 0.683, p < 0.001). In univariate Cox regression analysis, elevated KLK5 mRNA expression was remarkably associated with reduced progression-free survival (PFS; p = 0.047), but not with overall survival (OS). Association of KLK5 mRNA expression with PFS was validated in silico using The Cancer Genome Atlas. For this, Affymetrix-based mRNA data (n = 377) were analyzed applying the Kaplan-Meier Plotter tool (p = 0.027). In multivariable Cox analysis, KLK5 mRNA values revealed a trend towards statistical significance for PFS (p = 0.095), whereas residual tumor mass (0 mm vs. > 0 mm), but not ascites fluid volume (≤500 ml vs. > 500 ml), remained an independent indicator for both OS and PFS (p < 0.001, p = 0.005, respectively). CONCLUSIONS: These results obtained with a homogenous patient group with all patients suffering from advanced high-grade serous ovarian cancer support previous results suggesting elevated KLK5 mRNA levels as an unfavorable marker in ovarian cancer.

The meteoric rise and dramatic fall of Theranos: lessons learned for the diagnostic industry.

In this piece we discuss and reflect on the conclusion of the Theranos saga in the light of its fraud conviction. Theranos (founded in 2003 by Elizabeth Holmes) was supposed to disrupt the diagnostic testing industry by developing technology which could perform dozens of tests using a tiny amount of blood from a finger-prick. As a result, Ms. Holmes rose to fame, becoming the world's youngest female self-made billionaire and was plastered across magazine covers. However, in 2014, Theranos began to fall apart following increasingly damaging revelations regarding its lack of expertise, technology, framework, extreme secrecy and inaccurate test results. This led to the closure of two of its laboratories, investor and patient lawsuits and the devaluation of Ms. Holmes's wealth to nothing. In March 2018, the United States Security Exchange Commission ordered Ms. Holmes to pay $500,000 to settle the charge of massive fraud and barred her from being a director of a publicly owned company for 10 years, likely concluding Theranos's endeavors. We conclude our series of articles on this topic by reflecting on the lessons the laboratory medicine community can learn from Theranos.

Assessment of kallikrein 6 as a cross-sectional and longitudinal biomarker for Alzheimer's disease.

BACKGROUND: Kallikrein 6 (KLK6) is known to be an age-related protease expressed at high levels in the central nervous system. It was previously shown to be involved in proteolysis of extracellular proteins implicated in neurodegenerative diseases such as Alzheimer's disease (AD), prompting validation of KLK6 as a potential biomarker of disease. However, analyses of both plasma and cerebrospinal fluid (CSF) levels of KLK6 in patients with AD have been inconclusive. We present a detailed analysis of KLK6 in plasma and CSF in two separate cohorts in a cross-sectional and a longitudinal clinical setting. METHODS: The cross-sectional cohort included control subjects without dementia and patients with AD, and the longitudinal cohort included patients with MCI and patients with AD followed over a 2-year period. Plasma and CSF levels of KLK6 were quantified by use of a previously developed and validated enzyme-linked immunosorbent assay. Statistical analyses were performed to compare KLK6 levels between diagnostic groups and to identify potential associations between KLK6 level, age, apolipoprotein E (APOE) genotype, total apoE level and the classical CSF AD biomarkers. RESULTS: In the cross-sectional setting, KLK6 levels in plasma but not in CSF were significantly higher in the AD group than in control subjects. CSF but not plasma KLK6 levels were positively correlated with age in both the cross-sectional and longitudinal settings. In both cohorts, the CSF KLK6 levels were significantly and positively correlated with the CSF levels of core AD biomarkers. Total plasma and CSF apoE levels were positively associated with KLK6 in the cross-sectional study. Finally, during the 2-year monitoring period of the longitudinal cohort, CSF KLK6 levels increased with disease progression over time in the investigated patient groups. CONCLUSIONS: In two separate cohorts we have confirmed the previously reported correlation between age and CSF levels of KLK6. Increased plasma KLK6 levels in patients with AD with a more advanced disease stage suggest KLK6 as a potential biomarker in patients with AD with more severe dementia. Significant correlations between KLK6 levels and core CSF AD biomarkers suggest molecular links between KLK6 and AD-related pathological processes.

The emerging landscape of scientific publishing.

We present emerging models of publishing which have grown from the phenomenon of open access, the changing role of peer review in the scientific process and the new position of the impact factor. We juxtapose the new models of paid review, eponymous review, no review, post publication review and light review with the classic model which has dominated for a century, detailing advantages, problems and examples of each model to provide a comprehensive overview of the changing landscape of scientific publishing.

An integrated proteomic and peptidomic assessment of the normal human urinome.

BACKGROUND: Urine represents an ideal source of clinically relevant biomarkers as it contains a large number of proteins and low molecular weight peptides. The comprehensive characterization of the normal urinary proteome and peptidome can serve as a reference for future biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases. METHODS: We developed an integrated proteomic and peptidomic analytical protocol in normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer. RESULTS: By analyzing six urines from healthy individuals with advanced age, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma (RCC). CONCLUSIONS: We utilized a highly sensitive proteomics approach that enabled us to identify one of the largest sets of protein identifications documented in normal human urine. The raw proteomics and peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003595.

Assessment of kallikrein-related peptidase 5 (KLK5) protein expression in tumor tissue of advanced ovarian cancer patients by immunohistochemistry and ELISA: correlation with clinical outcome.

Members of the human kallikrein-related peptidase (KLK) family, including KLK5, have been reported to play an important role in ovarian cancer progression. In the present study, we assessed KLK5 protein expression in ovarian cancer tissues by immunohistochemistry (IHC) and ELISA, and analyzed its association with clinicopathologic parameters and disease outcome in 95 patients with advanced ovarian cancer FIGO stage III/IV. KLK5 immunoexpression was evaluated in ovarian cancer tissue microarrays by IHC using a manual semiquantitative scoring system. KLK5 antigen levels were determined in ovarian cancer tumour tissue extracts by ELISA. KLK5 protein is expressed in ovarian cancer tissue by stromal and tumor cells. Mean KLK5 immunoscore values in tumor cells (KLK5-Tc; 5.7, range 0 to 12) were higher compared to stromal cells (KLK5-Sc; 1.2, range 0 to 9) but the correlation between KLK5-Tc and KLK5-Sc was rather low (rs = 0.34, P < 0.05). No significant associations of clinicopathological parameters with KLK5-Tc, KLK5-Sc, the combined overall score KLK5-Tc+Sc, or ELISA (KLK5-E) expression values were determined, except for KLK5-E protein expression with advanced age and high nuclear grade (G3). In univariate Cox regression analysis, elevated expression levels of KLK5-Sc are significantly linked with both prolonged overall survival (OS) (hazard ratio [HR] = 0.6, P = 0.046) and progression-free survival (PFS) (HR = 0.54, P = 0.032) of advanced ovarian cancer patients. KLK5-Tc and KLK5-Tc+Sc scores as well as the KLK5-E values were not associated with patients' outcome. In multivariable analysis, KLK5-Sc expression was found to be statistically significant for PFS. Patients with elevated KLK5-Sc had a two-fold lower risk of disease recurrence (HR = 0.53, P = 0.037) as compared to patients with low KLK5-Sc. For KLK5-Sc and OS, a trend towards statistical significance was observed (HR = 0.62, P = 0.077). These results indicate that KLK5 overexpression by stromal cells (KLK5-Sc) may be a positive modulator lowering aggressiveness of ovarian cancer.

Theranos phenomenon - part 2.

Theranos' Chief Executive Officer recently published a paper in The Wall Street Journal committing to submit all of their tests for FDA approval and renewing her promise that self-testing by the general public will empower people to detect asymptomatic disease early, which will lead to life-saving therapeutic or preventive measures. This opinion paper provides additional information on the benefits and harms of self-testing and self-interpretation of laboratory tests by asymptomatic individuals. We conclude that the health benefit claims of Theranos are hypothetical and they are not supported by evidence. Until such evidence is provided, self-testing of the healthy population should be discouraged.

Theranos phenomenon: promises and fallacies.

Recently, spectacular advances in diagnostic technologies, genomics, etc. offer unprecedented opportunities for widespread testing of asymptomatic individuals, in the hope that this testing will unravel early disease signs which could lead to preventative or more effective therapeutic measures. In particular, one commercial organization, Theranos, promises to revolutionize diagnostics by offering multi-analyte testing at low prices in commercial outlets, thus challenging the current paradigm of targeted and centralized diagnostic testing. In this paper, I analyze the Theranos technology and their promises, and contrast this information with the currently used technologies, to show that most of the company's claims are exaggerated. While it remains to be seen if this technology will revolutionize diagnostics, in this Opinion Paper, I also draw attention of associated issues, such as self-testing and self-interpretation of results, over-testing, over-diagnosis and over-treatment, along with their associated harms. As the public is bombarded daily with new and revolutionary health-related advances, it is time to balance the enthusiasm of the seemingly obvious huge gains, by also explaining the associated possible harms.

Validation of new cancer biomarkers: a position statement from the European group on tumor markers.

BACKGROUND: Biomarkers are playing increasingly important roles in the detection and management of patients with cancer. Despite an enormous number of publications on cancer biomarkers, few of these biomarkers are in widespread clinical use. CONTENT: In this review, we discuss the key steps in advancing a newly discovered cancer candidate biomarker from pilot studies to clinical application. Four main steps are necessary for a biomarker to reach the clinic: analytical validation of the biomarker assay, clinical validation of the biomarker test, demonstration of clinical value from performance of the biomarker test, and regulatory approval. In addition to these 4 steps, all biomarker studies should be reported in a detailed and transparent manner, using previously published checklists and guidelines. Finally, all biomarker studies relating to demonstration of clinical value should be registered before initiation of the study. SUMMARY: Application of the methodology outlined above should result in a more efficient and effective approach to the development of cancer biomarkers as well as the reporting of cancer biomarker studies. With rigorous application, all stakeholders, and especially patients, would be expected to benefit.

Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.

Whole genome sequencing as a diagnostic test: challenges and opportunities.

BACKGROUND: Extraordinary technological advances and decreases in the cost of DNA sequencing have made the possibility of whole genome sequencing (WGS) as a highly accessible clinical test for numerous indications feasible. There have been many recent, successful applications of WGS in establishing the etiology of complex diseases and guiding therapeutic decision-making in neoplastic and nonneoplastic diseases and in various aspects of reproductive health. However, there are major, but not insurmountable, obstacles to the increased clinical implementation of WGS, such as hidden costs, issues surrounding sequencing and analysis, quality assurance and standardization protocols, ethical dilemmas, and difficulties with interpretation of the results. CONTENT: The widespread use of WGS in routine clinical practice remains a distant proposition. Prospective trials will be needed to establish if, and for whom, the benefits of WGS will outweigh the likely substantial costs associated with follow-up tests, the risks of overdiagnosis and overtreatment, and the associated emotional distress. SUMMARY: WGS should be carefully implemented in the clinic to allow the realization of its potential to improve patient health in specific indications. To minimize harm the use of WGS for all other reasons must be carefully evaluated before clinical implementation.