Abstract
Sensitive and specific urinary biomarkers can improve patient outcomes in many diseases through informing early diagnosis. Unfortunately, to date, the accuracy and translation of diagnostic urinary biomarkers into clinical practice has been disappointing. We believe this may be due to inappropriate standardization of diagnostic urinary biomarkers. Our objective was therefore to characterize the effects of standardizing urinary levels of IL-6, IL-8, and VEGF using the commonly applied standards namely urinary creatinine, osmolarity and protein. First, we report results based on the biomarker levels measured in 120 hematuric patients, 80 with pathologically confirmed bladder cancer, 27 with confounding pathologies and 13 in whom no underlying cause for their hematuria was identified, designated “no diagnosis”. Protein levels were related to final diagnostic categories (p = 0.022, ANOVA). Osmolarity (mean = 529 mOsm; median = 528 mOsm) was normally distributed, while creatinine (mean = 10163 µmol/l, median = 9350 µmol/l) and protein (0.3297, 0.1155 mg/ml) distributions were not. When we compared AUROCs for IL-6, IL-8 and VEGF levels, we found that protein standardized levels consistently resulted in the lowest AUROCs. The latter suggests that protein standardization attenuates the “true” differences in biomarker levels across controls and bladder cancer samples. Second, in 72 hematuric patients; 48 bladder cancer and 24 controls, in whom urine samples had been collected on recruitment and at follow-up (median = 11 (1 to 20 months)), we demonstrate that protein levels were approximately 24% lower at follow-up (Bland Altman plots). There was an association between differences in individual biomarkers and differences in protein levels over time, particularly in control patients. Collectively, our findings identify caveats intrinsic to the common practice of protein standardization in biomarker discovery studies conducted on urine, particularly in patients with hematuria.
Highlights
Advances in proteomics have enhanced our understanding of the urinary proteome [1,2,3,4] and subsequently encouraged biomarker discovery screens in a range of complex diseases [2,3], including bladder cancer [5,6]
We have presented evidence that the high prevalence of proteinuria in hematuric patients introduces a caveat with respect to using protein as the standardiser of urinary biomarker levels
The origin of proteins shed into the urine of patients with proteinuria is dependent on the specific disorder that the patient has [7]
Summary
Advances in proteomics have enhanced our understanding of the urinary proteome [1,2,3,4] and subsequently encouraged biomarker discovery screens in a range of complex diseases [2,3], including bladder cancer [5,6]. Urine has the advantage of ease of access and is relatively stable thermodynamically [3]. Despite these encouraging developments, no biomarker or biomarker combination to date, has achieved widespread clinical application as a diagnostic assay. No biomarker or biomarker combination to date, has achieved widespread clinical application as a diagnostic assay Perhaps this is partly attributable to the range of methodologies used to standardise urinary biomarker levels which introduces a lack of consistency in reported levels and inhibits cross study comparisons. As there is no standard methodology, the normalization method employed for any given study is still very much at the discretion of the project investigator, the accessibility of equipment and the available technical expertise. There is the potential both for biased data and masking detection of valuable biomarkers secreted into urine at low levels [7]
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