Abstract
Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); however, inadequate diagnostic accuracy remains a major challenge. We performed mass spectrometry-based proteomic analysis of urine samples of ten patients with BLCA and ten paired patients with benign urothelial lesion (BUL) to identify ancillary proteomic markers for use in liquid-based cytology (LBC). A total of 4,839 proteins were identified and 112 proteins were confirmed as expressed at significantly different levels between the two groups. We also performed an independent proteomic profiling of tumor tissue samples where we identified 7,916 proteins of which 758 were differentially expressed. Cross-platform comparisons of these data with comparative mRNA expression profiles from The Cancer Genome Atlas identified four putative candidate proteins, AHNAK, EPPK1, MYH14 and OLFM4. To determine their immunocytochemical expression levels in LBC, we examined protein expression data from The Human Protein Atlas and in-house FFPE samples. We further investigated the expression of the four candidate proteins in urine cytology samples from two independent validation cohorts. These analyses revealed AHNAK as a unique intracellular protein differing in immunohistochemical expression and subcellular localization between tumor and non-tumor cells. In conclusion, this study identified a new biomarker, AHNAK, applicable to discrimination between BLCA and BUL by LBC. To our knowledge, the present study provides the first identification of a clinical biomarker for LBC based on in-depth proteomics.
Highlights
Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); inadequate diagnostic accuracy remains a major challenge
After exclusion of antibodies with indistinguishable expression between the benign urothelial lesion and bladder urothelial carcinoma groups based on micrographs from the data set, four candidate proteins were subsequently validated by immunohistochemistry of 25 formalin fixed paraffin embedded (FFPE) cases and four immunocytochemistry of voided-urine liquid-based cytology samples
Quantitative Proteomic and Bioinformatic Analyses Workflow—In this study, we developed a workflow to achieve identification of reproducible biomarkers with high ability to discriminate between benign urothelial lesion and bladder urothelial carcinoma in voided liquid-based cytology urine samples
Summary
Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); inadequate diagnostic accuracy remains a major challenge. Bladder urothelial carcinoma RNA sequencing data were sourced independently for comparative analysis with protein expression data obtained using MS-based proteomic assays to evaluate reliable ancillary biomarkers for bladder urothelial carcinoma diagnosis using liquid-based cytology and FFPE samples (supplemental Fig. S2).
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