Research over the past several years has uncovered potentially important urine biomarkers and tests for BC, including BTA and NMP22. BTA-stat, an FDA-approved urine biomarker, is used clinically to detect bladder tumor-associated antigen (human complement factor H-related protein) in the urine. A meta-analysis of BTA stat reported a specificity of 67% and a sensitivity of 75% after reviewing 13 studies [8]. The sensitivity levels of BTA-stat have been shown to positively correlate with increasing grade of BC [8]. However, BTA-stat has several limitations. These include lower specificity values and issues relating to false-positive results in benign conditions [6]. Hence, urine BTA-stat may have limitations in the diagnosis and monitoring of disease progression. Similarly, NNP22 is an FDA-approved urine biomarker designed to detect the NMP22 protein levels which are high due to cell turnover from tumor apoptosis. A meta-analysis of 19 studies has identified this marker to have a pooled specificity of 88% and a sensitivity of 56% [7].

As opposed to studies looking at an isolated protein in the urine, a few screens have been reported where multiple proteins were examined simultaneously. Summarized below are a couple of studies documenting biomarkers with both sensitivity and specificity values greater than or equal to 85%. Goodison et al. performed a validation study for the urinary concentrations of 14 proteins (A1AT, APOE, ANG, CA9, CCL18, CD44, IL-8, MMP-9, MMP-10, OPN, PAI-1, PTX3, SDC1, and VEGF) using an ELISA [30]. An 8-biomarker panel (ANG, APOE, CA-9, IL-8, MMP-9, MMP-10, PAI-1, and VEGF) achieved the most accurate BC diagnosis with a sensitivity of 92% and a specificity of 97%. However, a panel of 3 biomarkers (APOE, IL-8, and VEGF) also performed well with a sensitivity of 90% and a specificity of 97% for the detection of BC [30]. Kumar et al. identified a 5-biomarker panel consisting of Apolipoprotein A4, Coronin-1A, DJ-1/PARK7, Gamma synuclein, and Semenogelin-2. ELISA and western blot data obtained an AUC of 0.92 and 0.98, respectively, in diagnosing Ta/T1 BC (sensitivity 79.2% and 93.9% for ELISA; specificity 100% and 96.7% for western blot) [31]. For the diagnosis of T2/T3 BC, the panel of markers achieved an AUC of 0.94 and 1, respectively, using the same methods (sensitivity 86.4% and 100%; specificity 100%) [31].

Low-grade BC has a high recurrence rate; therefore, identifying biomarkers for the surveillance of BC is essential for the potential clinical management of the disease. Rosser et al. identified 10 biomarkers (ANG, APOE, CA9, IL-8, MMP-9, MMP-10, SDC1, SERPINA1, SERPINE1, and VEFGA) using ELISA for monitoring urine for recurrent BC. The complete panel achieved an AUC of 0.90, a sensitivity of 79%, and a specificity of 88% [32]. De Paoli et al. identified a panel of 6 biomarkers (cadherin-1, EN2, ErbB2, IL-6, IL-8, and VEGF-A) and three clinical parameters including BCG therapies, stage at the time of diagnosis, and past recurrences. The panel achieved an AUC of 0.91 and was identified through microarray and ELISA analysis [33].

There are several reasons to discriminate patients with bladder cancer from benign conditions. In patients with hematuria, it would be helpful to identify who needs cystoscopic evaluation which is invasive. Given that urine proteins are easily measurable and are compatible with point-of-care monitoring, a quick urine test could dramatically impact triage and workflow in urology outpatient clinics. Likewise, in bladder cancer surveillance, a reliable urine biomarker can help determine if cancer (like CIS) was missed or to avoid cystoscopy in marker-negative patients. Similarly, urine biomarkers that can reliably distinguish MIBC from NMIBC can inform us as to who has the more aggressive disease. When used as a routine point-of-care test (either at home or at outpatient visits), these urinary biomarkers may facilitate earlier identification of aggressive disease and design of tailored therapy.

The present work represents the first attempt to screen>1000 urine proteins for urine biomarker candidates in BC, using a relatively novel aptamer-based screen. Systems biology analysis implicated molecular functions related to the extracellular matrix, collagen, integrin, heparin, and transmembrane tyrosine kinase signaling in BC susceptibility, with HNF4A and NFKB1 being key regulators. STEM analysis of the dysregulated pathways implicated a functional role for the immune system, complement, and interleukins in BC disease progression (Fig.3D). This study has also uncovered urine proteins that outperform current FDA-approved markers in many respects. Several urine proteins (d-dimer, Apolipoprotein A1, MMP-1, Properdin, Calgranulin B) significantly discriminate BC from UC with AUC values from 0.85 to 0.96 (p-value<0.0001). As a single biomarker, urine d-dimer was able to discriminate BC from UC with 96% accuracy (sensitivity=95%; specificity=90%). Likewise, several urine proteins (IL-8, IgA, Fibronectin, C2, Proteinase 3) significantly discriminate MIBC from NMIBC with AUC 0.840.99 (p-value<0.001). Interestingly, several of the proteins described above have been documented to be elevated in bladder cancer tissue (at the RNA or protein level) and/or implicated in tumor biology at some level, as summarized in Additional file 1: Table S5. Considering their biomarker potential and functional properties based on the literature (Additional file 1: Table S5), these urine proteins warrant further investigation, including, d-dimer [34], Apolipoprotein A1 [35, 36], Apolipoprotein L1, Calgranulin B [37, 38], complement C2 [39], Fibronectin [40,41,42,43], Ficolin-3, IL-8 [44,45,46,47,48,49], IgA [50], MMP-1 [51, 52], Properdin, and Proteinase 3 [53]. A summary of previous research on these proteins can be found in Additional file 1: Table S6. Additional markers increased in tissues are described in Additional file 1: Table S7.d-dimer is a specific cleavage product of fibrin and a symbol of hyperfibrinolysis [34]. It is the primary diagnostic tool in various diseases, such as deep venous thrombosis, systemic illness, and cancers [54]. Previous studies have reported that molecules in the coagulation/fibrinolysis system, especially plasma fibrinogen and d-dimer, are abnormal in cancer patients [34]. In the present study, urine d-dimer levels show a significant ability to differentiate BC from UC (AUC=0.96) (p<0.0001). After correcting for patient demographics, urine d-dimer is still eligible for inclusion within the 5-biomarker panel for best distinguishing BC from UC. Perhaps most impressive is the observation that urine d-dimer demonstrates a high sensitivity for the detection of BC (95%), and at a fixed specificity of 0.8, it can achieve a sensitivity of 0.97. Hence, as a single biomarker, urine d-dimer outperforms current FDA-approved biomarkers and competing biomarkers in the research literature as a sensitive biomarker for BC detection.

Apolipoproteins (Apolipoprotein A1 and Apolipoprotein L1) are proteins known to interact with the lipids of the lipoprotein core and also the aqueous environment of the plasma. Apolipoprotein A1 is the primary protein component of high-density lipoprotein while Apolipoprotein L1 is a minor component. Previous studies have validated Apolipoprotein A1 as a novel urinary biomarker for BC [35, 36]. In the current research, Apolipoprotein A1 was the second-best performing protein in terms of the AUC value (0.91) in distinguishing BC from UC.

After adjusting for demographics, this protein ranked within a 5-marker panel for distinguishing BC from UC. Similarly, Apolipoprotein L1 also ranked within the 5-marker panel for distinguishing BC from UC and MIBC from NMIBC.

Calgranulin B (S100A9) is a zinc- and calcium-binding protein that plays a prominent role in regulating inflammatory and immune responses. Several S100 proteins, including S100A9, have received attention regarding their possible role in tumor development and progression and studies report an increased expression in a variety of tumors, including ovarian, colon, gastric, and prostate cancer [37]. Increased expression of S100A9 protein in the serum has been previously associated with tumor grade [37]. Current validation results of Calgranulin B are promising as it was among the top markers in discriminating BC from UC with an AUC of 0.85.

Complement proteins may promote tumor growth in the context of chronic inflammation [39]. Complement C2s relation to BC at this time is unknown. However, the present study identified this protein as the fourth best single protein for differentiating MIBC from the NMIBC stage. Properdin is also a member of the complement system, controlling the alternate pathway of complement activation. Research on properdin in BC is limited. However, in the current study, properdin demonstrated the third highest AUC value (AUC=0.89, p<0.0001) in discriminating BC from UC. These biomarker findings are consistent with the observation that changes in complement activation constitute one of the major pathways that predict BC disease progression, based on STEM analysis (Fig.3D).

Fibronectin is a glycoprotein component of the extracellular matrix. Tumor cells can attach to fibronectin via integrins or other cell surface receptors [55]. Its effectiveness as a urine biomarker for BC has been explored in a variety of studies [40,41,42,43]. Here, fibronectin showed the third best discriminatory ability in identifying MIBC compared to NMIBC. The marker exhibited an AUC value of 0.87 (p<0.0001).

IL-8 is a proinflammatory CXC chemokine. It has previously been associated with the promotion of neutrophil chemotaxis and degranulation [56]. Increased expression of IL-8 has been associated with endothelial cells, infiltrating neutrophils, tumor-associated macrophages, and cancer cells [56]. Therefore, IL-8 may be a significant regulatory factor within the tumor microenvironment. Previous studies have identified urinary IL-8 as a potential marker for BC [44,45,46,47,48,49]. In the present study, urine IL-8 was the best-performing protein in the MIBC vs NMIBC comparison, with an AUC of 0.99 (p<0.0001), although its specificity was modest at a fixed sensitivity of 80%. Taken together with a wealth of supporting literature, this marker has the potential to be a monitoring tool for BC disease progression and warrants further analysis in this context.

IgA is an immunoglobulin and is often the first line of defense in the resistance against infections, particularly in mucosal tissues. A correlation of intra-tumor IgA1 and poor overall survival in BC patients has been identified in a previous study [50]. However, research regarding IgA in BC urine is limited. The data presented in this study indicated that urinary IgA may differentiate MIBC from NMIBC (AUC=0.89, p<0.0001). Overall, IgA performed 2nd best out of a total of 30 urinary markers validated for this comparison. Of particular note, urine IgA exhibited the highest specificity of 80% for MIBC, at 80% sensitivity, out-performing IL-8.

Matrix metalloproteinases (MMP) are a group of zinc-dependent proteolytic enzymes. Their role involves remodeling of the extracellular matrix. Many studies have evaluated the levels of MMPs in cancer patients and have reported the vital roles of some MMPs as potential diagnostic and prognostic biomarkers in tumorigenesis [57]. The current study has uncovered MMP-1 as the fourth best-performing molecule for distinguishing BC from UC (AUC=0.89, p<0.0001). This protein was also included in a 5-marker panel for distinguishing BC from UC. At the mechanistic level, one can envision tissue matrix remodeling as an important pre-requisite for cancer progression.

View original post here:
Comprehensive proteomics and platform validation of urinary ... - BMC Medicine