The gold standard for diagnosis of BCa is the cystoscopy. However, the cystoscopy is an invasive procedure for patients. Urine cytology is the most widely used conventional method in predicting BCa. The sensitivity of urine cytology is poor for detecting low grade BCa although it is useful for detecting high grade BCa and carcinoma in situ. Therefore, identification of additional potent urinary biomarkers are sought to establish earlier detection of BCa.
Recently, Reg family proteins have been focused as candidates of serum biomarkers for detecting some cancers. [13–19] To our knowledge, there are no prior reports describing the expression of HIP/PAP in relation to the occurrence of BCa. In this study, we demonstrated HIP/PAP protein was significantly expressed in BCa specimens and BCa cell lines but not in normal bladder tissues (Figure1). Furthermore, urinary levels of HIP/PAP in BCa patients were significantly higher than those in the control group including healthy volunteers and patients with benign urological diseases (Figure3A). Interestingly, urinary levels of HIP/PAP also correlated positively with bladder tumor size (T stage), recurrence- and progression-risk classifications of non-muscle invasive BCa (Figures 3B and 4). Although two previous reports proposed that Reg-I could be a marker for BCa, [20, 24] the ELISA used in this study showed virtually no cross-reactivates with Reg-Iα and -Iβ proteins (Figure2), indicating that the urinary signal detected in the ELISA was HIP/PAP and not Reg-I. In this study, we investigated six cases of non-interstitial severe cystitis patients in the group of benign urological disease, and two in the six cases were false positive. Similar to the cases of other urinary markers such as NMP-22 and BTA, the false positive for HIP/PAP may be possibly identified in patients with benign inflammatory conditions. In addition, to eliminate the potential impact of hematuria in this ELISA kit, we investigated two cases of patients with Nutcracker syndrome in the group of benign urological disease. Although the above two patients had gross hematuria, the concentration of HIP/PAP in the urine of both two cases was less than 8.5 pg/mL. We therefore assume that this ELISA kit is not affected by hematuria.
Approximately 80% of newly-diagnosed BCa patients have non-muscle invasive tumors.  Regardless of endoscopic complete resection of such tumors, about 50% and 15% of these cases have the possibility of recurrence and progression within 5 years, respectively.  The 5-year overall survival (OS) of patients with non-muscle invasive BCa ranges from 82% to 95%, whereas that of cases with muscle invasion is only 50%. [27–29] Therefore, both diagnosis of BCa at an early stage and strict surveillance after initial treatments are crucial for improving the outcomes of BCa patients. According to the EORTC risk criteria for non-muscle invasive BCa, patients treated with an initial transurethral tumor resection are divided into groups at low, intermediate, and high risk for recurrence and progression. The current study showed urinary HIP/PAP levels in the intermediate recurrence-risk group to be significantly higher than those in the low recurrence-risk group (Figure4A). Furthermore, urinary HIP/PAP levels correlated positively with the grade of progression-risk groups (low, intermediate, high) (Figure4B). On the basis of these results, pre-treatment urinary HIP/PAP levels may be applied as a prognostic factor for recurrence and progression of non-muscle invasive BCa. On the other hand, the reliability of predictive biomarkers is attributed to high PPV and NPV. However, routine urinary markers (e.g., NMP-22, BTA) are not sufficiently accurate to detect BCa. As shown in Figure5, the AUC of HIP/PAP and NMP-22 on ROC analysis were 0.863 (95% CI, 0.814-0.912) and 0.729 (95% CI, 0.655-0.803), respectively. This suggests that urinary HIP/PAP could be a better urinary marker for BCa than the urinary NMP-22. Furthermore, as shown in Table2, the statistical analysis to estimate the precisions of the three urinary markers revealed that HIP/PAP was superior to NMP-22 and BTA in terms of sensitivity (80.2% vs. 52.1% and 34.7%) and NPV (82.3% vs. 65.2% and 54.1%). This characteristic of urine HIP/PAP may be useful for reducing false negative cases of suspected BCa. In particular, it may contribute to strict follow-up for higher risk groups (e.g., intermediate and high) among non-muscle invasive BCa. However, false positive cases may undergo unnecessary endoscopic examination due to the low PPV of HIP/PAP. Based on the potential of current urinary markers, we propose that examinations combining a number of urinary biomarkers would provide a higher accuracy in predicting BCa.