ITIH5 and ECRG4 DNA Methylation Biomarker Test (EI-BLA) for Urine-Based Non-Invasive Detection of Bladder Cancer.

Bladder cancer is one of the more common malignancies in humans and the most expensive tumor for treating in the Unites States (US) and Europe due to the need for lifelong surveillance. Non-invasive tests approved by the FDA have not been widely adopted in routine diagnosis so far. Therefore, we aimed to characterize the two putative tumor suppressor genes ECRG4 and ITIH5 as novel urinary DNA methylation biomarkers that are suitable for non-invasive detection of bladder cancer. While assessing the analytical performance, a spiking experiment was performed by determining the limit of RT112 tumor cell detection (range: 100-10,000 cells) in the urine of healthy donors in dependency of the processing protocols of the RWTH cBMB. Clinically, urine sediments of 474 patients were analyzed by using quantitative methylation-specific PCR (qMSP) and Methylation Sensitive Restriction Enzyme (MSRE) qPCR techniques. Overall, ECRG4-ITIH5 showed a sensitivity of 64% to 70% with a specificity ranging between 80% and 92%, i.e., discriminating healthy, benign lesions, and/or inflammatory diseases from bladder tumors. When comparing single biomarkers, ECRG4 achieved a sensitivity of 73%, which was increased by combination with the known biomarker candidate NID2 up to 76% at a specificity of 97%. Hence, ITIH5 and, in particular, ECRG4 might be promising candidates for further optimizing current bladder cancer biomarker panels and platforms.

Plasma Inter-Alpha-Trypsin Inhibitor Heavy Chains H3 and H4 Serve as Novel Diagnostic Biomarkers in Human Colorectal Cancer.

OBJECTIVE: Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) are heavy chains of protein members belonging to the ITI family, which was associated with inflammation and carcinogenesis. However, the diagnostic value of ITIH3 and ITIH4 in human colorectal cancer (CRC) remains unknown. METHODS: In total, 101 CRC patients and 156 healthy controls were enrolled. The concentrations of ITIH3 and ITIH4 proteins in plasma samples of participants were assessed using enzyme-linked immunosorbent assay. ITIH3 and ITIH4 expressions in human CRC tissues were additionally assessed via immunohistochemical staining (IHC). Receiver operating characteristic (ROC) was applied to estimate the diagnostic power of the two proteins, and the net reclassification improvement (NRI) was adopted to evaluate the incremental predictive ability of ITIH3/ITIH4 when added to the tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: The plasma concentration of ITIH3 in CRC patients (median: 4.370 µg/mL; range: 2.152-8.170 µg/mL) was significantly lower than that in healthy subjects (median: 4.715 µg/mL; range: 2.665-10.257 µg/mL; p < 0.001), while the ITIH4 plasma level in subjects with CRC (median: 0.211 µg/mL; range: 0.099-0.592 µg/mL) was markedly increased relative to that in the control group (median: 0.134 µg/mL; range: 0.094-0.460 µg/mL, p < 0.001). Consistently, IHC score assessment showed a dramatic reduction in ITIH3 expression and, conversely, upregulation of ITIH4 in colorectal carcinoma specimens relative to adjacent normal colorectal tissues (p < 0.001 in both cases). The area under the curve (AUC) of the ROC for ITIH4 (AUC = 0.801, 95% CI: 0.745-0.857) was higher than that for ITIH3 (AUC = 0.638, 95% CI: 0.571-0.704, both p values < 0.001). The AUC of the ROC for combined ITIH3 and ITIH4 was even higher than that for carcinoembryonic antigen. NRI results showed that combining ITIH3 and ITIH4 with TIMP-1 significantly improved diagnostic accuracy (NRI = 17.12%, p = 0.002) for CRC patients compared to TIMP-1 alone. CONCLUSIONS: Circulating ITIH3 and ITIH4 levels are associated with carcinogenesis in CRC, supporting their potential diagnostic utility as surrogate biomarkers for colorectal cancer detection.