Promising Urinary Protein Biomarkers for the Early Detection of Hepatocellular Carcinoma among High-Risk Hepatitis C Virus Egyptian Patients

Abstract

Hepatocellular Carcinoma is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. There are 130 million Hepatitis C virus infected patients worldwide who are at a high-risk for developing Hepatocellular Carcinoma. Due to the fact that reliable parameters and/or tools for the early detection of Hepatocellular Carcinoma among high-risk individuals are severely lacking, Hepatocellular Carcinoma patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Urine was collected from 106 Hepatitis C infected patients patients, 32 of whom had already developed Hepatocellular Carcinoma and 74 patients who were diagnosed as Hepatocellular Carcinoma -free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins were isolated from the urine samples and LC-MS/MS was used to identify potential protein HCC biomarker candidates. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and Heat Shock Protein 60 (HSP60), was a characteristic event among Hepatocellular Carcinoma - post Hepatitis C virus infected patients. As a single-based Hepatocellular Carcinoma biomarker, CAF-1 over-expression identified Hepatocellular Carcinoma among Hepatitis C virus infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-1/HSP60 tandem identified Hepatocellular Carcinoma among Hepatitis C virus infected patients with a specificity of 92%, sensitivity of 61% and with an overall diagnostic accuracy of 77%.

Heat shock protein 60 and chromatin assembly factor-1 mRNA levels in hepatitis C virus-related hepatocellular carcinoma and clinical significance

“…They explained the up-regulated HSP60 in chronic HCV by its anti-apoptotic effect rather than being death-promoting for HCC. 11 Moreover, in HCC group, we found significant positive correlation between HSP60 and CAF-1 (urine and blood) and tumor characters (size, number) and BCLC staging system (p˂0.05), but no significant correlation with AFP could be detected. These finding were in accordance with Li et al and Joo et al, the former documented that CAF-1 over-expression indicated poor HCC prognosis and the later found a strong positive correlation between HSPs and clinic-pathological grading of HCC.…”

“…38 In agreement with our study; Xu et al observed a significant positive correlation between CAF-1 expression and both tumor number and size but 40 In the present study, HSP60 mRNA in urine at cutoff 258354 RU had 85%, sensitivity and 66% specificity for HCC diagnosis, a result agreed with Abdalla and Haj-Ahmad, who found 83% sensitivity and 43% specificity for HSP60 in urine for diagnosis of HCC. 11 Also, HSP60 in blood was able to diagnosis HCC with 85% sensitivity and 60% specificity at cutoff 37576 RU with no supported published data. Regarding urine CAF-1, its sensitivity and specificity for diagnosis of HCC in the current study were 81% and 66% respectively at cutoff 137756 RU.…”

“…This result agreed with Abdalla and Haj-Ahmad who found significantly higher CAF-1 in urine of HCC versus chronic HCV and controls. 11 On the same hand, Li et al, found up-regulated CAF-1 expression in HCC. 39 In addition, Xu et al reported increased CAF-1 in HCC tumor tissues versus the surrounding non-tumor tissues by immunohistochemistry, mRNA and protein expression in HCC patients and cell lines, and they concluded that enhanced CAF-1 expression promoted tumorigenesis and progression by promoting HCC cell proliferation through the dysregulated DNA damage checkpoints or inappropriate assembling of nucleosomes affecting DNA replication.…”

“…11 CAF-1 is overexpressed in many cancers. 12 Extracellular nucleic acids, both DNA and mRNA, were found to exist in many biological media; serum, plasma, saliva, urine, semen, milk, bronchial lavage, and cell culture supernatants.…”

PUEPro: A Computational Pipeline for Prediction of Urine Excretory Proteins

“…Currently, the most useful disease markers in urine have been largely for urogenital diseases, such as urothelial cancer [4], renal cell carcinoma [5], prostate cancer [6], and bladder cancer [7]. A few recent studies have demonstrated the feasibility in using urinary proteins as disease markers in distal organs, such as ovarian carcinoma [8], lung cancer [9], hepatocellular carcinoma [10], and gastric cancer [11].…”