Cairo University Fibrosis Index (CUFI): A MicroRNA Based Score for Accurate Prediction of Hepatic Fibrosis: A Biopsy Controlled Study


 Background: The exact identification of liver fibrosis is mandatory for customizing therapy and controlling complications. The aim of the current study is to measure the ability to circulate miRNAs; in the diagnosis of hepatic fibrosis.Methods: Routine laboratory, liver biopsy, and histopathological investigations were done on seventy HCV patients. In addition, estimation of HCV-RNA, serum micro RNA (miRNA)-122, 221, 192, 224, 375, and 885 were done by PCR. To assess the ability of each miRNA, the area under the receiver operating characteristics (AUC) curves were plotted. Significant miRNA were analyzed by linear regression analysis in a stepwise pattern to develop a score for pointing out significant fibrosis. Moreover, the following scores were calculated: aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (AAR), aspartate to platelet ratio index (APRI), FIB-4 score, Hui index, Fibrosis Index (FI), Fibro-Q, Fibro-Alfa Biotechnology Research Center (BRC) score, and Gotebörg University Cirrhosis Index (GUCI).Results: Patients with significant and advanced fibrosis have significantly lower miR-122. MiR-122, FIB4, total bilirubin, and miR-855 proved to be independent predictors of significant fibrosis in univariate analysis. A novel score; Cairo University Fibrosis Index (CUFI) based on microRNA 122, FIB4, bilirubin, and microRNA 855 were formulated for the identification of significant liver fibrosis. The AUC of the score, for predicting significant and advanced hepatic fibrosis was 0.85 and 0.81. This AUC was higher than those of other common fibrosis scores.Conclusion: Cairo University Fibrosis Index proved to be better than other existing scores in assessing fibrosis in chronic hepatitis C (CHC) patients.


Introduction
Liver brosis induced by Hepatitis C virus (HCV) is a dynamic, wound healing, process that results from ongoing damage to the hepatocytes from direct viral cytopathic effect or due to immunological response with subsequent activation of hepatic stellate cell and changes in extracellular matrix formation and degradation, including deposition of collagen [1]. In the era of treatment of chronic HCV using highly e cient direct-acting antivirals (DAAs), hepatic brosis staging as a triage for therapy indication is no longer as crucial as once believed [2][3][4][5][6]. Rather, precise identi cation of advanced hepatic brosis as well as cirrhosis is highly required for adjusting HCV therapy, tracking possible regression of liver brosis after achieving sustained virological response, and proper prevention and treatment of HCV-related end-stage liver disease complications [7][8][9][10]. Histopathology is the traditional benchmark for the determination of liver brosis stage, but needle liver biopsy procedure is invasive and has some limitations including cost, risk of complications, sampling errors, inaccurate representation of unevenly distributed liver disease as the sample of the liver biopsy parallels a fraction of 1/50,000th of the whole liver [5]. Noninvasive methods for hepatic brosis staging in chronic HCV patients including physical (imaging techniques) and biological (serum biomarkers) have become increasingly available over the last few decades [7,8,[10][11][12][13][14].
Mi-RNA are small non-coding RNAs with an average length of 22 nucleotides that control the translation and transcription of many genes [15]. Mi-RNAs are crucial for the regulation of in ammatory diseases and are considered predictive biomarkers for liver diseases [16,17]. Scarce studies explored the potential role of circulating miRNAs in the prediction of liver brosis stage. Department reviewed and approved the protocol. Every patient signed informed consent before taking part in the study.

Statistical analysis
Results were examined utilizing SPSS software package version 25.0 (SPSS Inc., Chicago, IL). Numerical data were demonstrated as mean ± SD and differences were carried out using analysis of variance (ANOVA) or Student's t-test. Tests were signi cant when the P-value < 0.05. Patients were classi ed into 2 groups; signi cant liver brosis (F2-F4) and advanced liver brosis (F3-F4). Noninvasive hepatic brosis indexes were calculated; table 1. To assess the ability of each biomarkerfor differentiating between the two groups, the AUC curves were plotted; signi cant biomarkers were selected and analyzed by linear regression analysis in a stepwise pattern to develop a score that combined the independent factors for pointing out signi cant brosis. The best cutoff value of the predictive score was optimized for identifying signi cant brosis. On the other hand, the diagnostic performances of our developed score were compared with other common noninvasive liver brosis scores. p<0.05 is considered signi cant. P value < 0.01 is considered highly signi cant, p < 0.001 is considered very signi cant and p < 0.0001 is considered extremely signi cant.

Discussion
Liver disease is a considerable cause of mortality and morbidity [5,28]. Accurate determination of the disease stage is mandatory for clinical decision-making. Still, liver histopathology is considered the golden benchmark for the diagnosis of liver brosis [29]. However, the invasiveness of biopsy, the potential associated complications, and poor sample quality urge the search for noninvasive diagnostic markers for liver brosis [30]. The need to nd predictors of the hepatic brosis stage at the molecular level is increasing as hepatic brosis progression may result in hepatocellular carcinoma development [31,32]. The miRNAs are now considered to be important indicators of liver brosis [33], and liver carcinogenesis [32]. In the current study, we analyzed the performance of microRNAs as hepatic brosis biomarkers. Patients suffering signi cant and advanced brosis showed a signi cantly lower value of MiR-122. Our study showed more decrease in serum miR-122 in patients with advanced hepatic brosis (F3,4) when compared with signi cant brosis (F2-4) which agreed with Trebicka et al who stated that loss of functional hepatocytes in the late stages of brosis (F3-4) might cause decreased serum levels of miRNA-122 [34]. Moreover, the suppressive function of miR-122 that hinders the proliferation of hepatic stellate cells and brogenesis is absent in the late stages of brosis. [35]. Also, many studies proved an inverse relationship between miR-122 and the stage of brosis in HCV-based liver disease [36, 37]. Furthermore, we formulated a novel score (CUFI) which shows a greater diagnostic value for pointing to signi cant and advanced hepatic brosis taking histopathology as the gold standard ( AUR 0.85, 0.81 respectively). The score included microRNA 122, microRNA 855, FIB4, and bilirubin. It is well known that changes in miRNA-122 serum levels occur early in liver disease, suggesting that miRNA -122 can be a reliable predictor of hepatic injury [33]. Additionally, miR-122 induces HCV translation and stimulates viral genomic RNA replication [38]. Also, it is inversely correlated with brosis in HCV-infected patients [36,37]. miR-122 levels were higher levels in the non-signi cant liver followed by reduced levels in signi cant liver brosis in response to in ammation and damage of liver cells con rming previous studies. miR-885 was found to correlate with different liver pathologies [39-41]. Bilirubin is the lipophilic end product of heme breakdown [42]. Hyperbilirubinemia is common in chronic liver disease and serum bilirubin is routinely included in the biochemical assessment of such patients, Serial measurement of serum bilirubin is might be indicative of liver disease progression being an indirect marker of hepatic brosis [43].
Our novel score CUFI which is composed of serum bilirubin, miRNA -122, and miRNA -855 performed even better than common liver brosis scores. Its AUC was 0.85 for documenting signi cant liver brosis and 0.81 for detection of advanced brosis which was the greatest score among common liver brosis scores. This could be cleared up by the fact that most of the common liver brosis scores depend on multiple biochemical and serum parameters which may differ with many factors such as blood sampling time, the blood sample delivery time, and liver functional status [44]. On the other hand,miRNAs are stably present in the blood, making them suitable as biomarkers [45]. The limitation of our study included a small sample, so our results should be validated on larger samples.
In conclusion, Cairo University Fibrosis Index is more accurate than the existing brosis scores as it gives more indicative results for judging brosis.    Table 4. Diagnostic power of single and combined candidate biomarkers