Dr. A I Ladu, Consultant Haematologist / Lecturer, College of Medical Science University of Maiduguri, Nigeria

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1 AMERICAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH 2017, Science Huβ, ISSN: X, doi: /ajsir Review of thepotential role of three serum biomarkers: Tissue inhibitor of matrix metalloproteinase1 (TIMP1), aminoterminal peptide of type 111 procollagen (P111NP) and hyaluronic acid (HA) in the evaluation of patients with liver fibrosis. Dr. A I Ladu, Consultant Haematologist / Lecturer, College of Medical Science University of Maiduguri, Nigeria ABSTRACT Liver fibrosis is a major complication of alcoholic liver disease (ALD). It is also a side effect of long time therapy with methotrexate (MTX), a hepatotoxic agent used in individuals with psoriasis. It is important to have accurate information about the degree of fibrosis, considering that the condition has no specific symptoms and signs, and can ultimately progress to cirrhosis and hepatocellular carcinoma.existing guidelines and recommendation indicates that staging of liver fibrosis is the most important factor in defining prognosis and subsequent management. Liver biopsy has historically been considered the gold standard for assessing the stage of liver fibrosis. However, liver biopsy is not suitable as a screening test, given that of the millions of people affected worldwide by chronic liver disease, only 2040% will go on to develop liver fibrosis in their life time. In addition, it is an invasive procedure, associated with complication such as pain and haemorrhage. Given the drawbacks of liver biopsy, serum markers of liver fibrosis are been investigated, to determine if they can be used as potential biomarkers of fibrosis. The advantages of serum markers include, less invasiveness, more cost effective and can be used for ongoing monitoring of fibrosis. This review examined the potential role of three biomarkers, namely tissue inhibitor of matrix metalloproteinase1 (TIMP1), aminoterminal peptide of type 111 procollagen (P111NP) and hyaluronic acid (HA), singly and in combination (as panels) in the diagnosis and management of patients with liver fibrosis resulting from ALD and chronic treatment with MTX. Overall the performance of the panel markers in detecting cirrhosis and differentiating between early and advanced stages of liver fibrosis was significant compared with the single markers. However this conclusion need to be considered with caution given the multiple confounding factors that may have affected the outcomes of these studies. This include, limited studies evaluating the performance of these markers in ALD, and less so in MTX induced liver fibrosis, few participants, varying inclusion criteria and cutoff points. INTRODUCTION Liver fibrosis can be defined as the common response of the liver to injury whether the injury is caused by viruses, alcohol or fat deposition (1). The importance of early identification of liver fibrosis cannot be overemphasized given that the disease can progress to cirrhosis and hepatocellular carcinomas (1,2).The pathogenic mechanism involved in liver fibrosis can be divided into two stages; In the acute stage, there is recruitment of variety of cells at the sites of injury, such as immune cells, inflammatory cells and secretion of extra cellular matrix proteins; In the chronic stage, there is accumulation of excess fibrous tissue with distortion of the normal liver parenchyma and impaired function. Ultimately, septae and nodules are formed giving rise to a cirrhotic architecture (3). Liver fibrosis is a major complication of alcoholic liver disease (ALD). It is also a side effect of long time therapy with methotrexate (MTX), a hepatotoxic agent used in individuals with psoriasis. It is important to have accurate information about the degree of fibrosis, considering that, the condition have no specific symptoms and signs; this will assist the Clinician in the assessment of severity and prognosis of the disease. It will also help in the management and monitoring of response to treatment (4).

2 Am. J. Sci. Ind. Res., 2017, 8(2):1621 Existing guidelines and recommendation indicates that staging of liver fibrosis is the most important factor in defining prognosis and subsequent management. There are several methods for staging liver fibrosis, amongst them, the METAVIR score is a widely used scoring method, and classified fibrosis into five stages as, no fibrosis (F0); portal fibrosis without septa (F1); portal fibrosis with few septa (F2); septal fibrosis without cirrhosis (F3) and cirrhosis (F4) (5). In this regard, liver biopsy has historically been considered the gold standard for assessing the stage of liver fibrosis. However, liver biopsy is not suitable as a screening test, given that of the millions of people affected worldwide by chronic liver disease, only 2040% will go on to develop liver fibrosis in their life time (5). In addition, it is an invasive procedure, associated with complication such as pain and haemorrhage. Other limiting factors to its diagnostic abilities include sampling errors, length of biopsy, observer variability (5). Given the drawbacks of liver biopsy, serum markers of liver fibrosis are being investigated, to determine if they can be used as potential biomarkers of fibrosis. The diagnostic performance of a serum marker is evaluated by using the receiveroperatingcharacteristic (ROC) curves and calculating the area under the ROC curve (AUROC), this combines the sensitivity and specificity of a particular serum marker to determine the diagnosis of a specific stage of fibrosis. For a particular test, sensitivity and specificity are usually calculated by computing the correct diagnosis of advanced fibrosis versus minimal fibrosis (3). The advantages of serum markers include, less invasiveness, more cost effective, can be used for on going calibration and monitoring of fibrosis (4). In the light of these advantages of serum markers, this review sought to determine the potential role of three biomarkers, namely tissue inhibitor of matrix metalloproteinase1 (TIMP1), aminoterminal peptide of type 111 procollagen (P111NP) and hyaluronic acid (HA), singly and in combination, in the diagnosis and management of patients with liver fibrosis resulting from ALD and chronic treatment with MTX. SINGLE MARKERS Hyaluronic acid (HA): Hyaluronic acid is an extracellular matrix protein found in several tissues. In various inflammatory conditions of the liver, its synthesis increases in parallel with increasing collagen synthesis, thereby resulting in raised serum levels (6). It is the most frequently measured amongst the single markers. Several studies have evaluated its performance in patients with ALD and liver fibrosis (4). The diagnostic performance of HA in detecting cirrhosis using the AUROC determined by three studies showed discrepancies; Stickel, et al reported AUROC of 0.78 (7), Nguyenkhac 0.80 (8) and Naveau, et al., 0.93 (9). In their study, Plevris, et al., reported a sensitivity and specificity of 87% and 89% respectively and also noted that the serum HA increases in proportion to the severity of cirrhosis (10). This varied results could be attributed to the differences in the number of subjects used in these studies (Tables A). In terms of detecting fibrosis at each of the various stages, the performance of HA was not significant; in three studies, the AUROCs determined for stages F012 versus F34 was 0.76 (7), 0.83 (8) and 0.69 (11). These findings suggest that HA is poor in differentiating early fibrosis from advanced stages, and therefore not a good marker for screening in early disease. Comparison of diagnostic performance of HA in nonalcoholic related liver fibrosis revealed HA to be the best class 1 biomarker and was associated with AUROCs of 0.92 (6), 0.98 (12) and 0.97 (13) in three studies. This raises the question of whether aetiological factor influences the performance of a particular biomarker. Experiment in animal models has suggested that alcohol influences communication between liver cells thereby altering the clearance of HA; furthermore, alcohol causes dysfunction of the hepatic sinusoidal endothelial cells (4). Amino terminal peptide of type 111 procollagen (P111NP): This is an important component of the connective tissue. During hepatogenesis, the basement membrane concentration increases, and this is closely followed by an increase in its serum level (3). There are few studies on its use as a single marker in subjects with ALD. In two small studies, the performance of P111NP in differentiating between fibrosis(f14) and nonfibrosis cases(f0) was high; Gabrielli, et al., (14) reported a sensitivity and specificity of 90% and 59% respectively while Li, et al., (15) reported an AUROC of In contrast, the performance of P111NP in differentiating early fibrosis(f12) from advanced stages(f34) was not significant; Gabrielli, et al., (14) reported a sensitivity and specificity of 71% and 56%, while Lieber, et al., (11) reported an AUROC of 0.67(Table A). Review of the literature indicated that P111NP was the single most evaluated markers in liver fibrosis 17

3 Am. J. Sci. Ind. Res., 2017, 8(2):1621 following treatment with methotrexate (MTX). Risteli, et al., (16) evaluated 24 patients on methotrexate using liver biopsies and serum assay of P111NP, and found that 90% of patients with serum level above normal had liver fibrosis and cirrhosis. Similarly, Oogarah, et al., (17) reported highest level of serum P111NP in patients with hepatic fibrosis and cirrhosis, further screening of patients with high P111NP and normal liver histology using electron microscope revealed presence of fibrosis in the space of Disse, they concluded that MTX causes subtle liver damage in a majority of treated patients. Mitchell, et al., (18) corroborated this finding in their study (Table B). In a clinical audit, Khan, Subedi, and Chowdhury., (19), assessed guidelines produced by Manchester on P111NP monitoring in patients receiving MTX, in order to determine if the agreed level correlates with histological severity; they concluded that a higher cumulative dose of MTX correlates significantly with maximum levels of P111NP. Currently, the British, EU and German guidelines recommends P111NP as a baseline test in psoriasis patients receiving treatment with MTX (20). Tissue inhibitor of matrix metalloproteinase 1 (TIMP1):Tissue inhibitor of matrix metalloproteinase 1 are secreted proteins that interacts with matrix metalloprotein (MMP) thereby regulating their activities and function. Levels of this protein have been shown to be raised in chronic liver disease (3). In two studies that evaluated its performance in ALD, Li, et al., (15) reported a high AUROC of 0.98 in the detection of any fibrosis; However, this result needs careful interpretation giving the small number of subjects (Table A); Lieber, et al., (11) in their evaluation of 247 subjects reported the AUROC to be 0.68 in discriminating between stages F012 vs 34. Panelmarkers: There was paucity of studies that evaluated the test performance of the three markers as a panel in alcoholic liver disease and none in MTX induced liver fibrosis; One of such combination of the three markers is the Enhanced liver fibrosis test (ELF), first developed by a group of European researchers in 1997, their work led to the identification of a combination of direct markers (HA, PIIINP & TIMP1) that have been validated. Rosenberg, et al., (21) in a large international multicentre cohort study, to assess the performance of serum markers in detecting significant histologic fibrosis, evaluated a subset of 64 with alcoholic liver disease using the ELF test. The AUROC obtained was 0.94 (p<0.0001; 95% CI ) in differentiating F012 vs F34. In detection of fibrosis stages 3 or 4 using a threshold of 0.87, the sensitivity and NPV were both 100% (specificity 17%, PPV 75%), at a lower cutoff value of 0.43 for the prediction of fibrosis, the sensitivity and specificity was 93% and 100% respectively, PPV was 100%, and NPV 86% (Table C). In the only study that compared the performance of the markers singly and as a panel in ALD, Lieber, et al.,( 11 ) reported a lower AUROC for the individual markers (HA 0.69, TIMP1 0.68, P111NP 0.67) while the overall performance of the panel in predicting different stages of fibrosis showed a sensitivity of 76%, specificity of 68% (PPV 53%, NPV 86%). Overall the performance of the panel markers in detecting cirrhosis and differentiating between early and advanced stages of liver fibrosis was significant compared with the single markers. However this conclusion need to be considered with caution given the multiple confounding factors that may have affected the outcomes of these studies. This include, limited studies evaluating the performance of these markers in ALD, and less so in MTX induced liver fibrosis, few participants, varying inclusion criteria and cutoff points.the importance of early detection of fibrosis is that it provides the opportunity in planning of effective management and monitoring of these groups of patients; therefore the combined markers undoubtedly will be a useful test for both the patients and Clinicians. 18

4 Am. J. Sci. Ind. Res., 2017, 8(2):1621 Table A: Diagnostic performance of single markers (Table partly reproduced from: Parkes et al.,2012. Comparative Hepatology) Degree of fibrosis tested Study Subjects AUROC SEN (%) SPE (%) PPV (%) NPV (%) HYALURONIC ACID Cirrhosis Plevris(2000) Stickel(2003) Naveau(2005) Nguyen Khac(2008) F012 vs 34 Stickel(2003) Nguyen Khac(2008) Lieber(2008) F01vs234 Naveau(2005) Nguyen Khac(2008) P111NP F01vs34 Gabrielli (1989) Lieber(2005) F0vsF16 Gabrielli(1989) Li(1994) TIMP1 F012vs34 Lieber(2008) Any fibrosis Li(1994) 0.96 AUROC= Area under the receiver operating curve. SEN Sensitivity SPE Specificity PPV Positive predictive value NPVnegative predictive value no record 19

5 Am. J. Sci. Ind. Res., 2017, 8(2):1621 Table B : Summaries of studies evaluating serum PIIINP in Psoriasis patients on long term methotrexate treatment. Author/date of studies No of patients Recruitment details % abnormal liver histology Comments Ristelli (1989) 24 Consecutive,Prospective 33(cirrhosis + fibrosis) Oogarah (1995) 22 Consecutive,Prospective 82 (Electron microscope) P111NP can be used as a valuable marker of fibrosis in patients MTX causes subtle liver damage in the majority of treated patients Mitchell (1990) 51 Consecutive,Prospective Highest P111NP value found in groups with cirrhosis and fibrosis Khan ( ) 65 Retrospective High cumulative dose of MTX corr.significantly with high mean P111NP MTX methotrexate no record Table C: Diagnostic performance of panel markers (Table partly reproduced from:parkes et al.,2012. Comparative Hepatology) Study Fibrosis grade No of AUROC cutoff %SEN. %SPE. PPV NPV Subjects Rosenberg F012vs (2004) Lieber F012 vs (2008) Mild Fibrosis AUROC= Area under the receiver operating curve. SEN Sensitivity SPE Specificity PPV Positive predictive value NPVnegative predictive value 20

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