Serum YKL-40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and mildly elevated ALT

Serum YKL-40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and... Purpose YKL-40 is a chitinase-like protein expressed in multiple tissues including liver and is reported as a fibrosis marker. This study aimed to determine whether YKL-40 could serve as a diagnostic marker for the assessment of liver fibrosis in chronic hepatitis B patients with normal and mildly elevated ALT. Methods Six hundred and eighty-five patients with chronic hepatitis B infection were enrolled in this study from October 2013 to March 2016. All patients underwent liver biopsy and then staged based on Ishak histological system. Serum YKL- 40 levels were measured by Human Magnetic Luminex Assays. Results Among chronic hepatitis B patients with normal and mildly elevated ALT, almost more than 30% of patients have significant liver fibrosis. Serum YKL-40 levels increased significantly in parallel with the progression of fibrosis in patients with ALT less than two times the upper limit of normal range (P < 0.0001). Multivariate analysis revealed that serum YKL- 40, hyaluronic acid, PLT, and AST were independently associated with significant fibrosis. We established a novel YKL- 40-based fibrosis model for patients with ALT less than two times the upper limit of normal range (ULN). YKL-40 model was superior to APRI, FIB-4, Forns’ index, and Hui model for diagnosis of significant fibrosis in patients with ALT < 2ULN, with AUROCs of 0.786 [95% confidence interval (CI) 0.726–0.846] in the training group, 0.831 (95%CI 0.752–0.910) in the validation group and 0.801 (95%CI 0.753–0.849) in the entire cohort. Conclusion Serum YKL-40 is a feasible biomarker of liver fibrosis in chronic hepatitis B patients. YKL-40 model was superior to APRI, FIB-4, Forns’ index and Hui model for diagnosis of significant fibrosis in patients with normal and mildly elevated ALT. Keywords YKL-40 · Chronic hepatitis B · Liver fibrosis Abbreviations HBsAg Hepatitis B surface antigen HBV Hepatitis B virus HBeAg Hepatitis B e antigen CHB Chronic hepatitis B BMI Body mass index ALT Alanine transaminase AST Aspartate transaminase Electronic supplementary material The online version of this ALP Alkaline phosphatase article (https ://doi.org/10.1007/s1501 0-018-1136-2) contains supplementary material, which is available to authorized users. * Guiqiang Wang Department of Infectious Disease, Center for Liver Disease, John131212@126.com Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China Linlin Yan ylinlin3@163.com The Department of Dermatology, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan, Yongqiong Deng China dengyongqiong1@126.com The Collaborative Innovation Center for Diagnosis Jiyuan Zhou and Treatment of Infectious Diseases, Zhejiang University, zhoujiyuan198703@163.com Hangzhou, Zhejiang, China Hong Zhao Peking University International Hospital, Beijing, China minmin2001@126.com Vol.:(0123456789) 1 3 386 L. Yan et al. GGT Gamma-glutamyl transpeptidase laboratory tests have been developed as surrogates to assess TBil Total bilirubin liver fibrosis, such as aspartate aminotransferase–platelet PT Prothrombin time index (APRI), fibrosis index based on the four factors (FIB- PLT Platelet counts 4), Forns’ index [9], and Hui model [10]. Gao et al. [11] HA Hyaluronic acid had reported a noninvasive model, consisting of aspartate LN Laminin transaminase (AST), HBsAg, platelet, and albumin, to pre- PIIINP N-terminal peptide of type III procollagen dict significant liver histology change [necroinflammatory YKL-40 Chitinase 3-like-1 activity grade (G) ≥ 2 or fibrosis stage ( S) ≥ 2] in HBeAg- sCD163 Soluble CD163 positive CHB with ALT ≤ 2ULN. Gao’s model had an area MMP Matrix metalloproteinase under the receiver operating characteristic curve of 0.868, TIMP-1 Tissue inhibitor of metalloproteinase 1 which was significantly higher than APRI and FIB-4. How - ULN Upper limit of normal ever, there is no noninvasive method to predict significant ROC Receiver operating characteristic curve fibrosis in CHB patients with ALT < 2ULN regardless of AUROC Area under the receiver operating characteristic HBeAg status and HBV DNA levels. curve YKL-40 (chitinase-3-like-1, or human cartilage glycopro- SD Standard deviation tein-39) is a member of the mammalian chitinase family [12] CI Confidence interval and is secreted by a variety of cells, including neutrophils, macrophages, and vascular smooth muscle cells [13]. YKL- 40 is thought to be involved in remodeling of the extracel- Introduction lular matrix and in inflammatory processes [14]. YKL-40, as the growth factor for fibroblasts and chemoattractant for Chronic hepatitis B (CHB) infection remains a major global endothelial cells, is also believed to modulate angiogenesis health burden; approximately, 350–400 million individuals during tissue damage [15, 16]. Recently, YKL-40 mRNA were infected [1]. The burden of chronic hepatitis B infec- expression was found in human liver [17], and serum YKL- tion is serious in China, with an estimated 120 million peo- 40 levels were associated with liver fibrosis in patients with ple infected, and 0.3 million deaths annually [2]. In China, a chronic liver disease [18]. Immunohistochemical studies proportion of CHB patients are asymptomatic accompanied have shown that YKL-40 is expressed in fibrotic areas of the by normal and mildly elevated alanine transaminase (ALT, liver [17, 19]. Based on these supporting evidences, serum ALT levels are less than two times the upper limit of nor- YKL-40 has been evaluated as a noninvasive marker of mal). The guidelines of American Association for the Study fibrotic liver diseases, including alcoholic liver disease [20 ], of Liver Diseases (AASLD) and Asian Pacific Association non-alcoholic fatty liver disease [21] and chronic hepatitis for the Study of the Liver for the management of CHB C-induced liver fibrosis [22, 23]. Therefore, we recently pro- recommend antiviral treatment when ALT levels are two posed a hypothesis that serum YKL-40 may be a potential times the upper limit of normal (ULN), and monitoring or biomarker for differentiating significant fibrosis in chronic performing liver biopsy (especially for patients > 40 years) hepatitis B patients with normal and mildly elevated ALT. to assess if significant histologic disease is present when In this study, we identified the proportion of significant ALT levels are less than two times the upper limit of nor- fibrosis in CHB patients with normal and mildly elevated mal (ULN) [3, 4]. However, CHB patients with normal ALT. We measured the serum levels of YKL-40 and com- and mildly elevated ALT levels may not have healthy liv- pared them with fibrosis stages to evaluate the feasibility ers. Several studies indicated that moderate inflammation of YKL-40 as a biomarker of liver fibrosis in patients with and/or advanced fibrosis was present in 28–37% of CHB normal and mildly elevated ALT levels. patients who had persistently normal ALT [5–7]. These stud- ies suggested that CHB patients with normal ALT might have histologically significant disease, an indication for Patients and methods antiviral treatment. Use of ALT without resorting to liver biopsy may miss a certain proportion of patients with histo- Patients logically significant disease who may benefit from antiviral therapy. Liver biopsy remains the gold standard for assess- A total of 685 patients with chronic HBV infection from 24 ing liver fibrosis in CHB patients. However, liver biopsy hospitals located in mainland China were enrolled in this has several limitations including invasiveness, risk of com- study between October 2013 and March 2016. Of which, plications, sampling error, and cost [8], which limited its 460 patients have ALT levels less than two times the upper application in assessing and dynamic monitoring of liver limit of normal range (ULN), and they were randomly fibrosis. Currently, multiple noninvasive methods based on divided into a training group (n = 307) and a validation 1 3 Serum YKL‑40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and… 387 group (n = 153). They all underwent liver biopsies. Inclu- variables, the differences between the groups were analyzed sion and exclusion criteria were described previously [24]. using Student t test and Mann–Whitney U test, respectively. All patients provided written informed consent for research For categorical variables, Chi-square test was used to com- use of their clinical data and specimens. This study was pare the differences in proportions. Spearman’s rank test was approved by the Ethics Committee of Peking University First used to analyze the correlations between different variables Hospital. The detailed protocol for the clinical trial was reg- and fibrosis stages. We performed multivariate backward istered at clinicaltrials.gov (NCT01962155) and chictr.org logistic regression analysis to determine the independent (ChiCTR-DDT-13003724). variables of significant fibrosis. Receiver operating charac- teristic curve (ROC) was used to assess the performance Histological staging of noninvasive models for staging significant fibrosis. The diagnostic performance of different variables was evaluated Ultrasonography-guided liver biopsies with a minimal based on the area under the receiver operating characteristic length of 20 mm (at least 11 portal tracts) were routinely curve (AUROC). SPSS 16.0 software (SPSS, Inc., Chicago, performed at each hospital according to a standardized pro- IL, USA) was used for statistical analyses. P < 0.05 were tocol after receiving the patient’s written informed consent. considered statistically significant. Pathological interpretations were conducted in the Depart- ment of Pathology at You An Hospital affiliated to the Capi- tal Medical University. The histopathological examination rules were previously reported [24]. Fibrosis stages were Results assessed according to Ishak criteria [25]. Significant fibrosis was defined as F3. Patient’s characteristics Examination of serum markers A total of 685 patients were enrolled in this study; seven patients were excluded because of unqualified liver tissue. The biochemical and hematological parameters were rou- The remaining 678 patients with chronic HBV infection tinely detected by standard methods in local hospitals. were analyzed, of which 460 patients with ALT less than two 1 8 Serum HBV DNA (range 2.0 × 10 –1.7 × 10  IU/ml) was times the upper limit of normal range (ULN). The baseline measured by the COBAS AmpliPrep/COBAS TaqMan characteristics of the study patients are shown in Table 1. (Roche Diagnostics, Basel, Switzerland). Serum HBsAg There were no significant distributional differences in fibro- (range of 20–52,000 IU/ml) was quantified using the Roche sis stages between the group of patients with ALT ≥ 2 × ULN Elecsys HBsAg II assay (Roche Diagnostics, Penzberg, and the group of patients with ALT < 2 × ULN (P = 0.312, Germany). The serum levels of YKL-40 were determined Table 1). This result indicated the presence of significant using Human Magnetic Luminex® Assays (LXSAHM-08, or more severe fibrosis in patients with ALT < 2 × ULN, R&D Systems, Inc, Minneapolis, MN, USA) according patients who do not meet the treatment criteria recom- to the manufacturer’s instructions. The serum concentra- mended by AASLD guideline. tions of hyaluronic acid (range of 2–200  μg/L), laminin (5–900 μg/L), were measured using a chemiluminescence immunoassay kit (Yuande Bio-Medical Engineering Co., ALT was not a perfect surrogate marker for liver Ltd, Beijing, China). histology Noninvasive fibrosis scores For patients with ALT < 2 × ULN, they were stratified (from G1 to G5) according to the status of HBeAg and the levels Noninvasive assessment of fibrosis, APRI, and FIB4 was cal- of HBV DNA, as shown in Table 1. In patients with normal culated according to the following formulae: APRI = [(AST/ ALT, differences in the proportion of significant fibrosis ULN)/platelet(× 10 /L)] × 100; FIB4 = (age × AST)/[plate- were statistically significant (P = 0.015, Fig. 1a). Overall, 9 1/2 let(× 10 /L) × ALT ]. Forns’ index [9] and Hui model [10] more than 30% of patients had significant fibrosis, besides were obtained from reported research. G1 (immuno-tolerant phase) with 17.8% incidence of sig- nificant fibrosis. Similar results were obtained in patients Statistical analysis with mildly elevated ALT (P <0.0001, Fig. 1b). Regarding the incidence of significant fibrosis between patients with Quantitative variables were expressed as mean ± standard normal ALT and patients with mildly elevated ALT, there deviation (SD) and categorical variables were expressed were no significant differences (data not shown). This result as proportions. For normally and non-normally distributed 1 3 388 L. Yan et al. Table 1 Patients’ characteristics ALT ≥ 2 × ULN (n = 218) ALT < 2 × ULN (n = 460) P value Age (median, ≥ 40 years %) 36, 77 (35.3%) 38, 205 (44.6%) 0.024 Gender (male %) 184 (84.4%) 345 (75.0%) 0.006 BMI (median, ≥ 24 kg/m  %) 23.3, 76 (34.9%) 23.0, 165 (35.9%) 0.864 HBsAg (log IU/mL) 3.59 ± 0.77 3.56 ± 0.88 0.409 AST (U/L) 116.55 ± 109.99 35.50 ± 17.84 < 0.001 ALP (U/L) 91.64 ± 29.93 77.31 ± 26.07 < 0.001 GGT (U/L) 82.62 ± 69.51 41.50 ± 47.42 < 0.001 Albumin (g/L) 43.67 ± 5.85 44.48 ± 5.26 0.002 TBil (µmol/L) 18.30 ± 15.33 16.94 ± 22.78 0.017 PT (s) 12.94 ± 1.51 12.56 ± 1.49 0.001 PLT (× 10 /L) 170.64 ± 52.76 172.33 ± 59.08 0.635 Hyaluronic acid (ug/L) 149.05 ± 102.82 115.26 ± 71.14 < 0.001 Laminin (ug/L) 179.54 ± 302.39 84.24 ± 177.79 < 0.001 PIIINP (ug/L) 5.65 ± 11.42 3.65 ± 5.04 < 0.001 Collagen IV (pg/mL) 1120.00 ± 628.19 896.98 ± 540.96 < 0.001 pg/mL) 4.46 ± 0.38 4.47 ± 0.38 0.718 YKL-40 (log sCD163(log pg/mL) 6.20 ± 0.36 6.01 ± 0.33 < 0.001 MMP-1 (log pg/mL) 3.47 ± 0.31 3.48 ± 0.32 0.566 MMP-2 (log pg/mL) 5.28 ± 0.10 5.26 ± 0.10 0.058 MMP-3 (log pg/mL) 4.17 ± 0.25 4.17 ± 0.26 0.624 MMP-9 (log pg/mL) 4.85 ± 0.41 4.87 ± 0.45 0.770 TIMP-1 (log pg/mL) 5.08 ± 0.12 5.06 ± 0.13 0.018 HBeAg status/HBV DNA(IU/mL) (n %) 0.020  G1 e + , HBV DNA ≥ 2 × 10 86 (39.4%) 128(27.6)  G2 e + , 20,000 ≤ HBV DNA < 2 × 10 54 (24.8) 116 (25.4)  G3 e + , HBV DNA < 20,000 9 (4.1) 25 (5.7)  G4 e − , HBV DNA ≥ 2000 59 (27.1) 152 (32.8)  G5 e − , HBV DNA < 2000 10 (4.6) 39 (8.5) Fibrosis stages (n %) 0.312  F0–2 128 (58.7%) 291 (63.2%)  F3 48 (22.0%) 85 (18.5%)  F4 33 (15.1%) 68 (14.8%)  F5–6 9 (4.1%) 16 (3.5%) Data presented as mean ± SD or no. (%) BMI body mass index, HBsAg hepatitis B surface antigen, AST aspartate transaminase, ALP alkaline phosphatase, GGT gamma-glutamyl transpeptidase, TBil total bilirubin, PT prothrombin time, PLT plate- let counts, PIIINP N-terminal peptide of type III procollagen, YKL-40 chitinase 3-like-1, sCD163 soluble CD163, MMP matrix metalloproteinase, TIMP-1 tissue inhibitor of metalloproteinase 1, HBeAg hepatitis B e antigen, HBV hepatitis B virus, ULN upper limit of normal suggested that ALT levels and fibrosis are not always con- showing significant difference between fibrosis stages (F01 sistent in CHB patients. vs F2–F56, F2 vs F3–F56) (P < 0.0001, Fig. 2a). In patients with ALT < 2 × ULN, similar results were obtained as in the Serum YKL‑40 levels increased with the progression total patients (Fig. 2b). In addition, serum YKL-40 levels of fibrosis were positively correlated with hyaluronic acid, laminin, PIIINP, Collagen, and AST, while they were negatively cor- Serum YKL-40 levels were measured to assess the feasibil- related with platelet count (Supplementary Table 1). ity of YKL-40 as a biomarker of fibrosis in CHB patients. Serum levels of YKL-40 throughout different fibrosis stages are shown in Fig. 2. In the total patients, serum YKL-40 levels increased in parallel with the progression of fibrosis, 1 3 44.0% Serum YKL‑40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and… 389 100 100 F0-2 F3 80 80 58.6% 55.6% 60 60 47.8% 43.8% 42.9% 38.3% 40 40 31.0% 19.3% 17.8% 20 20 0 0 G1(n=45) G2(n=46) G3(n=9)G4(n=71) G5(n=25) G1(n=83)G2(n=70)G3(n=16)G4(n=81)G5(n=14) Fig. 1 Proportion of patients with significant fibrosis in the group of G1–G5 in chronic hepatitis B patients. Patients with a normal ALT and b mildly elevated ALT *** **** *** A **** B ** **** *** 6 **** *** ** **** **** 4 4 3 3 2 2 F01F2F3F4F56 F01F2F3F4F56 Fig. 2 Associations between serum YKL-40 levels and liver fibrosis. a YKL-40 in total patients, b YKL-40 in patients with ALT < 2 × ULN. P < 0.0001 for all fibrosis stags. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 hyaluronic acid (HA), PLT, and AST were independent Development of YKL‑40‑based fibrosis model factors of significant fibrosis (Table  3). We performed in patients with ALT < 2 × ULN backward logistic regression analysis and established a novel YKL-40 based model for CHB patients with To determine the ability of YKL-40 to diagnose signifi- ALT < 2 ULN: cant fibrosis, all CHB patients with ALT < 2 × ULN were YKL -40 model = 0.032 × AST − 0.012 × PLT + 0.012 × divided into a training group and a validation group. There HA + 0.846 × log10 (YKL -40) − 4.752. was no statistical difference between training group and validation group about any parameters (Supplementary Diagnostic performance of YKL‑40 model Table 2). In the training group, univariate analysis found for significant fibrosis that serum YKL-40, hyaluronic acid, laminin, PIIINP, Col- lagen IV, sCD163, and MMP-2 were positively associ- YKL-40 model had an area of 0.786 (95%CI 0.726–0.846) ated with significant fibrosis (Table  2). PLT was inversely under the ROC curve in predicting significant fibrosis in associated with significant fibrosis (Table  2). Multivari- the training group, with 71.74% sensitivity, 72.85% speci- ate analysis revealed that YKL-40 [odd ratio (OR) 2.330, ficity, 61.68% PPV, and 80.88% NPV at the cut-off point 95% confidence interval (CI) 1.019–5.330, P = 0.045], 1 3 YKL-40 (log Pg/mL) Percentage (%) Percentage (%) YKL-40 (log Pg/mL) 10 390 L. Yan et al. Table 2 Univariate analysis of clinical parameters and biomarkers of − 0.56. It was superior to that of APRI [0.736 (95%CI with significant fibrosis in the training group (n = 307) 0.670–0.803)], FIB-4 [0.735 (95%CI 0.669–0.801)], Forns’ index [0.753 (95%CI 0.688–0.817)], and Hui model [0.734 F0–2 (n = 194) F ≥ 3 (n = 113) P value (95%CI 0.667–0.801)] (Fig. 3a, Table 4). The area under Age (≥ 40 years %) 37.54 ± 10.12 42.54 ± 10.92 < 0.0001 the ROC curve of YKL-40 model in the validation group Gender (male %) 141 (72.68%) 83 (73.45%) > 0.9999 was 0.831 (95%CI 0.752–0.910), with 71.79% sensitivity, BMI (≥ 24 kg/m  %) 22.88 ± 2.95 23.50 ± 2.74 0.096 85.33% specificity, 71.79% PPV, and 85.33% NPV at the cut- HBsAg (log IU/ 3.71 ± 0.88 3.28 ± 0.72 < 0.0001 off point of − 0.33, which was also higher than that of APRI, mL) FIB-4, Forns’ index, and Hui model (Fig. 3b, Table 4). In the ALT (U/L) 42.16 ± 17.61 44.55 ± 16.04 0.258 entire cohort, YKL-40 model had an area of 0.801 (95%CI AST (U/L) 32.52 ± 15.72 42.36 ± 21.75 < 0.0001 0.753–0.849) under the ROC curve in predicting significant ALP (U/L) 72.61 ± 19.21 85.90 ± 33.97 0.002 fibrosis (data not shown) . GGT (U/L) 35.60 ± 45.56 54.91 ± 47.21 < 0.0001 Albumin (g/L) 45.06 ± 4.53 43.58 ± 6.78 0.003 TBil (µmol/L) 15.45 ± 17.01 20.84 ± 38.75 0.002 Discussion PT (s) 12.32 ± 1.27 12.85 ± 1.43 0.002 PLT (× 10 /L) 187.06 ± 49.56 145.23 ± 53.21 < 0.0001 Serum ALT is commonly used to assess liver histology activ- Hyaluronic acid 93.89 ± 41.27 151.04 ± 96.27 < 0.0001 ity and to guide antiviral therapy in patients with liver dis- (ug/L) ease. However, results of the present study showed that, ALT Laminin (ug/L) 48.21 ± 91.38 126.52 ± 204.21 < 0.0001 levels and fibrosis are not always consistent in CHB patients. PIIINP (ug/L) 3.12 ± 6.14 4.53 ± 4.56 < 0.0001 We observed that a high proportion (> 30%) of CHB patients Collagen IV (pg/mL) 782.48 ± 387.26 1037.84 ± 584.14 < 0.0001 with normal and mildly elevated (1-2ULN) ALT have sig- YKL-40 (log10 pg/ 4.39 ± 0.35 4.62 ± 0.40 < 0.0001 mL) nificant fibrosis regardless of the state of HBeAg and the SCD163 (log10 pg/ 5.94 ± 0.32 6.12 ± 0.32 < 0.0001 levels of HBV DNA (Fig. 1, G2–G5). Even for patients in mL) the immunO-tolerant phase (Fig. 1, G1), 17.8 and 19.3%, MMP-1 (log10 pg/ 3.49 ± 0.32 3.46 ± 0.31 0.501 respectively, have significant fibrosis. Our present findings mL) are consistent with the previous reports that patients with MMP-2 (log10 pg/ 5.24 ± 0.10 5.28 ± 0.10 0.001 chronic HBV infection can display normal and mildly ele- mL) vated ALT levels despite significant histological injury [6 , MMP-3 (log10 pg/ 4.17 ± 0.27 4.18 ± 0.26 0.740 7, 26]. A meta-analysis [26] concluded that approximately mL) one-fifth of CHB patients with ALT ≤ 40 IU/L may have MMP-9 (log10 pg/ 4.86 ± 0.46 4.86 ± 0.40 0.752 mL) significant hepatic fibrosis. Lai et al. [7 ] found that 37% of TIMP-1 (log10 pg/ 5.05 ± 0.13 5.08 ± 0.13 0.134 CHB patients with persistently normal ALT had significant mL) fibrosis and inflammation. According to current guidelines, antiviral therapy should be initiated immediately for patients BMI body mass index, HBsAg hepatitis B surface antigen, ALT ala- with significant fibrosis [ 3, 4]. Our results confirmed that nine transaminase, AST aspartate transaminase, ALP alkaline phos- phatase, GGT gamma-glutamyl transpeptidase, TBil total bilirubin, ALT was not a perfect surrogate marker for liver histology, PT prothrombin time, PLT platelet counts, PIIINP N-terminal peptide because ALT failed to identify many patients who might of type III procollagen, YKL-40 chitinase 3-like-1, sCD163 soluble benefit from antiviral therapy. The “gray zone” patients were CD163, MMP matrix metalloproteinase, TIMP-1 tissue inhibitor of defined as those patients with normal and mildly elevated metalloproteinase 1 ALT. Because of the high proportion of significant liver disease in the “gray zone” patients, it is highly important Table 3 Multivariate logistic regression analysis of independent pre- to assess liver fibrosis. Liver biopsy, a gold standard for dictors for significant fibrosis in the training group (n = 307) assessing liver fibrosis, is not suitable for regular applica- tions due to the limitations of invasive, finite, complications, Coefficient OR 95%CI P value and cost [8]. Noninvasive models such as APRI and FIB-4 AST (U/L) 0.032 1.033 1.009–1.057 0.007 using biochemical laboratory index have been proposed to PLT (× 10 /L) − 0.012 0.988 0.982–0.995 < 0.0001 replace liver biopsy to assess liver fibrosis. Therefore, it is Hyaluronic acid (ug/L) 0.012 1.013 1.005–1.020 0.001 reasonable to evaluate “gray zone” patients based on such YKL-40 (log10 pg/ 0.846 2.330 1.019–5.330 0.045 noninvasive methods, and then to decide whether initiating mL) antiviral treatment or not. Constant − 4.758 0.009 – 0.018 Within the present study, we assessed the relationship YKL-40 model = 0.032 × AST − 0.012 × PLT + 0.012 × HA + 0.846 × l between serum markers, including YKL-40, hyaluronic og10 (YKL-40) − 4.752 1 3 Serum YKL‑40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and… 391 1.0 1.0 A B YKL40Model YKL40Model APRI APRI FIB-4 FIB-4 0.8 0.8 Hui model Hui model 0.6 0.6 0.4 0.4 0.2 0.2 Validation group Training group 0.0 0.0 0.00.2 0.40.6 0.81.0 0.00.2 0.40.6 0.81.0 1 - Specificity 1 - Specificity Fig. 3 Receiver operating characteristic curve (ROC) analysis show- ARPI, FIB4, Forns’ index and Hui model in the diagnosis of signif- ing the diagnostic performance of noninvasive models for significant icant fibrosis in CHB patients with ALT < 2ULN. a Training group fibrosis. Area under the ROC curves (AUROCs) of YKL-40 model, and b validation group Table 4 Receiver operating characteristics curve (ROC) analysis of noninvasive models for the diagnosis of significant fibrosis in CHB patients with ALT < 2ULN AUROC (95%CI) Cut-off value Sensitivity (%) Specificity (%) PPV (%) NPV (%) Training group  YKL-40 model 0.786 (0.726–0.846) − 0.56 71.74 72.85 61.68 80.88  APRI 0.736 (0.670–0.803) 0.76 42.39 93.38 79.60 72.68  FIB-4 0.735 (0.669–0.801) 1.33 50.00 88.08 71.88 74.30  Forns’ index 0.753 (0.688–0.817) 7.75 56.52 82.78 66.66 75.76  Hui model 0.734 (0.667–0.801) 0.12 69.57 70.20 58.72 79.11 Validation group  YKL-40 model 0.831 (0.752–0.910) − 0.33 71.79 85.33 71.79 85.33  APRI 0.762 (0.668–0.855) 0.50 71.79 73.33 58.33 83.33  FIB-4 0.743 (0.648–0.838) 1.14 79.49 64.00 53.45 85.72  Forns’ index 0.804 (0.723–0.886) 7.12 79.49 73.33 60.78 87.30  Hui model 0.771 (0.684–0.857) 0.08 87.18 54.67 50.00 89.13 acid, laminin, PIIINP, Collagen IV, sCD163 and metallo- fibrosis, a stage which represent an indication for antiviral proteinases, and liver fibrosis in patients with normal and therapy, from the “gray zone” patients. Our univariate analy- mildly elevated ALT. Of note, our results indicated that sis revealed that serum YKL-40, hyaluronic acid, laminin, serum YKl-40 levels significantly correlated with fibrosis PIIINP, Collagen IV, sCD163, and MMP-2 were associated stages as assessed by Ishak score. Serum levels of YKL-40 with significant fibrosis. However, multivariate analysis also increased in non-alcoholic fatty liver disease (NAFLD) showed that only YKL-40, hyaluronic acid and two labora- and chronic hepatitis C-induced liver fibrosis [21, 22, 27, tory parameters, PLT and AST, retained significance when 28]. It has been reported that YKL-40 is a growth factor combined with other clinical parameters. Series studies have for fibroblasts and is expressed in active liver fibrotic areas demonstrated that combination of multiple serum markers [29, 30]. In addition, the progression of fibrosis rate per could improve the sensitive, specific, and reproducible [31, year linearly correlates with the serum levels of YKL-40 32]. Based on our findings, a four-variable model including [23]. These observations further strengthen the possibility two serum fibrosis markers (log YKL-40, hyaluronic acid) that YKL-40 is involved in hepatic fibrogenesis in patients and two routinely laboratory tests (PLT, AST) was derived with HBV infection and is a useful biomarker for hepatic via backward logistic regression analysis to detect significant fibrosis. It is critical to discriminate patients with significant fibrosis. Hyaluronic acid is synthesized by stellate cells and 1 3 Sensitivity Sensitivity 392 L. Yan et al. is involved in fibrogenesis; it has been identified as one of ALT levels regardless of HBeAg status and HBV DNA the serum markers of liver fibrosis in non-alcoholic steato- levels. In CHB patients with ALT < 2ULN, serum YKL- hepatitis (NASH) and chronic hepatitis C [31–34]. Regard- 40 levels were independently associated with significant ing PLT, our finding is consistent with the previous stud- fibrosis and could be a feasible biomarker reflecting liver ies that found decreased platelet counts are associated with fibrosis. YKL-40 model was superior to existing scores more severe hepatic fibrosis [35, 36]. in diagnosing significant fibrosis in CHB patients with Identification of patients, who actually had significant normal and mildly elevated ALT. This finding offered a hepatic fibrosis, diagnosed as “none treatment required” promising method to identify those “gray zone” patients according to ALT levels, is very important. Significant who may benefit from antiviral therapy. fibrosis is an important endpoint of clinical antiviral ther - Acknowledgements We thank the members of China HepB-Related apy [37, 38]. The aim of this study was to develop an Fibrosis Assessment Research Group for assisting patient inclusion accurate noninvasive fibrosis model applied to “gray zone” and data acquisition. CHB patients. Over the past 20 years, various noninvasive fibrosis models have emerged. The most widely used two Funding This study was supported by China Mega-Project for Infec- scores, APRI and FIB-4, and Forns’ index, are based on tious Diseases (Grant Numbers 2013ZX10002005, 2012ZX10002006, 2013ZX10002004, 2012ZX10005005), Project of Beijing Science and patients with hepatitis C infection [39]. APRI and FIB-4 Technology Committee (Grant Number D121100003912002). have been validated and recommended for evaluation of liver fibrosis in CHB patients [39, 40]. Hui model is based Compliance with ethical standards on patients with HBV [10] while lacking of clinical valida- tion (Supplementary Table 3). Furthermore, the diagnostic Conflict of interest On behalf of all authors, the corresponding author performances of the above models for fibrosis assessment states that there is no conflict of interest. in CHB patients with normal and mildly elevated ALT Ethical approval This study was approved by the local ethics commit- have not been validated in large cohorts. A recent analy- tee of Peking University First Hospital. sis of APRI and FIB-4 in 231 HBV-infected patients with normal and mildly elevated ALT founded limited diag- Open Access This article is distributed under the terms of the Crea- nostic value for significant fibrosis [41]. In this study, we tive Commons Attribution 4.0 International License (http://creat iveco developed a YKL-40 model in 460 CHB patients with mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- normal and mildly elevated ALT, and then, we compared tion, and reproduction in any medium, provided you give appropriate the performances of the five noninvasive models to diag- credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. nose significant fibrosis. For the identification of patients with significant fibrosis, the AUROCs for patients with ALT < 2ULN were 0.736 for APRI and 0.735 for FIB-4 in the training group, compared with 0.762 for APRI and References 0.743 for FIB-4 in the validation group, showing simi- lar performance as previous reported [41]. We found that 1. Sorrell MF, Belongia EA, Costa J, Gareen IF, Grem JL, Inadomi YKL-40 model produced the best performances compared JM, et al. 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J Hepatol. 1995;22:696–9. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infection Springer Journals

Serum YKL-40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and mildly elevated ALT

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Medicine & Public Health; Infectious Diseases; General Practice / Family Medicine; Internal Medicine
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10.1007/s15010-018-1136-2
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Abstract

Purpose YKL-40 is a chitinase-like protein expressed in multiple tissues including liver and is reported as a fibrosis marker. This study aimed to determine whether YKL-40 could serve as a diagnostic marker for the assessment of liver fibrosis in chronic hepatitis B patients with normal and mildly elevated ALT. Methods Six hundred and eighty-five patients with chronic hepatitis B infection were enrolled in this study from October 2013 to March 2016. All patients underwent liver biopsy and then staged based on Ishak histological system. Serum YKL- 40 levels were measured by Human Magnetic Luminex Assays. Results Among chronic hepatitis B patients with normal and mildly elevated ALT, almost more than 30% of patients have significant liver fibrosis. Serum YKL-40 levels increased significantly in parallel with the progression of fibrosis in patients with ALT less than two times the upper limit of normal range (P < 0.0001). Multivariate analysis revealed that serum YKL- 40, hyaluronic acid, PLT, and AST were independently associated with significant fibrosis. We established a novel YKL- 40-based fibrosis model for patients with ALT less than two times the upper limit of normal range (ULN). YKL-40 model was superior to APRI, FIB-4, Forns’ index, and Hui model for diagnosis of significant fibrosis in patients with ALT < 2ULN, with AUROCs of 0.786 [95% confidence interval (CI) 0.726–0.846] in the training group, 0.831 (95%CI 0.752–0.910) in the validation group and 0.801 (95%CI 0.753–0.849) in the entire cohort. Conclusion Serum YKL-40 is a feasible biomarker of liver fibrosis in chronic hepatitis B patients. YKL-40 model was superior to APRI, FIB-4, Forns’ index and Hui model for diagnosis of significant fibrosis in patients with normal and mildly elevated ALT. Keywords YKL-40 · Chronic hepatitis B · Liver fibrosis Abbreviations HBsAg Hepatitis B surface antigen HBV Hepatitis B virus HBeAg Hepatitis B e antigen CHB Chronic hepatitis B BMI Body mass index ALT Alanine transaminase AST Aspartate transaminase Electronic supplementary material The online version of this ALP Alkaline phosphatase article (https ://doi.org/10.1007/s1501 0-018-1136-2) contains supplementary material, which is available to authorized users. * Guiqiang Wang Department of Infectious Disease, Center for Liver Disease, John131212@126.com Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China Linlin Yan ylinlin3@163.com The Department of Dermatology, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan, Yongqiong Deng China dengyongqiong1@126.com The Collaborative Innovation Center for Diagnosis Jiyuan Zhou and Treatment of Infectious Diseases, Zhejiang University, zhoujiyuan198703@163.com Hangzhou, Zhejiang, China Hong Zhao Peking University International Hospital, Beijing, China minmin2001@126.com Vol.:(0123456789) 1 3 386 L. Yan et al. GGT Gamma-glutamyl transpeptidase laboratory tests have been developed as surrogates to assess TBil Total bilirubin liver fibrosis, such as aspartate aminotransferase–platelet PT Prothrombin time index (APRI), fibrosis index based on the four factors (FIB- PLT Platelet counts 4), Forns’ index [9], and Hui model [10]. Gao et al. [11] HA Hyaluronic acid had reported a noninvasive model, consisting of aspartate LN Laminin transaminase (AST), HBsAg, platelet, and albumin, to pre- PIIINP N-terminal peptide of type III procollagen dict significant liver histology change [necroinflammatory YKL-40 Chitinase 3-like-1 activity grade (G) ≥ 2 or fibrosis stage ( S) ≥ 2] in HBeAg- sCD163 Soluble CD163 positive CHB with ALT ≤ 2ULN. Gao’s model had an area MMP Matrix metalloproteinase under the receiver operating characteristic curve of 0.868, TIMP-1 Tissue inhibitor of metalloproteinase 1 which was significantly higher than APRI and FIB-4. How - ULN Upper limit of normal ever, there is no noninvasive method to predict significant ROC Receiver operating characteristic curve fibrosis in CHB patients with ALT < 2ULN regardless of AUROC Area under the receiver operating characteristic HBeAg status and HBV DNA levels. curve YKL-40 (chitinase-3-like-1, or human cartilage glycopro- SD Standard deviation tein-39) is a member of the mammalian chitinase family [12] CI Confidence interval and is secreted by a variety of cells, including neutrophils, macrophages, and vascular smooth muscle cells [13]. YKL- 40 is thought to be involved in remodeling of the extracel- Introduction lular matrix and in inflammatory processes [14]. YKL-40, as the growth factor for fibroblasts and chemoattractant for Chronic hepatitis B (CHB) infection remains a major global endothelial cells, is also believed to modulate angiogenesis health burden; approximately, 350–400 million individuals during tissue damage [15, 16]. Recently, YKL-40 mRNA were infected [1]. The burden of chronic hepatitis B infec- expression was found in human liver [17], and serum YKL- tion is serious in China, with an estimated 120 million peo- 40 levels were associated with liver fibrosis in patients with ple infected, and 0.3 million deaths annually [2]. In China, a chronic liver disease [18]. Immunohistochemical studies proportion of CHB patients are asymptomatic accompanied have shown that YKL-40 is expressed in fibrotic areas of the by normal and mildly elevated alanine transaminase (ALT, liver [17, 19]. Based on these supporting evidences, serum ALT levels are less than two times the upper limit of nor- YKL-40 has been evaluated as a noninvasive marker of mal). The guidelines of American Association for the Study fibrotic liver diseases, including alcoholic liver disease [20 ], of Liver Diseases (AASLD) and Asian Pacific Association non-alcoholic fatty liver disease [21] and chronic hepatitis for the Study of the Liver for the management of CHB C-induced liver fibrosis [22, 23]. Therefore, we recently pro- recommend antiviral treatment when ALT levels are two posed a hypothesis that serum YKL-40 may be a potential times the upper limit of normal (ULN), and monitoring or biomarker for differentiating significant fibrosis in chronic performing liver biopsy (especially for patients > 40 years) hepatitis B patients with normal and mildly elevated ALT. to assess if significant histologic disease is present when In this study, we identified the proportion of significant ALT levels are less than two times the upper limit of nor- fibrosis in CHB patients with normal and mildly elevated mal (ULN) [3, 4]. However, CHB patients with normal ALT. We measured the serum levels of YKL-40 and com- and mildly elevated ALT levels may not have healthy liv- pared them with fibrosis stages to evaluate the feasibility ers. Several studies indicated that moderate inflammation of YKL-40 as a biomarker of liver fibrosis in patients with and/or advanced fibrosis was present in 28–37% of CHB normal and mildly elevated ALT levels. patients who had persistently normal ALT [5–7]. These stud- ies suggested that CHB patients with normal ALT might have histologically significant disease, an indication for Patients and methods antiviral treatment. Use of ALT without resorting to liver biopsy may miss a certain proportion of patients with histo- Patients logically significant disease who may benefit from antiviral therapy. Liver biopsy remains the gold standard for assess- A total of 685 patients with chronic HBV infection from 24 ing liver fibrosis in CHB patients. However, liver biopsy hospitals located in mainland China were enrolled in this has several limitations including invasiveness, risk of com- study between October 2013 and March 2016. Of which, plications, sampling error, and cost [8], which limited its 460 patients have ALT levels less than two times the upper application in assessing and dynamic monitoring of liver limit of normal range (ULN), and they were randomly fibrosis. Currently, multiple noninvasive methods based on divided into a training group (n = 307) and a validation 1 3 Serum YKL‑40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and… 387 group (n = 153). They all underwent liver biopsies. Inclu- variables, the differences between the groups were analyzed sion and exclusion criteria were described previously [24]. using Student t test and Mann–Whitney U test, respectively. All patients provided written informed consent for research For categorical variables, Chi-square test was used to com- use of their clinical data and specimens. This study was pare the differences in proportions. Spearman’s rank test was approved by the Ethics Committee of Peking University First used to analyze the correlations between different variables Hospital. The detailed protocol for the clinical trial was reg- and fibrosis stages. We performed multivariate backward istered at clinicaltrials.gov (NCT01962155) and chictr.org logistic regression analysis to determine the independent (ChiCTR-DDT-13003724). variables of significant fibrosis. Receiver operating charac- teristic curve (ROC) was used to assess the performance Histological staging of noninvasive models for staging significant fibrosis. The diagnostic performance of different variables was evaluated Ultrasonography-guided liver biopsies with a minimal based on the area under the receiver operating characteristic length of 20 mm (at least 11 portal tracts) were routinely curve (AUROC). SPSS 16.0 software (SPSS, Inc., Chicago, performed at each hospital according to a standardized pro- IL, USA) was used for statistical analyses. P < 0.05 were tocol after receiving the patient’s written informed consent. considered statistically significant. Pathological interpretations were conducted in the Depart- ment of Pathology at You An Hospital affiliated to the Capi- tal Medical University. The histopathological examination rules were previously reported [24]. Fibrosis stages were Results assessed according to Ishak criteria [25]. Significant fibrosis was defined as F3. Patient’s characteristics Examination of serum markers A total of 685 patients were enrolled in this study; seven patients were excluded because of unqualified liver tissue. The biochemical and hematological parameters were rou- The remaining 678 patients with chronic HBV infection tinely detected by standard methods in local hospitals. were analyzed, of which 460 patients with ALT less than two 1 8 Serum HBV DNA (range 2.0 × 10 –1.7 × 10  IU/ml) was times the upper limit of normal range (ULN). The baseline measured by the COBAS AmpliPrep/COBAS TaqMan characteristics of the study patients are shown in Table 1. (Roche Diagnostics, Basel, Switzerland). Serum HBsAg There were no significant distributional differences in fibro- (range of 20–52,000 IU/ml) was quantified using the Roche sis stages between the group of patients with ALT ≥ 2 × ULN Elecsys HBsAg II assay (Roche Diagnostics, Penzberg, and the group of patients with ALT < 2 × ULN (P = 0.312, Germany). The serum levels of YKL-40 were determined Table 1). This result indicated the presence of significant using Human Magnetic Luminex® Assays (LXSAHM-08, or more severe fibrosis in patients with ALT < 2 × ULN, R&D Systems, Inc, Minneapolis, MN, USA) according patients who do not meet the treatment criteria recom- to the manufacturer’s instructions. The serum concentra- mended by AASLD guideline. tions of hyaluronic acid (range of 2–200  μg/L), laminin (5–900 μg/L), were measured using a chemiluminescence immunoassay kit (Yuande Bio-Medical Engineering Co., ALT was not a perfect surrogate marker for liver Ltd, Beijing, China). histology Noninvasive fibrosis scores For patients with ALT < 2 × ULN, they were stratified (from G1 to G5) according to the status of HBeAg and the levels Noninvasive assessment of fibrosis, APRI, and FIB4 was cal- of HBV DNA, as shown in Table 1. In patients with normal culated according to the following formulae: APRI = [(AST/ ALT, differences in the proportion of significant fibrosis ULN)/platelet(× 10 /L)] × 100; FIB4 = (age × AST)/[plate- were statistically significant (P = 0.015, Fig. 1a). Overall, 9 1/2 let(× 10 /L) × ALT ]. Forns’ index [9] and Hui model [10] more than 30% of patients had significant fibrosis, besides were obtained from reported research. G1 (immuno-tolerant phase) with 17.8% incidence of sig- nificant fibrosis. Similar results were obtained in patients Statistical analysis with mildly elevated ALT (P <0.0001, Fig. 1b). Regarding the incidence of significant fibrosis between patients with Quantitative variables were expressed as mean ± standard normal ALT and patients with mildly elevated ALT, there deviation (SD) and categorical variables were expressed were no significant differences (data not shown). This result as proportions. For normally and non-normally distributed 1 3 388 L. Yan et al. Table 1 Patients’ characteristics ALT ≥ 2 × ULN (n = 218) ALT < 2 × ULN (n = 460) P value Age (median, ≥ 40 years %) 36, 77 (35.3%) 38, 205 (44.6%) 0.024 Gender (male %) 184 (84.4%) 345 (75.0%) 0.006 BMI (median, ≥ 24 kg/m  %) 23.3, 76 (34.9%) 23.0, 165 (35.9%) 0.864 HBsAg (log IU/mL) 3.59 ± 0.77 3.56 ± 0.88 0.409 AST (U/L) 116.55 ± 109.99 35.50 ± 17.84 < 0.001 ALP (U/L) 91.64 ± 29.93 77.31 ± 26.07 < 0.001 GGT (U/L) 82.62 ± 69.51 41.50 ± 47.42 < 0.001 Albumin (g/L) 43.67 ± 5.85 44.48 ± 5.26 0.002 TBil (µmol/L) 18.30 ± 15.33 16.94 ± 22.78 0.017 PT (s) 12.94 ± 1.51 12.56 ± 1.49 0.001 PLT (× 10 /L) 170.64 ± 52.76 172.33 ± 59.08 0.635 Hyaluronic acid (ug/L) 149.05 ± 102.82 115.26 ± 71.14 < 0.001 Laminin (ug/L) 179.54 ± 302.39 84.24 ± 177.79 < 0.001 PIIINP (ug/L) 5.65 ± 11.42 3.65 ± 5.04 < 0.001 Collagen IV (pg/mL) 1120.00 ± 628.19 896.98 ± 540.96 < 0.001 pg/mL) 4.46 ± 0.38 4.47 ± 0.38 0.718 YKL-40 (log sCD163(log pg/mL) 6.20 ± 0.36 6.01 ± 0.33 < 0.001 MMP-1 (log pg/mL) 3.47 ± 0.31 3.48 ± 0.32 0.566 MMP-2 (log pg/mL) 5.28 ± 0.10 5.26 ± 0.10 0.058 MMP-3 (log pg/mL) 4.17 ± 0.25 4.17 ± 0.26 0.624 MMP-9 (log pg/mL) 4.85 ± 0.41 4.87 ± 0.45 0.770 TIMP-1 (log pg/mL) 5.08 ± 0.12 5.06 ± 0.13 0.018 HBeAg status/HBV DNA(IU/mL) (n %) 0.020  G1 e + , HBV DNA ≥ 2 × 10 86 (39.4%) 128(27.6)  G2 e + , 20,000 ≤ HBV DNA < 2 × 10 54 (24.8) 116 (25.4)  G3 e + , HBV DNA < 20,000 9 (4.1) 25 (5.7)  G4 e − , HBV DNA ≥ 2000 59 (27.1) 152 (32.8)  G5 e − , HBV DNA < 2000 10 (4.6) 39 (8.5) Fibrosis stages (n %) 0.312  F0–2 128 (58.7%) 291 (63.2%)  F3 48 (22.0%) 85 (18.5%)  F4 33 (15.1%) 68 (14.8%)  F5–6 9 (4.1%) 16 (3.5%) Data presented as mean ± SD or no. (%) BMI body mass index, HBsAg hepatitis B surface antigen, AST aspartate transaminase, ALP alkaline phosphatase, GGT gamma-glutamyl transpeptidase, TBil total bilirubin, PT prothrombin time, PLT plate- let counts, PIIINP N-terminal peptide of type III procollagen, YKL-40 chitinase 3-like-1, sCD163 soluble CD163, MMP matrix metalloproteinase, TIMP-1 tissue inhibitor of metalloproteinase 1, HBeAg hepatitis B e antigen, HBV hepatitis B virus, ULN upper limit of normal suggested that ALT levels and fibrosis are not always con- showing significant difference between fibrosis stages (F01 sistent in CHB patients. vs F2–F56, F2 vs F3–F56) (P < 0.0001, Fig. 2a). In patients with ALT < 2 × ULN, similar results were obtained as in the Serum YKL‑40 levels increased with the progression total patients (Fig. 2b). In addition, serum YKL-40 levels of fibrosis were positively correlated with hyaluronic acid, laminin, PIIINP, Collagen, and AST, while they were negatively cor- Serum YKL-40 levels were measured to assess the feasibil- related with platelet count (Supplementary Table 1). ity of YKL-40 as a biomarker of fibrosis in CHB patients. Serum levels of YKL-40 throughout different fibrosis stages are shown in Fig. 2. In the total patients, serum YKL-40 levels increased in parallel with the progression of fibrosis, 1 3 44.0% Serum YKL‑40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and… 389 100 100 F0-2 F3 80 80 58.6% 55.6% 60 60 47.8% 43.8% 42.9% 38.3% 40 40 31.0% 19.3% 17.8% 20 20 0 0 G1(n=45) G2(n=46) G3(n=9)G4(n=71) G5(n=25) G1(n=83)G2(n=70)G3(n=16)G4(n=81)G5(n=14) Fig. 1 Proportion of patients with significant fibrosis in the group of G1–G5 in chronic hepatitis B patients. Patients with a normal ALT and b mildly elevated ALT *** **** *** A **** B ** **** *** 6 **** *** ** **** **** 4 4 3 3 2 2 F01F2F3F4F56 F01F2F3F4F56 Fig. 2 Associations between serum YKL-40 levels and liver fibrosis. a YKL-40 in total patients, b YKL-40 in patients with ALT < 2 × ULN. P < 0.0001 for all fibrosis stags. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 hyaluronic acid (HA), PLT, and AST were independent Development of YKL‑40‑based fibrosis model factors of significant fibrosis (Table  3). We performed in patients with ALT < 2 × ULN backward logistic regression analysis and established a novel YKL-40 based model for CHB patients with To determine the ability of YKL-40 to diagnose signifi- ALT < 2 ULN: cant fibrosis, all CHB patients with ALT < 2 × ULN were YKL -40 model = 0.032 × AST − 0.012 × PLT + 0.012 × divided into a training group and a validation group. There HA + 0.846 × log10 (YKL -40) − 4.752. was no statistical difference between training group and validation group about any parameters (Supplementary Diagnostic performance of YKL‑40 model Table 2). In the training group, univariate analysis found for significant fibrosis that serum YKL-40, hyaluronic acid, laminin, PIIINP, Col- lagen IV, sCD163, and MMP-2 were positively associ- YKL-40 model had an area of 0.786 (95%CI 0.726–0.846) ated with significant fibrosis (Table  2). PLT was inversely under the ROC curve in predicting significant fibrosis in associated with significant fibrosis (Table  2). Multivari- the training group, with 71.74% sensitivity, 72.85% speci- ate analysis revealed that YKL-40 [odd ratio (OR) 2.330, ficity, 61.68% PPV, and 80.88% NPV at the cut-off point 95% confidence interval (CI) 1.019–5.330, P = 0.045], 1 3 YKL-40 (log Pg/mL) Percentage (%) Percentage (%) YKL-40 (log Pg/mL) 10 390 L. Yan et al. Table 2 Univariate analysis of clinical parameters and biomarkers of − 0.56. It was superior to that of APRI [0.736 (95%CI with significant fibrosis in the training group (n = 307) 0.670–0.803)], FIB-4 [0.735 (95%CI 0.669–0.801)], Forns’ index [0.753 (95%CI 0.688–0.817)], and Hui model [0.734 F0–2 (n = 194) F ≥ 3 (n = 113) P value (95%CI 0.667–0.801)] (Fig. 3a, Table 4). The area under Age (≥ 40 years %) 37.54 ± 10.12 42.54 ± 10.92 < 0.0001 the ROC curve of YKL-40 model in the validation group Gender (male %) 141 (72.68%) 83 (73.45%) > 0.9999 was 0.831 (95%CI 0.752–0.910), with 71.79% sensitivity, BMI (≥ 24 kg/m  %) 22.88 ± 2.95 23.50 ± 2.74 0.096 85.33% specificity, 71.79% PPV, and 85.33% NPV at the cut- HBsAg (log IU/ 3.71 ± 0.88 3.28 ± 0.72 < 0.0001 off point of − 0.33, which was also higher than that of APRI, mL) FIB-4, Forns’ index, and Hui model (Fig. 3b, Table 4). In the ALT (U/L) 42.16 ± 17.61 44.55 ± 16.04 0.258 entire cohort, YKL-40 model had an area of 0.801 (95%CI AST (U/L) 32.52 ± 15.72 42.36 ± 21.75 < 0.0001 0.753–0.849) under the ROC curve in predicting significant ALP (U/L) 72.61 ± 19.21 85.90 ± 33.97 0.002 fibrosis (data not shown) . GGT (U/L) 35.60 ± 45.56 54.91 ± 47.21 < 0.0001 Albumin (g/L) 45.06 ± 4.53 43.58 ± 6.78 0.003 TBil (µmol/L) 15.45 ± 17.01 20.84 ± 38.75 0.002 Discussion PT (s) 12.32 ± 1.27 12.85 ± 1.43 0.002 PLT (× 10 /L) 187.06 ± 49.56 145.23 ± 53.21 < 0.0001 Serum ALT is commonly used to assess liver histology activ- Hyaluronic acid 93.89 ± 41.27 151.04 ± 96.27 < 0.0001 ity and to guide antiviral therapy in patients with liver dis- (ug/L) ease. However, results of the present study showed that, ALT Laminin (ug/L) 48.21 ± 91.38 126.52 ± 204.21 < 0.0001 levels and fibrosis are not always consistent in CHB patients. PIIINP (ug/L) 3.12 ± 6.14 4.53 ± 4.56 < 0.0001 We observed that a high proportion (> 30%) of CHB patients Collagen IV (pg/mL) 782.48 ± 387.26 1037.84 ± 584.14 < 0.0001 with normal and mildly elevated (1-2ULN) ALT have sig- YKL-40 (log10 pg/ 4.39 ± 0.35 4.62 ± 0.40 < 0.0001 mL) nificant fibrosis regardless of the state of HBeAg and the SCD163 (log10 pg/ 5.94 ± 0.32 6.12 ± 0.32 < 0.0001 levels of HBV DNA (Fig. 1, G2–G5). Even for patients in mL) the immunO-tolerant phase (Fig. 1, G1), 17.8 and 19.3%, MMP-1 (log10 pg/ 3.49 ± 0.32 3.46 ± 0.31 0.501 respectively, have significant fibrosis. Our present findings mL) are consistent with the previous reports that patients with MMP-2 (log10 pg/ 5.24 ± 0.10 5.28 ± 0.10 0.001 chronic HBV infection can display normal and mildly ele- mL) vated ALT levels despite significant histological injury [6 , MMP-3 (log10 pg/ 4.17 ± 0.27 4.18 ± 0.26 0.740 7, 26]. A meta-analysis [26] concluded that approximately mL) one-fifth of CHB patients with ALT ≤ 40 IU/L may have MMP-9 (log10 pg/ 4.86 ± 0.46 4.86 ± 0.40 0.752 mL) significant hepatic fibrosis. Lai et al. [7 ] found that 37% of TIMP-1 (log10 pg/ 5.05 ± 0.13 5.08 ± 0.13 0.134 CHB patients with persistently normal ALT had significant mL) fibrosis and inflammation. According to current guidelines, antiviral therapy should be initiated immediately for patients BMI body mass index, HBsAg hepatitis B surface antigen, ALT ala- with significant fibrosis [ 3, 4]. Our results confirmed that nine transaminase, AST aspartate transaminase, ALP alkaline phos- phatase, GGT gamma-glutamyl transpeptidase, TBil total bilirubin, ALT was not a perfect surrogate marker for liver histology, PT prothrombin time, PLT platelet counts, PIIINP N-terminal peptide because ALT failed to identify many patients who might of type III procollagen, YKL-40 chitinase 3-like-1, sCD163 soluble benefit from antiviral therapy. The “gray zone” patients were CD163, MMP matrix metalloproteinase, TIMP-1 tissue inhibitor of defined as those patients with normal and mildly elevated metalloproteinase 1 ALT. Because of the high proportion of significant liver disease in the “gray zone” patients, it is highly important Table 3 Multivariate logistic regression analysis of independent pre- to assess liver fibrosis. Liver biopsy, a gold standard for dictors for significant fibrosis in the training group (n = 307) assessing liver fibrosis, is not suitable for regular applica- tions due to the limitations of invasive, finite, complications, Coefficient OR 95%CI P value and cost [8]. Noninvasive models such as APRI and FIB-4 AST (U/L) 0.032 1.033 1.009–1.057 0.007 using biochemical laboratory index have been proposed to PLT (× 10 /L) − 0.012 0.988 0.982–0.995 < 0.0001 replace liver biopsy to assess liver fibrosis. Therefore, it is Hyaluronic acid (ug/L) 0.012 1.013 1.005–1.020 0.001 reasonable to evaluate “gray zone” patients based on such YKL-40 (log10 pg/ 0.846 2.330 1.019–5.330 0.045 noninvasive methods, and then to decide whether initiating mL) antiviral treatment or not. Constant − 4.758 0.009 – 0.018 Within the present study, we assessed the relationship YKL-40 model = 0.032 × AST − 0.012 × PLT + 0.012 × HA + 0.846 × l between serum markers, including YKL-40, hyaluronic og10 (YKL-40) − 4.752 1 3 Serum YKL‑40 as a biomarker for liver fibrosis in chronic hepatitis B patients with normal and… 391 1.0 1.0 A B YKL40Model YKL40Model APRI APRI FIB-4 FIB-4 0.8 0.8 Hui model Hui model 0.6 0.6 0.4 0.4 0.2 0.2 Validation group Training group 0.0 0.0 0.00.2 0.40.6 0.81.0 0.00.2 0.40.6 0.81.0 1 - Specificity 1 - Specificity Fig. 3 Receiver operating characteristic curve (ROC) analysis show- ARPI, FIB4, Forns’ index and Hui model in the diagnosis of signif- ing the diagnostic performance of noninvasive models for significant icant fibrosis in CHB patients with ALT < 2ULN. a Training group fibrosis. Area under the ROC curves (AUROCs) of YKL-40 model, and b validation group Table 4 Receiver operating characteristics curve (ROC) analysis of noninvasive models for the diagnosis of significant fibrosis in CHB patients with ALT < 2ULN AUROC (95%CI) Cut-off value Sensitivity (%) Specificity (%) PPV (%) NPV (%) Training group  YKL-40 model 0.786 (0.726–0.846) − 0.56 71.74 72.85 61.68 80.88  APRI 0.736 (0.670–0.803) 0.76 42.39 93.38 79.60 72.68  FIB-4 0.735 (0.669–0.801) 1.33 50.00 88.08 71.88 74.30  Forns’ index 0.753 (0.688–0.817) 7.75 56.52 82.78 66.66 75.76  Hui model 0.734 (0.667–0.801) 0.12 69.57 70.20 58.72 79.11 Validation group  YKL-40 model 0.831 (0.752–0.910) − 0.33 71.79 85.33 71.79 85.33  APRI 0.762 (0.668–0.855) 0.50 71.79 73.33 58.33 83.33  FIB-4 0.743 (0.648–0.838) 1.14 79.49 64.00 53.45 85.72  Forns’ index 0.804 (0.723–0.886) 7.12 79.49 73.33 60.78 87.30  Hui model 0.771 (0.684–0.857) 0.08 87.18 54.67 50.00 89.13 acid, laminin, PIIINP, Collagen IV, sCD163 and metallo- fibrosis, a stage which represent an indication for antiviral proteinases, and liver fibrosis in patients with normal and therapy, from the “gray zone” patients. Our univariate analy- mildly elevated ALT. Of note, our results indicated that sis revealed that serum YKL-40, hyaluronic acid, laminin, serum YKl-40 levels significantly correlated with fibrosis PIIINP, Collagen IV, sCD163, and MMP-2 were associated stages as assessed by Ishak score. Serum levels of YKL-40 with significant fibrosis. However, multivariate analysis also increased in non-alcoholic fatty liver disease (NAFLD) showed that only YKL-40, hyaluronic acid and two labora- and chronic hepatitis C-induced liver fibrosis [21, 22, 27, tory parameters, PLT and AST, retained significance when 28]. It has been reported that YKL-40 is a growth factor combined with other clinical parameters. Series studies have for fibroblasts and is expressed in active liver fibrotic areas demonstrated that combination of multiple serum markers [29, 30]. In addition, the progression of fibrosis rate per could improve the sensitive, specific, and reproducible [31, year linearly correlates with the serum levels of YKL-40 32]. Based on our findings, a four-variable model including [23]. These observations further strengthen the possibility two serum fibrosis markers (log YKL-40, hyaluronic acid) that YKL-40 is involved in hepatic fibrogenesis in patients and two routinely laboratory tests (PLT, AST) was derived with HBV infection and is a useful biomarker for hepatic via backward logistic regression analysis to detect significant fibrosis. It is critical to discriminate patients with significant fibrosis. Hyaluronic acid is synthesized by stellate cells and 1 3 Sensitivity Sensitivity 392 L. Yan et al. is involved in fibrogenesis; it has been identified as one of ALT levels regardless of HBeAg status and HBV DNA the serum markers of liver fibrosis in non-alcoholic steato- levels. In CHB patients with ALT < 2ULN, serum YKL- hepatitis (NASH) and chronic hepatitis C [31–34]. Regard- 40 levels were independently associated with significant ing PLT, our finding is consistent with the previous stud- fibrosis and could be a feasible biomarker reflecting liver ies that found decreased platelet counts are associated with fibrosis. YKL-40 model was superior to existing scores more severe hepatic fibrosis [35, 36]. in diagnosing significant fibrosis in CHB patients with Identification of patients, who actually had significant normal and mildly elevated ALT. This finding offered a hepatic fibrosis, diagnosed as “none treatment required” promising method to identify those “gray zone” patients according to ALT levels, is very important. Significant who may benefit from antiviral therapy. fibrosis is an important endpoint of clinical antiviral ther - Acknowledgements We thank the members of China HepB-Related apy [37, 38]. The aim of this study was to develop an Fibrosis Assessment Research Group for assisting patient inclusion accurate noninvasive fibrosis model applied to “gray zone” and data acquisition. CHB patients. Over the past 20 years, various noninvasive fibrosis models have emerged. The most widely used two Funding This study was supported by China Mega-Project for Infec- scores, APRI and FIB-4, and Forns’ index, are based on tious Diseases (Grant Numbers 2013ZX10002005, 2012ZX10002006, 2013ZX10002004, 2012ZX10005005), Project of Beijing Science and patients with hepatitis C infection [39]. APRI and FIB-4 Technology Committee (Grant Number D121100003912002). have been validated and recommended for evaluation of liver fibrosis in CHB patients [39, 40]. Hui model is based Compliance with ethical standards on patients with HBV [10] while lacking of clinical valida- tion (Supplementary Table 3). Furthermore, the diagnostic Conflict of interest On behalf of all authors, the corresponding author performances of the above models for fibrosis assessment states that there is no conflict of interest. in CHB patients with normal and mildly elevated ALT Ethical approval This study was approved by the local ethics commit- have not been validated in large cohorts. A recent analy- tee of Peking University First Hospital. sis of APRI and FIB-4 in 231 HBV-infected patients with normal and mildly elevated ALT founded limited diag- Open Access This article is distributed under the terms of the Crea- nostic value for significant fibrosis [41]. In this study, we tive Commons Attribution 4.0 International License (http://creat iveco developed a YKL-40 model in 460 CHB patients with mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- normal and mildly elevated ALT, and then, we compared tion, and reproduction in any medium, provided you give appropriate the performances of the five noninvasive models to diag- credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. nose significant fibrosis. For the identification of patients with significant fibrosis, the AUROCs for patients with ALT < 2ULN were 0.736 for APRI and 0.735 for FIB-4 in the training group, compared with 0.762 for APRI and References 0.743 for FIB-4 in the validation group, showing simi- lar performance as previous reported [41]. We found that 1. Sorrell MF, Belongia EA, Costa J, Gareen IF, Grem JL, Inadomi YKL-40 model produced the best performances compared JM, et al. 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InfectionSpringer Journals

Published: Mar 29, 2018

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