Serum YKL-40 as a marker of liver fibrosis in patients with non-alcoholic fatty

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1 Supplementary Information Serum YKL- as a marker of liver fibrosis in patients with non-alcoholic fatty liver disease Erina Kumagai 1,,#, Yohei Mano 1,#, Sachiyo Yoshio 1, Hirotaka Shoji 1, Masaya Sugiyama 1, Masaaki Korenaga 1, Tsuyoshi Ishida, Taeang Arai, Norio Itokawa, Masanori Atsukawa, Hideyuki Hyogo 5,6, Kazuaki Chayama 5, Tomohiko Ohashi 7, Kiyoaki Ito 7, Masashi Yoneda 7, Takumi Kawaguchi 8, Takuji Torimura 8, Yuichi Nozaki 9, Sumio Watanabe, Masashi Mizokami 1 and Tatsuya Kanto 1 1 The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan. Department of Gastroenterology, Juntendo University School of Medicine, Hongo, Bunkyo, Tokyo, Japan. Department of Pathology and Laboratory Medicine, Kohnodai Hospital, National Center for Global Health and Medicine, Ichikawa, Japan. Division of Gastroenterology, Department of Internal Medicine, Nippon Medical

2 School Chiba Hokusoh Hospital, Inzai, Japan. 5 Department of Gastroenterology and Metabolism, Hiroshima University, Hiroshima, Japan. 6 Department of Gastroenterology and Hepatology, JA Hiroshima General Hospital, Hiroshima, Japan. 7 Division of Gastroenterology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Japan. 8 Division of Gastroenterology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan. 9 Department of Gastroenterology, National Center for Global Health and Medicine, Central Hospital, Tokyo, Japan. # Erina Kumagai and Yohei Mano contributed equally to this work. Corresponding author: Tatsuya Kanto, M.D., Ph.D. The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1, Kohnodai, Ichikawa , Japan. kantot@hospk.ncgm.go.jp Telephone: , Fax:

3 Supplementary Table S1. NAFLD Activity Score (NAS) and Background of Patients in Each Stage of Fibrosis NAS Stage -1 (n = 1) Stage (n= 1) Stage (n = ) Stage (n=9) Male/Female /8 8/1 7/16 /5 Age (year) 61 (-7) 59 (-81) 61 (1-79) 67 (56-8) BMI (kg/m²) 8.6 (.8-.) 8.6 (.8-5.) 8. (.7-.) 7. (.1-9.) Platelet (x1 /mm ). ( ). (1.-.5) 1. ( ) 1. (.9-5.1) Albumin (g/dl).6 (.1-.8).5 (.9-5.). (.5-.9).8 (.6-.6) Total bilirubin (mg/dl).7 (.-1.6).8 (.-.).9 (.-.) 1. (.-.5) AST (IU/U) (19-5) 5(17-5) 8 (1-186) 5 (7-1) ALT (IU/L) 56 (1-1) 96 (1-59) 116 (-18) 5 (1-18) Type IV collagen 7s (ng/ml). (.-5.) 5. (.7-8.1) 6.1 (.8-8.9) 7.7 ( ) Hyaluronic acid (ng/ml) (1-9) 115 (1-751) 8 (1-1) 19 (9-51) APRI.5 (.7-.91).7 (.5-.86) 1. (.58-.1) 1.69 (.-.57) FIB- index 1.5(.6-.) 1.5 (.55-.8). (.7-.9) 6.57 ( ) Steatosis (/1//) 1///6 /8/5/8 /11/7/5 1///1 Hepatocyte Balloning (/1/) /9/ 1/11/9 /1/1 /5/ Lobular Inflammation (/1//) /9// /1/7/1 /9/1/ 1///1

4 Supplementary Table S. Clinical Background of Chronic Hepatitis for HCV Patients in Each Stage of Fibrosis Chronic hepatitis with HCV Stage -1 (n = 18) Stage (n= 1) Stage (n = ) Stage (n=15) HCC (n=1) Male/Female 9/9 7/5 1/ /11 19/1 Age (year) 5 (18-7) 65 (9-7) 65 (5-76) 67 (6-89) 7 (58-85) Platelet (x1 /mm ) 18.1 (1.-.1) 15.7 (8.-1.) 8.8 ( ) 1. ( ) 9.1 (.-.7) Albumin (g/dl). (.8-.9).1 (.9-.6).8 (.5-.).7 (.5-.1).5 (1.9-.) Total bilirubin (mg/dl).6 (.-1.).8 (.-1.1).8 (.7-1.5).9 (.6-.7).8 (.-16.7) AST (IU/U) 1 (17-99) 51 (1-9) 71 (7-6) 56 (-11) 6 (1-15) ALT (IU/L) 5 (17-176) 55 (18-116) 8 (1-9) 7 (1-9) 9 (1-16) Type IV collagen 7s (ng/ml).9 (.-5) 5.7 (-9.8) 7. (6.-8.5) 11 ( ) 8.9 (.-5.) APRI.5 (.-.).85 (.-.). (1.-5.). (1.-9.5) 1.1 (.6-1.5) FIB- index 1.6 ( ).6 ( ) 5.59 ( ) 6.59 (.69-.) 5.7 (.6-.5) Histological Activity (/1//) /15// //9/ ///1 /7/7/1 AFP (ng/ml) 17.5 (-1859) PIVKA-II (mau/ml) (8-56) Data are presented as the median values (range) BMI, APRI see Table1

5 Supplementary Figure Legends Supplementary Figure S1: Type IV collagen 7s, hyaluronic acid, WFA + -MBP, APRI, FIB- index in patients with NAFLD with different stages of fibrosis. The serum levels of type IV collagen 7s (a), hyaluronic acid (b), and WFA + -MBP (c) APRI (d), and FIB- index (e) are shown., p<.5;, p<.1 Kruskal-Wallis test with Dunn s multiple comparison test. The stage of fibrosis was assessed according to Brunt classification. Supplementary Figure S: Serum levels of YKL- in patients with HCV infection with different stages of fibrosis. Serum YKL- levels are shown in patients with HCV stratified by the stage of fibrosis (METAVIR classification). p<.5, p<.1, by Kruskal-Wallis test with Dunn s multiple comparison test. HV, healthy volunteers Supplementary Figure S: ROC analyses of serum YKL-, other markers and fibrosis scores for the diagnosis of advanced fibrosis in patients with NAFLD. This figure shows ROC curves of YKL-, type IV collagen 7s, hyaluronic acid, WFA + - MBP, APRI, FIB- index, and the prediction model for NAFLD established by YKL- and type IV collagen 7s as a diagnostic marker of advanced fibrosis (Stage - vs. Stage -). Supplementary Figure S: Expression and production of YKL- from macrophages in vitro.

6 Primary macrophages were generated from monocytes obtained from healthy donors as described in Materials and Methods. a: The mrna levels of YKL- in M-CSFdifferentiated macrophages were examined on day 5, 6 and 7 of the culture. b: The supernatants were collected from day 7 macrophages stimulated with (conc.) TNF-α and/or (conc.) IL-1β. The YKL- levels in the supernatants were assayed by ELISA. p<.5, p<.1 by Student t-test. Supplementary Figure S5: Serum YKL- levels were not correlated with serum TNF levels and serum IL-1 levels. The correlations between serum YKL- levels and serum TNF or IL-1 levels were assessed by Spearman s rank-correlation coefficient. The p-values and correlation coefficients are depicted in each plot. Supplementary Figure S6: Serum YKL- levels in CHC patients with or without HCC. Serum YKL- levels in chronic hepatitis and liver cirrhosis patients with or without HCC are shown. p<.5, by Kruskal-Wallis test with Dunn s multiple comparison test

7 Supplementary Figure S1 a A b B Cc ng/m l COI Type IV collagen 7s H yaluronic acid ng/m l 8 6 WFA+ MBP stage stage stage d D e E ng/m l APRI 1 FIB - index stage stage

8 Supplementary Figure S ng/ml 6 YKL- HV -1 stage

9 Supplementary Figure S sensitivity.6.. YKL- APRI FIB- index specificity sensitivity.6.. YKL- Type IV collagen 7s Hyaluronic acid WFA+ MBP specificity 1.8 sensitivity.6.. YKL- and type IV collagen 7s YKL- Type IV collagen 7s specificity

10 Supplementary Figure S a A YKL- mrna fold induction 1 day5 day6 day7 day in culture YK L- B b ng/ml 5 1 control P=.7 T N F α IL1β TNFα+IL1β

11 Supplementary Figure S5 (pg/ml) (ng/ml) 15 P=.17 R= P=.98 R= TNF-a IL-1b YKL- (µg/ml) 1 5 YKL- (µg/ml)

12 Supplementary Figure S6 ng/m l 6 YKL- CH HCC- CH HCC+ LC HCC- LC HCC+