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The Laennec staging system for histological sub-classification of cirrhosis is useful for stratification of prognosis in patients with liver cirrhosis
Research Article
The Laennec staging system for histological sub-classification of cirrhosis is useful for stratification of prognosis in patients with liver cirrhosis Seung Up Kim1,2,4, , Hyun Jung Oh1, , Ian R. Wanless7, Sarah Lee3, Kwang-Hyub Han1,2,4,5,⇑, Young Nyun Park3,4,5,6,⇑ 1
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea; 2Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea; 3Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea; 4Liver Cirrhosis Clinical Research Center, Seoul, South Korea; 5Brain Korea 21 Project of Medical Science, South Korea; 6Integrated Genomic Research Center for Metabolic Regulation, Seoul, South Korea; 7Department of Laboratory Medicine, Queen Elizabeth II Health Sciences Centre, Halifax, Canada
Backgrounds & Aims: The clinical severity of cirrhosis varies widely. We investigated whether histological sub-classification of cirrhosis using the Laennec system can discriminate different outcomes among patients with cirrhosis. Methods: One hundred and seventy-five patients with chronic liver disease who underwent liver biopsy and showed stage 3 or 4 fibrosis between January 2001 and December 2008 were prospectively enrolled. Cirrhosis was sub-classified into three groups (4A, 4B, and 4C) according to the Laennec system. The end point was liver-related event (LRE) occurrence, including decompensation, hepatocellular carcinoma, and liver-related death. Results: The median age of the patients (110 men, 65 women) was 55 years. Stages 3, 4A, 4B, and 4C were identified in 46 (26.3%), 16 (9.1%), 82 (46.9%), and 31 (17.7%) patients, respectively. During the follow-up period, LREs occurred in 32 (18.3%) patients: 4 (8.7%) with stage 3, 2 (12.5%) with stage 4A, 17 (20.7%) with stage 4B, and 9 (29.0%) with stage 4C. In a multivariate analysis, histological sub-classification of cirrhosis independently predicted LRE occurrence. While patients with stage 4A tended to be at higher risk of LRE occurrence than those with stage 3, patients with stages 4B and 4C had significantly higher risks of LRE occurrence, with hazard ratios of 6.158 (p = 0.016) and 8.945 (p = 0.004), respectively.
Keywords: Cirrhosis; Fibrosis; Laennec staging system; Liver biopsy; Liver-related event. Received 14 November 2011; received in revised form 11 April 2012; accepted 27 April 2012; available online 19 May 2012 ⇑ Corresponding authors. Addresses: Department of Internal Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun–gu, Seoul 120–752, Korea. Tel: +82 2 2228 1949; fax: +82 2 363 6994 (K.-H. Han), Department of Pathology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun–gu, Seoul 120–752, Korea. Tel.: +82 2 2228 1768; fax: +82 2 362 0860 (Y.N. Park). E-mail addresses: [email protected] (K.-H. Han), [email protected] (Y.N. Park). These authors contributed equally to this work. Abbreviations: HCC, hepatocellular carcinoma; LRE, liver related event; LB, liver biopsy; TE, transient elastography; LS, liver stiffness; AFP, alpha-fetoprotein; HR, hazard ratio; CI, confidence interval; INR, international normalized ratio.
Conclusions: Histological sub-classification of cirrhosis using the Laennec system can be used to assess the risk of LRE occurrence among patients with cirrhosis. Our study provides a solid basis for further studies of non-invasive methods for monitoring the risk of LRE occurrence and will help physicians to establish optimum treatment strategies. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Liver cirrhosis is histologically defined as a diffuse state with replacement of the normal lobular architecture by abnormal nodules and fibrous septa [1]. Although cirrhosis is widely considered to be an end-stage liver disease, clinical severity, and prognosis within this diagnosis vary widely [2]. Clinically, cirrhosis can be categorized into compensated and decompensated states [3]. While this approach may give a clear indication for liver transplantation [4], it is of no use in predicting the development of hepatic decompensation. Furthermore, it does not provide prognostic information for the bulk of cirrhotic patients who are compensated and might benefit from tailored management to prevent decompensation. Therefore, the identification of prognostic factors to predict the development of liver-related events (LREs), such as hepatic decompensation, hepatocellular carcinoma (HCC), and liver-related mortality in patients with compensated cirrhosis is important. Among the characteristics of patients with compensated cirrhosis, the degree of liver fibrosis on biopsy may be important for predicting the development of hepatic decompensation. However, currently used histological staging systems, such as the Ishak [5], METAVIR [6], and Batts systems [7], group all patients with cirrhosis into a single category without taking account the severity of cirrhosis. Furthermore, because recent antiviral and anti-fibrotic treatments can contribute to the regression of fibrosis in patients with histological cirrhosis [8–10], the simple onestage description for cirrhosis is not able to stratify patients for
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JOURNAL OF HEPATOLOGY treatment assignment or evaluation of treatment response [3]. Thus, a more refined histological sub-classification of cirrhosis is needed [11]. The Laennec staging system, a modification of the METAVIR system, was proposed to meet this need [12,13]. This system subdivides cirrhosis into three groups (4A, 4B, and 4C) based on the thickness of the fibrous septa and the size of nodules. Although recent cross-sectional studies reported the usefulness of the Laennec system in correlating clinical stage and grade of portal hypertension [12,14], no prospective longitudinal study has documented the predictive values of the Laennec system using LRE occurrence, including hepatic decompensation, HCC, and liverrelated death, as solid end points. In this study, we evaluated the Laennec staging system for predicting LRE occurrence in patients with cirrhosis and therefore stratifying these patients according to prognosis.
fication of 20. The thickness of the fibrous septa and the size of the nodules (maximum distance between two adjacent septa) were measured and the mean value of each case was calculated.
Measurement of liver stiffness values After transient elastography (TE) using FibroScanÒ (EchoSens, Paris, France) was introduced in our institute, liver stiffness (LS) values by TE were obtained for all patients who underwent LB. LS measurement using TE was performed according to the manufacturer’s recommendations [17]. All LS measurements were obtained by one experienced technician who was blinded to the clinical and histologic data. In this study, only LS values with at least 10 validated measurements and a success rate of at least 60%, and interquartile range to median value ratio less than 0.3, were considered reliable. The median interval between TE and LB was 12 (range, 0–18) days.
Laboratory tests In addition to demographic data, the following laboratory parameters were measured at the time of LB: serum albumin level, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alpha-fetoprotein (AFP), prothrombin time, and platelet count.
Patients and methods Patients Between January 2001 and December 2008, a total of 712 patients with chronic hepatitis who underwent liver biopsy (LB) at Severance Hospital, Yonsei University College of Medicine, Seoul, Korea were consecutively enrolled in this prospective study. Informed consent was obtained from all patients before enrollment. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board of our institute. Because we planned to calculate the relative risk of LRE occurrence in patients with cirrhosis (stage 4A, 4B, or 4C), as compared to stage 3, we first excluded 481 patients with Laennec stage 0–2 on biopsy and a further 56 patients if the biopsy was less than 10 mm in length or if other exclusion criteria were present. The remaining 175 patients with stage 3, 4A, 4B, or 4C were selected for statistical analysis (Supplementary Fig. 1).
Follow-up Each patient was screened for HCC by ultrasonography at their initial visit. Six patients were excluded due to the presence of HCC at enrollment. If no evidence of HCC was found, patients were subjected to periodic surveillance by ultrasonography and laboratory tests, including measurement of AFP levels every 3 or 6 months. Furthermore, the development of hepatic decompensation was monitored during the study period. If LREs occurred, patients were admitted for immediate and appropriate management. The last follow-up month of our study was April 2011 and the median follow-up duration was 52.4 (range, 9.7–125.2) months. The follow-up duration was similar among fibrosis stages (69.2 ± 30.2, 51.6 ± 11.7, 51.2 ± 15.0, and 54.2 ± 20.1 months in stage 3, 4A, 4B, and 4C, respectively; all p >0.05).
Liver biopsy and histological assessment The indication of LB was to assess the severity of liver fibrosis and necroinflammation. LB was performed before starting antiviral treatment, especially in patients with chronic hepatitis B or C virus infection. All patients underwent ultrasoundguided percutaneous LB. LB specimens were formalin fixed and paraffin embedded. Then, 4-lm-thick sections were stained with hematoxylin-eosin and Masson’s trichrome. All liver tissue samples were evaluated by an experienced hepatopathologist (Y.N. Park) who had no access to clinical data for the study population. The degree of liver fibrosis was evaluated semi-quantitatively according to the Laennec system (Supplementary Fig. 2) [13]. Fibrosis was first scored as follows: 0, no definite fibrosis; 1, minimal fibrosis (no septa or rare thin septum; may have portal expansion or mild sinusoidal fibrosis); 2, mild fibrosis (occasional thin septa); 3, moderate fibrosis (moderate thin septa; up to incomplete cirrhosis); and 4, cirrhosis. Stage 4 was distinguished from stage 3 by more numerous septa per unit length of biopsy sufficient to display nodules with rounded contours. Then, stage 4 was sub-classified into three groups: 4A, mild cirrhosis, definite or probable; 4B, moderate cirrhosis (at least two broad septa); and 4C, severe cirrhosis (at least one very broad septum or many minute nodules). The terms ‘broad septum’ and ‘very broad septum’ were defined by comparing the relative thickness of fibrous septa and sizes of nodules [14]. Activity grade (degree of necroinflammatory activity in the lobules and periportal area) was scored as follows: A0, none; A1, mild activity; A2, moderate activity; and A3, severe activity. Activity grade was defined as lobule or periportal activity, whichever was the higher. The median length of biopsies in the study set was 16 mm (range, 10– 25 mm). The mean biopsy length of each fibrosis stage was statistically equivalent (15.0 ± 3.6, 16.1 ± 3.1, 16.0 ± 3.2, and 16.6 ± 3.5 mm in stages 3, 4A, 4B, and 4C, respectively; all p >0.05 among fibrosis stages). Morphometry Morphometry was performed as described [15,16]. Briefly, trichrome-stained sections of all liver biopsies were examined without knowledge of the histological score. Digital images of 1–4 fields of each biopsy were captured at a final magni-
Definitions of liver-related events The primary end point was LRE occurrence, defined as a cirrhosis-related complication including hepatic decompensation, HCC, and liver-related death. Hepatic decompensation comprised ascites, variceal bleeding, hepatic encephalopathy, spontaneous bacterial peritonitis, and hepatorenal syndrome. Ascites was diagnosed clinically and was confirmed by ultrasonography or computed tomography [18]. Variceal bleeding was diagnosed endoscopically [19]. Hepatic encephalopathy was diagnosed based on the presence of temporospatial disorientation, altered level of consciousness, or asterixis in the absence of other possible causes [20]. Spontaneous bacterial peritonitis (SBP) was defined as an ascitic fluid infection without an apparent intra-abdominal surgically treatable source with confirmation by culture and an elevated ascitic fluid absolute polymorphonuclear leukocyte count (P250 cells/mm3) [21]. Hepatorenal syndrome was diagnosed when acute renal failure developed in association with advanced chronic liver disease, after exclusion of other causes of renal failure [22]. For patients enrolled before 2005, diagnosis of HCC was based on the guidelines of the European Association for the Study of the Liver (EASL) [23]. Briefly, for nodules larger than 2 cm, HCC can be diagnosed by the coincident findings from at least two imaging techniques showing typical features of arterial hypervascularization, or findings from one imaging technique and an AFP level of >400 ng/ ml. For nodules between 1 and 2 cm in size, biopsy is recommended. For patients who were enrolled after 2005, HCC was diagnosed based on the guidelines of the American Association for the Study of Liver Diseases (AASLD) [24]. Briefly, for tumors larger than 2 cm, HCC was diagnosed when typical features of HCC were detected (hypervascularity and washout in the portal/venous phase) by one dynamic imaging technique and the AFP level was >200 ng/ml. For nodules between 1 and 2 cm in size, it was necessary to detect typical features of HCC using two dynamic imaging techniques. For tumors smaller than 1 cm, ultrasonography was repeated after 3 months. Liver-related deaths included death caused by hepatic failure (regardless of etiology), HCC progression, variceal bleeding, and acute infections such as SBP [25].
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Research Article of grade 3 necroinflammatory activity was high in patients with stage 3 (n = 20, 43.5%) but low in stage 4 (n = 4, 3.1%).
Statistical analysis Data are expressed as mean ± SD, median (range), or n (%) as appropriate. Characteristics of patients with and without LRE occurrence and HCC were compared using Chi-square test or Fisher’s exact test (for categorical data), or unpaired Student’s t-test or Mann–Whitney U test (continuous variables). The proportions of patients with LRE occurrence based on Laennec stage were compared using Mantel–Haenszel tests. Univariate and multivariate Cox proportional hazard regression analyses were used to estimate independent predictors of LRE occurrence. The time factor in univariate and multivariate analyses was defined as the time period from enrollment to first LRE occurrence. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were calculated. The Kaplan–Meier method was used to examine the cumulative incidence of LREs based on histological fibrosis stage. A p value of <0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 18.0 (Chicago, IL, USA).
Results Baseline characteristics The baseline characteristics of the study population at enrollment are summarized in Table 1. The median age of the patients (110 men and 65 women) was 55 years. The etiology of liver disease was mostly hepatitis B virus infection (n = 147, 84%). The majority of patients had Child–Pugh scores of 5 (n = 158, 90.3%). After LB, antiviral treatment was initiated using oral nucleos(t)ide analogues or pegylated interferon for patients with chronic hepatitis B and the combination of pegylated interferon and ribavirin for those with chronic hepatitis C.
Comparisons of patients with and without LREs or HCC During the follow-up period, LREs occurred in 32 (18.3%) patients. In particular, HCC developed in 22 (12.6%) patients. The baseline characteristics of patients with and without LREs or HCC are shown in Table 3. Patients in whom LREs occurred were significantly older, had higher rates of diabetes mellitus and Child–Pugh score, and had lower serum albumin level, prothrombin time, and platelet count, than those in whom LREs did not occur (all p <0.05). Patients who developed HCC were more likely to be male, and had lower BMI value, prothrombin time, and platelet count, than those who did not develop HCC (all p <0.05). When cirrhosis was histologically sub-classified using the Laennec system, the distribution of stages did not differ between patients with LREs or HCC and those without (Table 3). However, when we stratified our study population into two groups (patients with stage 3 and 4A vs. those with 4B and 4C), we found that rates of stage 4B and 4C were significantly higher in patients with LREs or HCC than in those without LREs or HCC (all p <0.05) (Fig. 1). Incidence of LREs based on Laennec stage
Values
The proportion of patients with LRE occurrence significantly correlated with the histological sub-classification of cirrhosis (p = 0.016, Fig. 2). During the follow-up period, LREs occurred in 8.7% of patients with stage 3, 12.5% of patients with stage 4A, 20.7% of patients with stage 4B, and 29.0% of patients with stage 4C. Moreover, while the incidence of liver-related death tended to be associated with stage (p = 0.081), the incidence of hepatic decompensation and HCC significantly correlated with stage (p = 0.013 and 0.041, respectively, Fig. 2).
Age, yr
55 (18-73)
Risk assessment of LRE occurrence according to Laennec stage
Male gender
110 (62.9)
Diabetes mellitus
24 (13.7)
Body mass index, kg/m2
23.4 (17.2-34.0)
Liver histology Stage 3, 4A, 4B, and 4C was identified in 46 (26.3%), 16 (9.1%), 82 (46.9%), and 31 (17.7%) patients, respectively (Table 2). The rate Table 1. Baseline characteristics (n = 175).
Characteristics Demographic data
In multivariate analysis, diabetes mellitus, etiology of liver disease (non-viral vs. viral), and histological sub-classification of
Etiology of liver disease HBV/HCV/others
147 (84.0)/17 (9.7)/11 (6.3)
Child-Pugh score 5/6
158 (91.3)/15 (8.7)
Table 2. Histological information.
Laennec stage
n (%)
Activity grade n
3
46 (26.3)
1
16
2
10
Laboratory data Serum albumin, g/dl
4.2 (3.5-5.1) 4A
16 (9.1)
3
20
1
15
Total bilirubin, mg/dl
0.8 (0.3-2.0)
AST, IU/L
39 (13-445)
2
1
ALT, IU/L
46 (11-609)
3
0
Prothrombin time, %
92.5 (77-100)
1
55
Platelet count, 109/L
141 (102-314)
2
26
5.0 (1.2-190)
3
1
1
14
2
14
3
3
Alpha fetoprotein, ng/ml
4B
4C
Biopsy length, mm
16 (10-25)
Spleen size, cm
10.5 (6.3-14.5)
Variables are expressed as median (range) or n (%). HBV, hepatitis B virus; HCV, hepatitis C virus.
558
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82 (46.9)
31 (17.7)
JOURNAL OF HEPATOLOGY Table 3. Comparison of baseline characteristics between patients with and without LRE or HCC.
Characteristics
Patients with LRE occurrence
Patients without LRE occurrence
(n = 32, 17.8%)
(n = 143, 82.2%)
Age, yr
51.2 ± 10.3
46.4 ± 11.6
Male gender
24 (75.0)
86 (60.1)
Diabetes mellitus
9 (28.1) 23.0 ± 3.1
p value
p value
Patients with HCC development
Patients without HCC development
(n = 22, 12.6%)
(n = 153, 87.4%)
0.033*
51.1 ± 9.3
46.7 ± 11.7
0.089
0.116
19 (86.4)
91 (59.5)
0.015*
15 (10.5)
0.009*
5 (22.7)
19 (12.4)
0.189
23.9 ± 2.8
0.136
22.4 ± 2.7
24.0 ± 2.8
0.023*
19 (86.4)/2 (9.1)/ 1 (4.5)
128 (83.7)/15 (9.8)/ 10 (6.5)
19 (86.4)/3 (13.6)
139 (92.1)/12 (7.9)
Demographic data
2
Body mass index, kg/m
Etiology of liver disease HBV/HCV/ others Child-Pugh score
0.550
0.929
24 (75.0)/3 (9.4)/ 5 (15.6)
123 (86.0)/14 (9.8)/ 6 (4.2)
26 (81.3)/6 (18.8)
132 (93.6)/9 (6.4)
Serum albumin, g/dl
3.9 ± 0.5
4.2 ± 0.4
0.004*
4.0 ± 0.5
4.1 ± 0.5
0.171
Total bilirubin, mg/dl
0.9 ± 0.3
0.9 ± 0.4
0.496
0.9 ± 0.3
0.9 ± 0.4
0.629
AST, IU/L
49.8 ± 38.5
54.5 ± 52.9
0.505
45.6 ± 36.8
56.6 ± 52.0
0.341
ALT, IU/L
55.6 ± 52.5
74.6 ± 86.4
0.204
55.8 ± 53.8
73.2 ± 84.4
0.349
Prothrombin time, %
89.0 ± 9.0
93.5 ± 6.2
<0.001* 89.4 ± 6.2
93.8 ± 6.9
0.017*
Platelet count, 10 /L
156 ± 44
174 ± 50
0.005*
154 ± 41
172 ± 52
0.016*
Alpha fetoprotein, ng/ml
13.0 ± 12.5
10.9 ± 23.8
0.638
14.9 ± 14.0
10.8 ± 23.1
0.432
11.0 ± 1.8
10.6 ± 1.9
0.327
10.5 ± 1.6
10.7 ± 1.9
0.623
0.025*
5/6
0.376
Laboratory data
9
Spleen size, cm Laennec stage on liver biopsy
0.116
0.152
3
4 (12.5)
42 (29.4)
2 (9.1)
44 (28.8)
4A
2 (6.3)
14 (9.8)
1 (4.5)
15 (9.8)
4B
17 (53.1)
65 (45.5)
14 (63.6)
68 (44.4)
4C
9 (28.1)
22 (15.4)
5 (22.7)
26 (17.0)
Variables are expressed as mean ± SD or n (%). HBV, hepatitis B virus; HCV, hepatitis C virus. ⁄ p <0.05.
p = 0.029*
100 90 15.4 80 70 45.5 60 50 40 9.8 30 20 29.4 10 0
Stage 3 Stage 4A Stage 4B Stage 4C
p = 0.022*
17.0
22.7
28.1
44.4 63.6
53.1 9.8 6.3 12.5
-
+
Liver-related event
28.8
4.5 9.1
-
+
Hepatocellular carcinoma
Fig. 1. Laennec stage patterns in patients with liver-related events and HCC and in those without. The rates of stage 4B and C were significantly higher in patients with liver-related event and HCC than in those without (p = 0.029 and 0.022, respectively). ⁄p <0.05.
cirrhosis using the Laennec system independently predicted LRE occurrence (all p <0.05) (Table 4). Moreover, while patients with
stage 4A tended to be at higher risk of suffering LREs (p = 0.271; HR, 2.924; 95% CI, 0.433–19.705) than those with stage 3, those with 4B or 4C had a significantly higher risk of LRE occurrence, with HRs of 6.158 (95% CI, 1.403–27.022; p = 0.016) and 8.945 (95% CI, 2.018–39.646; p = 0.004), respectively. When patients with chronic hepatitis B virus (n = 147, 84.0%) were sub-analyzed, the Laennec system was also selected as an independent predictor of LRE occurrence (p = 0.816, HR 1.318, 95% CI 0.129–13.470 in stage 4A; p = 0.050, HR 3.879, 95% CI, 0.896–16.793 in stage 4B; p = 0.035, HR 4.193, 95% CI 1.106– 15.886 in stage 4C with the reference to stage 3). Cumulative incidence of LREs according to Laennec stage The cumulative incidence rates of LREs increased significantly with increasing fibrosis stage, as shown in Fig. 3 (p = 0.018, logrank test). The 1-, 2-, and 3-year cumulative incidence rates for LREs were 0.0%, 2.2%, and 4.4%, respectively, in patients with stage 3; 0.0%, 6.2%, and 12.5%, respectively, in patients with stage 4A; 6.1%, 12.2%, and 13.6%, respectively, in patients with stage 4B; and 16.1%, 16.1%, and 26.1%, respectively, in patients with stage 4C.
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559
Research Article Table 4. Independent predictors of LRE occurrence.
Stage 3 Stage 4A Stage 4B Stage 4C
Characteristics
p = 0.0128*
p = 0.0411*
p = 0.0805
p = 0.0163* 29.0
Demographic data
25.8 20.7 17.1 16.1 12.5 12.2
12.5 9.7 6.3
4.3
6.3
4.3
8.7
2.4
Li v ev eren rel t ( ate % d )
Li v de erat rel h at (% ed )
Age, yr
0.003*
Male gender
0.242
0.703
-
-
Diabetes mellitus
0.003*
0.040* 2.519
1.041-6.093
Body mass index, 0.112 kg/m2 Etiology of liver disease Non-viral vs. viral 0.028*
0.029* 4.385
1.159-16.586
0.010*
0.548
-
-
Serum albumin, g/dl Total bilirubin, mg/dl AST, IU/L
0.006*
0.718
-
-
ALT, IU/L
0.085 <0.001*
0.110
-
-
0.003*
0.236
-
-
Child-Pugh score 6 vs. 5 Laboratory data
D
ec
H ca ep rc ato in c om el a lula (% r )
pe
ns
at
io
n
(%
)
0
om
Univariate Multivariate p value p value Hazard 95% CI ratio
Fig. 2. Proportion of patients who suffered from hepatic decompensation, HCC, liver-related death, and liver-related events according to Laennec stages (Mantel–Haenszel test). The proportion of patients who developed liver-related events was significantly correlated with the histological sub-classification of liver cirrhosis (p = 0.016). While the incidence of liver-related death only tended to be correlated with fibrosis stage (p = 0.081), the incidence of hepatic decompensation and HCC was significantly correlated with fibrosis stage (p = 0.013 and 0.041, respectively). ⁄p <0.05.
Prothrombin time, % Platelet count, 109/L Alpha fetoprotein, ng/ml Spleen size, cm
Liver stiffness values according to Laennec stage Among patients in the study population, reliable LS values were obtained in 144 (82.3%) patients. The median LS values for patients with stage 3 (n = 29), 4A (n = 16), 4B (n = 75), and 4C (n = 24) were 9.6 (range, 3.3–21.8), 7.7 (range, 5.1–39.8), 13.1 (range, 5.2–51.4), and 18.9 (range, 7.4–67.8) kPa, respectively (Fig. 4). While LS values did not differ between stage 3 and 4A (p = 0.145), LS values significantly increased as advanced stage from 4A to 4C (4A vs. 4B, p = 0.001 and 4B vs. 4C, p = 0.014, respectively).
Laennec stage on liver biopsy 3 (Reference)
0.754 0.264
0.609 0.287 0.116 1
4A
0.295
0.271
2.924
0.433-19.765
4B
0.014*
0.016* 6.158
1.403-27.022
4C
0.004*
0.004* 8.945
2.018-39.646
Reference values: viral etiology and Child–Pugh score 5.
Morphometric analysis
⁄
A significant increment in the mean septal thickness (46.4 ± 16.7 lm in 4A, 247.4 ± 209.7 lm in 4B, and 438.8 ± 299.7 lm in 4C; Supplementary Fig. 3A) and a decrement in the mean nodule size (3532.6 ± 1941.9 lm in 4A, 2299.8 ± 1066.6 lm in 4B, and 1201.1 ± 340.2 lm in 4C; Supplementary Fig. 3B) were identified as stage increased (p <0.01 for all comparisons among stages). Furthermore, the histological subclassification of cirrhosis showed a significant positive correlation with septal thickness (r = 0.616, p <0.001) and a negative correlation with nodule size (r = 0.647, p <0.001; Spearman’s correlation analysis).
Discussion Chronic liver disease has often been regarded as an illness that progresses to an end stage known as cirrhosis. In support of this view, the most commonly used histologic grading systems assign cirrhosis a single category at the most severe end of a series of categories [5–7]. In recent years, this viewpoint has been modi560
p <0.05.
fied by observations that cirrhosis may regress substantially on withdrawal of activity. Furthermore, patients with histologic cirrhosis vary greatly in their clinical severity and prognosis [3,26]. This point has long been recognized, as patients with cirrhosis are routinely subdivided into mild and severe categories based on blood tests, clinical features of portal hypertension, and more exact pressure measurements. These clinical criteria, embedded in the Child–Pugh and MELD classifications, have proven useful in assessing the need for transplantation and the response to medical therapy [1,2]. This refinement in the clinical assessment of patients with cirrhosis has been matched by the recent introduction of the Laennec staging system that subdivides cirrhosis into three degrees of histological severity. This classification is important since it captures clinically useful histologic information that would be lost if a unitary staging system was employed. The Laennec staging system is a modification of the METAVIR system with cirrhosis (F4) being subclassified into stages 4A, 4B, and 4C based on thickness of septa and size of nodules [13]. This system was validated by showing that the histologic severity of
Journal of Hepatology 2012 vol. 57 j 556–563
JOURNAL OF HEPATOLOGY Cumulative incidence rate of liver-related events
0.7 0.6
Stage 4B
p = 0.018
0.5 0.4
Stage 4C
0.3 0.2
Stage 4A
Stage 3
0.1 0
No. at risk Stage 3 Stage 4A Stage 4B Stage 4C
0
1
2
3
4 5 6 7 8 Year of enrollment
46 16 82 31
45 16 77 26
44 15 72 26
43 12 57 19
37 7 35 11
30 3 18 6
9 10 11
24 22 16 10 3 0 2 1 1 0 3 3 2 0
0
Fig. 3. Cumulative incidence of liver-related events according to Laennec stages (Kaplan–Meier plot). The cumulative incidence rates increased significantly with increasing fibrosis stage (p = 0.018, log-rank test).
Liver stiffness value (kPa)
p = 0.145
p = 0.001*
p = 0.014*
60 * 40 *
20 0 Stage 3
Stage 4A
Stage 4B
Stage 4C
Fig. 4. The distribution of liver stiffness values using transient elastography. While liver stiffness values did not differ between stage 3 and 4A (p = 0.145), liver stiffness values significantly increased as advanced stage from 4A to C (4A vs. 4B, p = 0.001 and 4B vs. 4C, p = 0.014, respectively). ⁄p <0.05.
cirrhosis was significantly correlated with hepatic vein pressure gradient, ascites, international normalized ratio (INR), bilirubin, and Child–Pugh class, and inversely correlated with albumin [12]. Several other studies have confirmed the importance of histological subdivision of cirrhosis by showing correlations between histologic and clinical severity in patient populations that included those with predominantly alcohol- or hepatitis Brelated disease [14,18,26,27]. Based on these cross-sectional studies, we designed this prospective longitudinal study to investigate whether histological sub-classification of cirrhosis can stratify the different clinical outcomes among patients with cirrhosis using LRE occurrence as the solid end point. In our cross-sectional analyses, we focused on HCC among LREs because HCC development is a significant watershed in the disease course of cirrhosis, an important cause of death, and an indicator of poor prognosis [2,28]. The incidence of LREs and HCC significantly increased as histological sub-classification of cirrhosis became more severe (between stage 3 and 4C). Furthermore, longitudinal, multivariate analysis showed histological sub-classification of cirrhosis to be an independent predictor of LRE occurrence. Taken together, our findings indicate that histological sub-classification of cirrhosis is useful in predicting LRE occurrence in patients with compensated cirrhosis. The lack of correlation between stage and incidence of liver-related death can be explained, in part, by the small number of patients in each
group, the small number of deaths, and potential sampling error in evaluation of needle biopsies [29]. Thus, further large scale studies are needed to evaluate the prediction of mortality using histologic subclassification of cirrhosis. In addition to histological sub-classification of cirrhosis, diabetes mellitus, and etiology of liver disease (non-viral vs. viral) were also identified as independent predictors of LRE occurrence in our study. Consistent with our results, diabetes mellitus is known to be a risk factor for chronic liver disease and to be associated with an increased risk of HCC and cirrhosis-related mortality [30]. In contrast to viral cirrhosis, which may be treated with antiviral agents, non-viral cirrhosis, for which no effective treatments have been established, was associated with a higher risk of LRE occurrence. Interestingly, age, Child–Pugh score, serum albumin level, prothrombin time, and platelet count, which have been identified as predictors of poor prognosis in cirrhotic patients [2], were only significant predictors of LRE occurrence in univariate analyses, their prognostic value being attenuated in multivariate analyses. Because our study population only comprised patients with compensated cirrhosis at enrollment, minute differences in variables related to liver function and portal hypertension may not have been sufficient to discriminate the different risks of LRE occurrence. This, in turn, further supports the notion that histological sub-classification of cirrhosis can independently stratify patients with compensated cirrhosis into specific groups with different risks of LRE occurrence, even when clinical parameters cannot. Although our study proved the usefulness of histological subclassification of cirrhosis, some issues still remain. First, histological sub-classification of cirrhosis, which adds two further categories to the grading system, can potentially aggravate the interpretational variability of LB specimens [11,31]. Thus, further studies investigating whether the clinical benefit of histological sub-classification of cirrhosis outweighs the fundamental pitfalls of LB, including interpretational variability, are required prior to adoption of the Laennec system in clinical practice. Second, although our study was performed based on histological evaluation, it is not feasible in clinical practice to obtain histological information on all patients with chronic liver disease who are suspected to have advanced liver fibrosis or cirrhosis because LB is an invasive procedure and cannot be performed repeatedly. However, because LB is the gold standard in the evaluation of liver fibrosis [11], we first tried to confirm whether sub-classification of cirrhosis based on LB specimens is a useful prognostic factor. As a next step, future studies may seek alternative noninvasive methods for monitoring the dynamic changes in the risk of LRE occurrence. Recently, transient elastography using FibroScanÒ was shown to sub-grade cirrhosis using the continuous expression of LS values after sufficient validation [32]. Indeed, LS values of stage 4B and 4C were significantly higher than those of stage 3, although LS values did not differ between stage 3 and 4A. Thus, it should be tested whether transient elastography with optimal cut-off values can predict LRE occurrence with similar or superior accuracy to histological sub-classification of cirrhosis using the Laennec system. Third, differences in treatment strategies, such as antiviral therapy, exist and can differently influence the incidence of LREs according to the etiology of chronic liver disease. Thus, incorporation of variables relating to intervening treatments according to the etiology of chronic liver disease should be investigated to provide more solid evidence in support of the usefulness of histological sub-classification of cirrhosis,
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Research Article although the usefulness of histological sub-classification of cirrhosis was also identified in a sub-group of patients with chronic hepatitis B virus infection in our study. Lastly, morphometric analysis also showed that each Laennec stage had a significant correlation to fibrosis thickness and nodule size. However, it should be further validated whether objective morphometric analysis of liver fibrosis more accurately predicts long-term prognosis. In conclusion, this prospective study showed that histological sub-classification of cirrhosis using the Laennec staging system can be useful in assessing risk of LRE occurrence among patients with cirrhosis. Our study provides a solid basis for further studies of non-invasive methods for monitoring the risk of LRE occurrence and should ultimately help physicians to establish optimum strategies for the management of patients with cirrhosis.
Grant support This study was supported in part by a Grant of the Korea Healthcare technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A102065) and by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MEST) (Nos. 2010-0008075, 2011-0030707) to Y.N. Park.
Author contributions Hyun Jung Oh: acquisition of data, analysis and interpretation of data, drafting of manuscript, and statistical analysis; Seung Up Kim: study concept and design, analysis and interpretation of data, drafting of manuscript, and study supervision; Ian R. Wanless: critical revision of manuscript; Sarah Lee: acquisition of data and technical support; Kwang-Hyub Han: critical revision of manuscript and study supervision; Young Nyun Park: acquisition of data, technical support, critical revision of manuscript, and study supervision.
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2012.04. 029.
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JOURNAL OF HEPATOLOGY [28] Ripoll C, Groszmann RJ, Garcia Tsao G, Bosch J, Grace N, Burroughs A, et al. Hepatic venous pressure gradient predicts development of hepatocellular carcinoma independently of severity of cirrhosis. J Hepatol 2009;50:923–928. [29] Hytiroglou P, Snover DC, Alves V, Balabaud C, Bhathal PS, Bioulac-Sage P, et al. Beyond ‘‘cirrhosis’’: a proposal from the International Liver Pathology Study Group. Am J Clin Pathol 2012;137:5–9. [30] Garcia Compean D, Jaquez Quintana JO, Gonzalez Gonzalez JA, Maldonado Garza H. Liver cirrhosis and diabetes: risk factors, pathophysiology, clinical implications and management. World J Gastroenterol 2009;15:280–288.
[31] Goldin RD, Goldin JG, Burt AD, Dhillon PA, Hubscher S, Wyatt J, et al. Intraobserver and inter-observer variation in the histopathological assessment of chronic viral hepatitis. J Hepatol 1996;25:649–654. [32] Jung KS, Kim SU, Ahn SH, Park YN, Kim DY, Park JY, et al. Risk assessment of hepatitis B virus-related hepatocellular carcinoma development using liver stiffness measurement (FibroScan). Hepatology 2011;53:885–894.
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The Laennec staging system for histological sub-classification of cirrhosis is useful for stratification of prognosis in patients with liver cirrhosis Seung Up Kim1,2,4, , Hyun Jung Oh1, , Ian R. Wanless7, Sarah Lee3, Kwang-Hyub Han1,2,4,5,⇑, Young Nyun Park3,4,5,6,⇑ 1
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea; 2Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea; 3Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea; 4Liver Cirrhosis Clinical Research Center, Seoul, South Korea; 5Brain Korea 21 Project of Medical Science, South Korea; 6Integrated Genomic Research Center for Metabolic Regulation, Seoul, South Korea; 7Department of Laboratory Medicine, Queen Elizabeth II Health Sciences Centre, Halifax, Canada
Backgrounds & Aims: The clinical severity of cirrhosis varies widely. We investigated whether histological sub-classification of cirrhosis using the Laennec system can discriminate different outcomes among patients with cirrhosis. Methods: One hundred and seventy-five patients with chronic liver disease who underwent liver biopsy and showed stage 3 or 4 fibrosis between January 2001 and December 2008 were prospectively enrolled. Cirrhosis was sub-classified into three groups (4A, 4B, and 4C) according to the Laennec system. The end point was liver-related event (LRE) occurrence, including decompensation, hepatocellular carcinoma, and liver-related death. Results: The median age of the patients (110 men, 65 women) was 55 years. Stages 3, 4A, 4B, and 4C were identified in 46 (26.3%), 16 (9.1%), 82 (46.9%), and 31 (17.7%) patients, respectively. During the follow-up period, LREs occurred in 32 (18.3%) patients: 4 (8.7%) with stage 3, 2 (12.5%) with stage 4A, 17 (20.7%) with stage 4B, and 9 (29.0%) with stage 4C. In a multivariate analysis, histological sub-classification of cirrhosis independently predicted LRE occurrence. While patients with stage 4A tended to be at higher risk of LRE occurrence than those with stage 3, patients with stages 4B and 4C had significantly higher risks of LRE occurrence, with hazard ratios of 6.158 (p = 0.016) and 8.945 (p = 0.004), respectively.
Keywords: Cirrhosis; Fibrosis; Laennec staging system; Liver biopsy; Liver-related event. Received 14 November 2011; received in revised form 11 April 2012; accepted 27 April 2012; available online 19 May 2012 ⇑ Corresponding authors. Addresses: Department of Internal Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun–gu, Seoul 120–752, Korea. Tel: +82 2 2228 1949; fax: +82 2 363 6994 (K.-H. Han), Department of Pathology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun–gu, Seoul 120–752, Korea. Tel.: +82 2 2228 1768; fax: +82 2 362 0860 (Y.N. Park). E-mail addresses: [email protected] (K.-H. Han), [email protected] (Y.N. Park). These authors contributed equally to this work. Abbreviations: HCC, hepatocellular carcinoma; LRE, liver related event; LB, liver biopsy; TE, transient elastography; LS, liver stiffness; AFP, alpha-fetoprotein; HR, hazard ratio; CI, confidence interval; INR, international normalized ratio.
Conclusions: Histological sub-classification of cirrhosis using the Laennec system can be used to assess the risk of LRE occurrence among patients with cirrhosis. Our study provides a solid basis for further studies of non-invasive methods for monitoring the risk of LRE occurrence and will help physicians to establish optimum treatment strategies. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Liver cirrhosis is histologically defined as a diffuse state with replacement of the normal lobular architecture by abnormal nodules and fibrous septa [1]. Although cirrhosis is widely considered to be an end-stage liver disease, clinical severity, and prognosis within this diagnosis vary widely [2]. Clinically, cirrhosis can be categorized into compensated and decompensated states [3]. While this approach may give a clear indication for liver transplantation [4], it is of no use in predicting the development of hepatic decompensation. Furthermore, it does not provide prognostic information for the bulk of cirrhotic patients who are compensated and might benefit from tailored management to prevent decompensation. Therefore, the identification of prognostic factors to predict the development of liver-related events (LREs), such as hepatic decompensation, hepatocellular carcinoma (HCC), and liver-related mortality in patients with compensated cirrhosis is important. Among the characteristics of patients with compensated cirrhosis, the degree of liver fibrosis on biopsy may be important for predicting the development of hepatic decompensation. However, currently used histological staging systems, such as the Ishak [5], METAVIR [6], and Batts systems [7], group all patients with cirrhosis into a single category without taking account the severity of cirrhosis. Furthermore, because recent antiviral and anti-fibrotic treatments can contribute to the regression of fibrosis in patients with histological cirrhosis [8–10], the simple onestage description for cirrhosis is not able to stratify patients for
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JOURNAL OF HEPATOLOGY treatment assignment or evaluation of treatment response [3]. Thus, a more refined histological sub-classification of cirrhosis is needed [11]. The Laennec staging system, a modification of the METAVIR system, was proposed to meet this need [12,13]. This system subdivides cirrhosis into three groups (4A, 4B, and 4C) based on the thickness of the fibrous septa and the size of nodules. Although recent cross-sectional studies reported the usefulness of the Laennec system in correlating clinical stage and grade of portal hypertension [12,14], no prospective longitudinal study has documented the predictive values of the Laennec system using LRE occurrence, including hepatic decompensation, HCC, and liverrelated death, as solid end points. In this study, we evaluated the Laennec staging system for predicting LRE occurrence in patients with cirrhosis and therefore stratifying these patients according to prognosis.
fication of 20. The thickness of the fibrous septa and the size of the nodules (maximum distance between two adjacent septa) were measured and the mean value of each case was calculated.
Measurement of liver stiffness values After transient elastography (TE) using FibroScanÒ (EchoSens, Paris, France) was introduced in our institute, liver stiffness (LS) values by TE were obtained for all patients who underwent LB. LS measurement using TE was performed according to the manufacturer’s recommendations [17]. All LS measurements were obtained by one experienced technician who was blinded to the clinical and histologic data. In this study, only LS values with at least 10 validated measurements and a success rate of at least 60%, and interquartile range to median value ratio less than 0.3, were considered reliable. The median interval between TE and LB was 12 (range, 0–18) days.
Laboratory tests In addition to demographic data, the following laboratory parameters were measured at the time of LB: serum albumin level, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alpha-fetoprotein (AFP), prothrombin time, and platelet count.
Patients and methods Patients Between January 2001 and December 2008, a total of 712 patients with chronic hepatitis who underwent liver biopsy (LB) at Severance Hospital, Yonsei University College of Medicine, Seoul, Korea were consecutively enrolled in this prospective study. Informed consent was obtained from all patients before enrollment. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board of our institute. Because we planned to calculate the relative risk of LRE occurrence in patients with cirrhosis (stage 4A, 4B, or 4C), as compared to stage 3, we first excluded 481 patients with Laennec stage 0–2 on biopsy and a further 56 patients if the biopsy was less than 10 mm in length or if other exclusion criteria were present. The remaining 175 patients with stage 3, 4A, 4B, or 4C were selected for statistical analysis (Supplementary Fig. 1).
Follow-up Each patient was screened for HCC by ultrasonography at their initial visit. Six patients were excluded due to the presence of HCC at enrollment. If no evidence of HCC was found, patients were subjected to periodic surveillance by ultrasonography and laboratory tests, including measurement of AFP levels every 3 or 6 months. Furthermore, the development of hepatic decompensation was monitored during the study period. If LREs occurred, patients were admitted for immediate and appropriate management. The last follow-up month of our study was April 2011 and the median follow-up duration was 52.4 (range, 9.7–125.2) months. The follow-up duration was similar among fibrosis stages (69.2 ± 30.2, 51.6 ± 11.7, 51.2 ± 15.0, and 54.2 ± 20.1 months in stage 3, 4A, 4B, and 4C, respectively; all p >0.05).
Liver biopsy and histological assessment The indication of LB was to assess the severity of liver fibrosis and necroinflammation. LB was performed before starting antiviral treatment, especially in patients with chronic hepatitis B or C virus infection. All patients underwent ultrasoundguided percutaneous LB. LB specimens were formalin fixed and paraffin embedded. Then, 4-lm-thick sections were stained with hematoxylin-eosin and Masson’s trichrome. All liver tissue samples were evaluated by an experienced hepatopathologist (Y.N. Park) who had no access to clinical data for the study population. The degree of liver fibrosis was evaluated semi-quantitatively according to the Laennec system (Supplementary Fig. 2) [13]. Fibrosis was first scored as follows: 0, no definite fibrosis; 1, minimal fibrosis (no septa or rare thin septum; may have portal expansion or mild sinusoidal fibrosis); 2, mild fibrosis (occasional thin septa); 3, moderate fibrosis (moderate thin septa; up to incomplete cirrhosis); and 4, cirrhosis. Stage 4 was distinguished from stage 3 by more numerous septa per unit length of biopsy sufficient to display nodules with rounded contours. Then, stage 4 was sub-classified into three groups: 4A, mild cirrhosis, definite or probable; 4B, moderate cirrhosis (at least two broad septa); and 4C, severe cirrhosis (at least one very broad septum or many minute nodules). The terms ‘broad septum’ and ‘very broad septum’ were defined by comparing the relative thickness of fibrous septa and sizes of nodules [14]. Activity grade (degree of necroinflammatory activity in the lobules and periportal area) was scored as follows: A0, none; A1, mild activity; A2, moderate activity; and A3, severe activity. Activity grade was defined as lobule or periportal activity, whichever was the higher. The median length of biopsies in the study set was 16 mm (range, 10– 25 mm). The mean biopsy length of each fibrosis stage was statistically equivalent (15.0 ± 3.6, 16.1 ± 3.1, 16.0 ± 3.2, and 16.6 ± 3.5 mm in stages 3, 4A, 4B, and 4C, respectively; all p >0.05 among fibrosis stages). Morphometry Morphometry was performed as described [15,16]. Briefly, trichrome-stained sections of all liver biopsies were examined without knowledge of the histological score. Digital images of 1–4 fields of each biopsy were captured at a final magni-
Definitions of liver-related events The primary end point was LRE occurrence, defined as a cirrhosis-related complication including hepatic decompensation, HCC, and liver-related death. Hepatic decompensation comprised ascites, variceal bleeding, hepatic encephalopathy, spontaneous bacterial peritonitis, and hepatorenal syndrome. Ascites was diagnosed clinically and was confirmed by ultrasonography or computed tomography [18]. Variceal bleeding was diagnosed endoscopically [19]. Hepatic encephalopathy was diagnosed based on the presence of temporospatial disorientation, altered level of consciousness, or asterixis in the absence of other possible causes [20]. Spontaneous bacterial peritonitis (SBP) was defined as an ascitic fluid infection without an apparent intra-abdominal surgically treatable source with confirmation by culture and an elevated ascitic fluid absolute polymorphonuclear leukocyte count (P250 cells/mm3) [21]. Hepatorenal syndrome was diagnosed when acute renal failure developed in association with advanced chronic liver disease, after exclusion of other causes of renal failure [22]. For patients enrolled before 2005, diagnosis of HCC was based on the guidelines of the European Association for the Study of the Liver (EASL) [23]. Briefly, for nodules larger than 2 cm, HCC can be diagnosed by the coincident findings from at least two imaging techniques showing typical features of arterial hypervascularization, or findings from one imaging technique and an AFP level of >400 ng/ ml. For nodules between 1 and 2 cm in size, biopsy is recommended. For patients who were enrolled after 2005, HCC was diagnosed based on the guidelines of the American Association for the Study of Liver Diseases (AASLD) [24]. Briefly, for tumors larger than 2 cm, HCC was diagnosed when typical features of HCC were detected (hypervascularity and washout in the portal/venous phase) by one dynamic imaging technique and the AFP level was >200 ng/ml. For nodules between 1 and 2 cm in size, it was necessary to detect typical features of HCC using two dynamic imaging techniques. For tumors smaller than 1 cm, ultrasonography was repeated after 3 months. Liver-related deaths included death caused by hepatic failure (regardless of etiology), HCC progression, variceal bleeding, and acute infections such as SBP [25].
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Research Article of grade 3 necroinflammatory activity was high in patients with stage 3 (n = 20, 43.5%) but low in stage 4 (n = 4, 3.1%).
Statistical analysis Data are expressed as mean ± SD, median (range), or n (%) as appropriate. Characteristics of patients with and without LRE occurrence and HCC were compared using Chi-square test or Fisher’s exact test (for categorical data), or unpaired Student’s t-test or Mann–Whitney U test (continuous variables). The proportions of patients with LRE occurrence based on Laennec stage were compared using Mantel–Haenszel tests. Univariate and multivariate Cox proportional hazard regression analyses were used to estimate independent predictors of LRE occurrence. The time factor in univariate and multivariate analyses was defined as the time period from enrollment to first LRE occurrence. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were calculated. The Kaplan–Meier method was used to examine the cumulative incidence of LREs based on histological fibrosis stage. A p value of <0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 18.0 (Chicago, IL, USA).
Results Baseline characteristics The baseline characteristics of the study population at enrollment are summarized in Table 1. The median age of the patients (110 men and 65 women) was 55 years. The etiology of liver disease was mostly hepatitis B virus infection (n = 147, 84%). The majority of patients had Child–Pugh scores of 5 (n = 158, 90.3%). After LB, antiviral treatment was initiated using oral nucleos(t)ide analogues or pegylated interferon for patients with chronic hepatitis B and the combination of pegylated interferon and ribavirin for those with chronic hepatitis C.
Comparisons of patients with and without LREs or HCC During the follow-up period, LREs occurred in 32 (18.3%) patients. In particular, HCC developed in 22 (12.6%) patients. The baseline characteristics of patients with and without LREs or HCC are shown in Table 3. Patients in whom LREs occurred were significantly older, had higher rates of diabetes mellitus and Child–Pugh score, and had lower serum albumin level, prothrombin time, and platelet count, than those in whom LREs did not occur (all p <0.05). Patients who developed HCC were more likely to be male, and had lower BMI value, prothrombin time, and platelet count, than those who did not develop HCC (all p <0.05). When cirrhosis was histologically sub-classified using the Laennec system, the distribution of stages did not differ between patients with LREs or HCC and those without (Table 3). However, when we stratified our study population into two groups (patients with stage 3 and 4A vs. those with 4B and 4C), we found that rates of stage 4B and 4C were significantly higher in patients with LREs or HCC than in those without LREs or HCC (all p <0.05) (Fig. 1). Incidence of LREs based on Laennec stage
Values
The proportion of patients with LRE occurrence significantly correlated with the histological sub-classification of cirrhosis (p = 0.016, Fig. 2). During the follow-up period, LREs occurred in 8.7% of patients with stage 3, 12.5% of patients with stage 4A, 20.7% of patients with stage 4B, and 29.0% of patients with stage 4C. Moreover, while the incidence of liver-related death tended to be associated with stage (p = 0.081), the incidence of hepatic decompensation and HCC significantly correlated with stage (p = 0.013 and 0.041, respectively, Fig. 2).
Age, yr
55 (18-73)
Risk assessment of LRE occurrence according to Laennec stage
Male gender
110 (62.9)
Diabetes mellitus
24 (13.7)
Body mass index, kg/m2
23.4 (17.2-34.0)
Liver histology Stage 3, 4A, 4B, and 4C was identified in 46 (26.3%), 16 (9.1%), 82 (46.9%), and 31 (17.7%) patients, respectively (Table 2). The rate Table 1. Baseline characteristics (n = 175).
Characteristics Demographic data
In multivariate analysis, diabetes mellitus, etiology of liver disease (non-viral vs. viral), and histological sub-classification of
Etiology of liver disease HBV/HCV/others
147 (84.0)/17 (9.7)/11 (6.3)
Child-Pugh score 5/6
158 (91.3)/15 (8.7)
Table 2. Histological information.
Laennec stage
n (%)
Activity grade n
3
46 (26.3)
1
16
2
10
Laboratory data Serum albumin, g/dl
4.2 (3.5-5.1) 4A
16 (9.1)
3
20
1
15
Total bilirubin, mg/dl
0.8 (0.3-2.0)
AST, IU/L
39 (13-445)
2
1
ALT, IU/L
46 (11-609)
3
0
Prothrombin time, %
92.5 (77-100)
1
55
Platelet count, 109/L
141 (102-314)
2
26
5.0 (1.2-190)
3
1
1
14
2
14
3
3
Alpha fetoprotein, ng/ml
4B
4C
Biopsy length, mm
16 (10-25)
Spleen size, cm
10.5 (6.3-14.5)
Variables are expressed as median (range) or n (%). HBV, hepatitis B virus; HCV, hepatitis C virus.
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82 (46.9)
31 (17.7)
JOURNAL OF HEPATOLOGY Table 3. Comparison of baseline characteristics between patients with and without LRE or HCC.
Characteristics
Patients with LRE occurrence
Patients without LRE occurrence
(n = 32, 17.8%)
(n = 143, 82.2%)
Age, yr
51.2 ± 10.3
46.4 ± 11.6
Male gender
24 (75.0)
86 (60.1)
Diabetes mellitus
9 (28.1) 23.0 ± 3.1
p value
p value
Patients with HCC development
Patients without HCC development
(n = 22, 12.6%)
(n = 153, 87.4%)
0.033*
51.1 ± 9.3
46.7 ± 11.7
0.089
0.116
19 (86.4)
91 (59.5)
0.015*
15 (10.5)
0.009*
5 (22.7)
19 (12.4)
0.189
23.9 ± 2.8
0.136
22.4 ± 2.7
24.0 ± 2.8
0.023*
19 (86.4)/2 (9.1)/ 1 (4.5)
128 (83.7)/15 (9.8)/ 10 (6.5)
19 (86.4)/3 (13.6)
139 (92.1)/12 (7.9)
Demographic data
2
Body mass index, kg/m
Etiology of liver disease HBV/HCV/ others Child-Pugh score
0.550
0.929
24 (75.0)/3 (9.4)/ 5 (15.6)
123 (86.0)/14 (9.8)/ 6 (4.2)
26 (81.3)/6 (18.8)
132 (93.6)/9 (6.4)
Serum albumin, g/dl
3.9 ± 0.5
4.2 ± 0.4
0.004*
4.0 ± 0.5
4.1 ± 0.5
0.171
Total bilirubin, mg/dl
0.9 ± 0.3
0.9 ± 0.4
0.496
0.9 ± 0.3
0.9 ± 0.4
0.629
AST, IU/L
49.8 ± 38.5
54.5 ± 52.9
0.505
45.6 ± 36.8
56.6 ± 52.0
0.341
ALT, IU/L
55.6 ± 52.5
74.6 ± 86.4
0.204
55.8 ± 53.8
73.2 ± 84.4
0.349
Prothrombin time, %
89.0 ± 9.0
93.5 ± 6.2
<0.001* 89.4 ± 6.2
93.8 ± 6.9
0.017*
Platelet count, 10 /L
156 ± 44
174 ± 50
0.005*
154 ± 41
172 ± 52
0.016*
Alpha fetoprotein, ng/ml
13.0 ± 12.5
10.9 ± 23.8
0.638
14.9 ± 14.0
10.8 ± 23.1
0.432
11.0 ± 1.8
10.6 ± 1.9
0.327
10.5 ± 1.6
10.7 ± 1.9
0.623
0.025*
5/6
0.376
Laboratory data
9
Spleen size, cm Laennec stage on liver biopsy
0.116
0.152
3
4 (12.5)
42 (29.4)
2 (9.1)
44 (28.8)
4A
2 (6.3)
14 (9.8)
1 (4.5)
15 (9.8)
4B
17 (53.1)
65 (45.5)
14 (63.6)
68 (44.4)
4C
9 (28.1)
22 (15.4)
5 (22.7)
26 (17.0)
Variables are expressed as mean ± SD or n (%). HBV, hepatitis B virus; HCV, hepatitis C virus. ⁄ p <0.05.
p = 0.029*
100 90 15.4 80 70 45.5 60 50 40 9.8 30 20 29.4 10 0
Stage 3 Stage 4A Stage 4B Stage 4C
p = 0.022*
17.0
22.7
28.1
44.4 63.6
53.1 9.8 6.3 12.5
-
+
Liver-related event
28.8
4.5 9.1
-
+
Hepatocellular carcinoma
Fig. 1. Laennec stage patterns in patients with liver-related events and HCC and in those without. The rates of stage 4B and C were significantly higher in patients with liver-related event and HCC than in those without (p = 0.029 and 0.022, respectively). ⁄p <0.05.
cirrhosis using the Laennec system independently predicted LRE occurrence (all p <0.05) (Table 4). Moreover, while patients with
stage 4A tended to be at higher risk of suffering LREs (p = 0.271; HR, 2.924; 95% CI, 0.433–19.705) than those with stage 3, those with 4B or 4C had a significantly higher risk of LRE occurrence, with HRs of 6.158 (95% CI, 1.403–27.022; p = 0.016) and 8.945 (95% CI, 2.018–39.646; p = 0.004), respectively. When patients with chronic hepatitis B virus (n = 147, 84.0%) were sub-analyzed, the Laennec system was also selected as an independent predictor of LRE occurrence (p = 0.816, HR 1.318, 95% CI 0.129–13.470 in stage 4A; p = 0.050, HR 3.879, 95% CI, 0.896–16.793 in stage 4B; p = 0.035, HR 4.193, 95% CI 1.106– 15.886 in stage 4C with the reference to stage 3). Cumulative incidence of LREs according to Laennec stage The cumulative incidence rates of LREs increased significantly with increasing fibrosis stage, as shown in Fig. 3 (p = 0.018, logrank test). The 1-, 2-, and 3-year cumulative incidence rates for LREs were 0.0%, 2.2%, and 4.4%, respectively, in patients with stage 3; 0.0%, 6.2%, and 12.5%, respectively, in patients with stage 4A; 6.1%, 12.2%, and 13.6%, respectively, in patients with stage 4B; and 16.1%, 16.1%, and 26.1%, respectively, in patients with stage 4C.
Journal of Hepatology 2012 vol. 57 j 556–563
559
Research Article Table 4. Independent predictors of LRE occurrence.
Stage 3 Stage 4A Stage 4B Stage 4C
Characteristics
p = 0.0128*
p = 0.0411*
p = 0.0805
p = 0.0163* 29.0
Demographic data
25.8 20.7 17.1 16.1 12.5 12.2
12.5 9.7 6.3
4.3
6.3
4.3
8.7
2.4
Li v ev eren rel t ( ate % d )
Li v de erat rel h at (% ed )
Age, yr
0.003*
Male gender
0.242
0.703
-
-
Diabetes mellitus
0.003*
0.040* 2.519
1.041-6.093
Body mass index, 0.112 kg/m2 Etiology of liver disease Non-viral vs. viral 0.028*
0.029* 4.385
1.159-16.586
0.010*
0.548
-
-
Serum albumin, g/dl Total bilirubin, mg/dl AST, IU/L
0.006*
0.718
-
-
ALT, IU/L
0.085 <0.001*
0.110
-
-
0.003*
0.236
-
-
Child-Pugh score 6 vs. 5 Laboratory data
D
ec
H ca ep rc ato in c om el a lula (% r )
pe
ns
at
io
n
(%
)
0
om
Univariate Multivariate p value p value Hazard 95% CI ratio
Fig. 2. Proportion of patients who suffered from hepatic decompensation, HCC, liver-related death, and liver-related events according to Laennec stages (Mantel–Haenszel test). The proportion of patients who developed liver-related events was significantly correlated with the histological sub-classification of liver cirrhosis (p = 0.016). While the incidence of liver-related death only tended to be correlated with fibrosis stage (p = 0.081), the incidence of hepatic decompensation and HCC was significantly correlated with fibrosis stage (p = 0.013 and 0.041, respectively). ⁄p <0.05.
Prothrombin time, % Platelet count, 109/L Alpha fetoprotein, ng/ml Spleen size, cm
Liver stiffness values according to Laennec stage Among patients in the study population, reliable LS values were obtained in 144 (82.3%) patients. The median LS values for patients with stage 3 (n = 29), 4A (n = 16), 4B (n = 75), and 4C (n = 24) were 9.6 (range, 3.3–21.8), 7.7 (range, 5.1–39.8), 13.1 (range, 5.2–51.4), and 18.9 (range, 7.4–67.8) kPa, respectively (Fig. 4). While LS values did not differ between stage 3 and 4A (p = 0.145), LS values significantly increased as advanced stage from 4A to 4C (4A vs. 4B, p = 0.001 and 4B vs. 4C, p = 0.014, respectively).
Laennec stage on liver biopsy 3 (Reference)
0.754 0.264
0.609 0.287 0.116 1
4A
0.295
0.271
2.924
0.433-19.765
4B
0.014*
0.016* 6.158
1.403-27.022
4C
0.004*
0.004* 8.945
2.018-39.646
Reference values: viral etiology and Child–Pugh score 5.
Morphometric analysis
⁄
A significant increment in the mean septal thickness (46.4 ± 16.7 lm in 4A, 247.4 ± 209.7 lm in 4B, and 438.8 ± 299.7 lm in 4C; Supplementary Fig. 3A) and a decrement in the mean nodule size (3532.6 ± 1941.9 lm in 4A, 2299.8 ± 1066.6 lm in 4B, and 1201.1 ± 340.2 lm in 4C; Supplementary Fig. 3B) were identified as stage increased (p <0.01 for all comparisons among stages). Furthermore, the histological subclassification of cirrhosis showed a significant positive correlation with septal thickness (r = 0.616, p <0.001) and a negative correlation with nodule size (r = 0.647, p <0.001; Spearman’s correlation analysis).
Discussion Chronic liver disease has often been regarded as an illness that progresses to an end stage known as cirrhosis. In support of this view, the most commonly used histologic grading systems assign cirrhosis a single category at the most severe end of a series of categories [5–7]. In recent years, this viewpoint has been modi560
p <0.05.
fied by observations that cirrhosis may regress substantially on withdrawal of activity. Furthermore, patients with histologic cirrhosis vary greatly in their clinical severity and prognosis [3,26]. This point has long been recognized, as patients with cirrhosis are routinely subdivided into mild and severe categories based on blood tests, clinical features of portal hypertension, and more exact pressure measurements. These clinical criteria, embedded in the Child–Pugh and MELD classifications, have proven useful in assessing the need for transplantation and the response to medical therapy [1,2]. This refinement in the clinical assessment of patients with cirrhosis has been matched by the recent introduction of the Laennec staging system that subdivides cirrhosis into three degrees of histological severity. This classification is important since it captures clinically useful histologic information that would be lost if a unitary staging system was employed. The Laennec staging system is a modification of the METAVIR system with cirrhosis (F4) being subclassified into stages 4A, 4B, and 4C based on thickness of septa and size of nodules [13]. This system was validated by showing that the histologic severity of
Journal of Hepatology 2012 vol. 57 j 556–563
JOURNAL OF HEPATOLOGY Cumulative incidence rate of liver-related events
0.7 0.6
Stage 4B
p = 0.018
0.5 0.4
Stage 4C
0.3 0.2
Stage 4A
Stage 3
0.1 0
No. at risk Stage 3 Stage 4A Stage 4B Stage 4C
0
1
2
3
4 5 6 7 8 Year of enrollment
46 16 82 31
45 16 77 26
44 15 72 26
43 12 57 19
37 7 35 11
30 3 18 6
9 10 11
24 22 16 10 3 0 2 1 1 0 3 3 2 0
0
Fig. 3. Cumulative incidence of liver-related events according to Laennec stages (Kaplan–Meier plot). The cumulative incidence rates increased significantly with increasing fibrosis stage (p = 0.018, log-rank test).
Liver stiffness value (kPa)
p = 0.145
p = 0.001*
p = 0.014*
60 * 40 *
20 0 Stage 3
Stage 4A
Stage 4B
Stage 4C
Fig. 4. The distribution of liver stiffness values using transient elastography. While liver stiffness values did not differ between stage 3 and 4A (p = 0.145), liver stiffness values significantly increased as advanced stage from 4A to C (4A vs. 4B, p = 0.001 and 4B vs. 4C, p = 0.014, respectively). ⁄p <0.05.
cirrhosis was significantly correlated with hepatic vein pressure gradient, ascites, international normalized ratio (INR), bilirubin, and Child–Pugh class, and inversely correlated with albumin [12]. Several other studies have confirmed the importance of histological subdivision of cirrhosis by showing correlations between histologic and clinical severity in patient populations that included those with predominantly alcohol- or hepatitis Brelated disease [14,18,26,27]. Based on these cross-sectional studies, we designed this prospective longitudinal study to investigate whether histological sub-classification of cirrhosis can stratify the different clinical outcomes among patients with cirrhosis using LRE occurrence as the solid end point. In our cross-sectional analyses, we focused on HCC among LREs because HCC development is a significant watershed in the disease course of cirrhosis, an important cause of death, and an indicator of poor prognosis [2,28]. The incidence of LREs and HCC significantly increased as histological sub-classification of cirrhosis became more severe (between stage 3 and 4C). Furthermore, longitudinal, multivariate analysis showed histological sub-classification of cirrhosis to be an independent predictor of LRE occurrence. Taken together, our findings indicate that histological sub-classification of cirrhosis is useful in predicting LRE occurrence in patients with compensated cirrhosis. The lack of correlation between stage and incidence of liver-related death can be explained, in part, by the small number of patients in each
group, the small number of deaths, and potential sampling error in evaluation of needle biopsies [29]. Thus, further large scale studies are needed to evaluate the prediction of mortality using histologic subclassification of cirrhosis. In addition to histological sub-classification of cirrhosis, diabetes mellitus, and etiology of liver disease (non-viral vs. viral) were also identified as independent predictors of LRE occurrence in our study. Consistent with our results, diabetes mellitus is known to be a risk factor for chronic liver disease and to be associated with an increased risk of HCC and cirrhosis-related mortality [30]. In contrast to viral cirrhosis, which may be treated with antiviral agents, non-viral cirrhosis, for which no effective treatments have been established, was associated with a higher risk of LRE occurrence. Interestingly, age, Child–Pugh score, serum albumin level, prothrombin time, and platelet count, which have been identified as predictors of poor prognosis in cirrhotic patients [2], were only significant predictors of LRE occurrence in univariate analyses, their prognostic value being attenuated in multivariate analyses. Because our study population only comprised patients with compensated cirrhosis at enrollment, minute differences in variables related to liver function and portal hypertension may not have been sufficient to discriminate the different risks of LRE occurrence. This, in turn, further supports the notion that histological sub-classification of cirrhosis can independently stratify patients with compensated cirrhosis into specific groups with different risks of LRE occurrence, even when clinical parameters cannot. Although our study proved the usefulness of histological subclassification of cirrhosis, some issues still remain. First, histological sub-classification of cirrhosis, which adds two further categories to the grading system, can potentially aggravate the interpretational variability of LB specimens [11,31]. Thus, further studies investigating whether the clinical benefit of histological sub-classification of cirrhosis outweighs the fundamental pitfalls of LB, including interpretational variability, are required prior to adoption of the Laennec system in clinical practice. Second, although our study was performed based on histological evaluation, it is not feasible in clinical practice to obtain histological information on all patients with chronic liver disease who are suspected to have advanced liver fibrosis or cirrhosis because LB is an invasive procedure and cannot be performed repeatedly. However, because LB is the gold standard in the evaluation of liver fibrosis [11], we first tried to confirm whether sub-classification of cirrhosis based on LB specimens is a useful prognostic factor. As a next step, future studies may seek alternative noninvasive methods for monitoring the dynamic changes in the risk of LRE occurrence. Recently, transient elastography using FibroScanÒ was shown to sub-grade cirrhosis using the continuous expression of LS values after sufficient validation [32]. Indeed, LS values of stage 4B and 4C were significantly higher than those of stage 3, although LS values did not differ between stage 3 and 4A. Thus, it should be tested whether transient elastography with optimal cut-off values can predict LRE occurrence with similar or superior accuracy to histological sub-classification of cirrhosis using the Laennec system. Third, differences in treatment strategies, such as antiviral therapy, exist and can differently influence the incidence of LREs according to the etiology of chronic liver disease. Thus, incorporation of variables relating to intervening treatments according to the etiology of chronic liver disease should be investigated to provide more solid evidence in support of the usefulness of histological sub-classification of cirrhosis,
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Research Article although the usefulness of histological sub-classification of cirrhosis was also identified in a sub-group of patients with chronic hepatitis B virus infection in our study. Lastly, morphometric analysis also showed that each Laennec stage had a significant correlation to fibrosis thickness and nodule size. However, it should be further validated whether objective morphometric analysis of liver fibrosis more accurately predicts long-term prognosis. In conclusion, this prospective study showed that histological sub-classification of cirrhosis using the Laennec staging system can be useful in assessing risk of LRE occurrence among patients with cirrhosis. Our study provides a solid basis for further studies of non-invasive methods for monitoring the risk of LRE occurrence and should ultimately help physicians to establish optimum strategies for the management of patients with cirrhosis.
Grant support This study was supported in part by a Grant of the Korea Healthcare technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A102065) and by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MEST) (Nos. 2010-0008075, 2011-0030707) to Y.N. Park.
Author contributions Hyun Jung Oh: acquisition of data, analysis and interpretation of data, drafting of manuscript, and statistical analysis; Seung Up Kim: study concept and design, analysis and interpretation of data, drafting of manuscript, and study supervision; Ian R. Wanless: critical revision of manuscript; Sarah Lee: acquisition of data and technical support; Kwang-Hyub Han: critical revision of manuscript and study supervision; Young Nyun Park: acquisition of data, technical support, critical revision of manuscript, and study supervision.
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2012.04. 029.
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