Background
Liver biopsy is currently recommended as the gold standard method of staging fibrosis in patients with chronic hepatitis C
Another non-invasive approach relies on the measurement of substances that regulate fibrosis or participate in the generation of the liver extracellular matrix. The most applicable include hyaluronic acid (HA)
The first aim of this study was to evaluate the diagnostic value of HA for significant fibrosis (F2-F4), severe fibrosis, (F3F4) and cirrhosis (F4), in patients with HCV infection. The second aim was to determine the serum HA level cut-off to predict both presence and absence of F2-F4, F3F4, and F4.
Results
Patients
The cohort included 405 patients. Table
Characteristics of the 405 patients at the time of liver biopsy (comparison between the training and the validation sets).
Characteristics
Training set (n = 151)
Validation set (n = 254)
All patients (n = 405)
Age (Mean ± SD)
51 ± 14
47 ± 12
49 ± 13
Male (n (%))
82 (54)
133 (52)
215 (53)
HA (μg/l) (Mean {95% CI})
63 {47;79}
53 {41;65}
57 {47;67}
Stage of fibrosis (n (%))
0
28 (19)
33 (13)
61 (15)
1
51 (34)
103 (41)
154 (38)
2
33 (22)
58 (23)
91 (23)
3
27 (18)
47 (18)
74 (18)
4
12 (7)
13 (5)
25 (6)
Hyaluronic acid and fibrosis in the training set
Figure
Box & Whisker plot representing the relation between the stage of fibrosis and HA level
Box & Whisker plot representing the relation between the stage of fibrosis and HA level. The line through the box is the median; the top and bottom edges of each box represent the 25th and 75th percentiles, giving the interquartile range; and the cross in the box is the mean. The vertical lines at each side of the box represent distribution from the quartile to the farthest observation. The curve represents the HA median value of each fibrosis stage (F0: 20 μg/l, F1: 25 μg/l, F2: 30 μg/l, F3: 58 μg/l, and F4: 180 μg/l). The relation between the stages of fibrosis and HA level was statistically significant (Kruskal-Wallis –
Fibrosis, severe fibrosis, and cirrhosis diagnosis
Figure
Receiver operating characteristic curves of HA for the prediction of significant fibrosis (F2-F4), severe fibrosis (F3F4), and cirrhosis (F4) in the training set
Receiver operating characteristic curves of HA for the prediction of significant fibrosis (F2-F4), severe fibrosis (F3F4), and cirrhosis (F4) in the training set.
Receiver operating characteristic curves of HA for the prediction of significant fibrosis (F2-F4), severe fibrosis (F3F4), and cirrhosis (F4) in the validation set
Receiver operating characteristic curves of HA for the prediction of significant fibrosis (F2-F4), severe fibrosis (F3F4), and cirrhosis (F4) in the validation set.
Fibrosis
Two cut-off values were chosen for identifying absence (less than 16 μg/l) and presence (greater than 121 μg/l) of significant fibrosis (F2-F4). Applying the lower cut-off, the presence of significant fibrosis could be excluded with a high certainty as only 3 (17%) of the 18 patients with an HA level below 16 μg/l had significant fibrosis, with a negative predictive value (NPV) of 83%. In the validation set, 11 (18%) of 60 patients with a score below 16 μg/l had significant fibrosis (NPV of 82%). Applying the high cut-off to the training group (HA greater than 121 μg/l), only 2 (13%) of the 15 patients with HA greater than 121 μg/l had no fibrosis, with a positive predictive value (PPV) of 87%. In the validation set, 16 of the 17 patients with HA greater than 121 μg/l had significant fibrosis (PPV of 94%) (Table
Diagnostic performance of HA in the validation set.
HA cut-off
Sensitivity (%)
Specificity (%)
NPV (%)
PPV (%)
Population involved (%)
Interpretation
<16
91
36
82
55
24
Absence of fibrosis (F0F1) (82% certainty)
>121
14
99
57
94
7
Presence of fibrosis (F2) (94% certainty)
≤ 25
78
53
89
34
46
Absence of severe fibrosis (F0F1F2) (89% certainty)
>160
22
100
81
100
5
Presence of severe fibrosis (F3) (100% certainty)
≤ 50
100
79
100
20
75
Absence of cirrhosis (F0F1F2F3) (100% certainty)
>237
31
99
96
57
3
Presence of cirrhosis (F4) (57% certainty)
Note 1: Accuracy = Sensitivity + Specificity + NPV + PPV. Note 2: Population involved stands for the proportion of patients who fall in the corresponding cut-off.
Severe fibrosis
As for significant fibrosis diagnosis, 2 cut-off values were chosen for identifying absence (less than 25 μg/l) and presence (greater than 160 μg/l) of severe fibrosis (F3F4). Applying the lower cut-off, only 3 (5%) of the 64 patients with HA lower than 25 μg/l had severe fibrosis (NPV of 95%). In the validation set, only 13 (11%) of the 123 patients with HA lower than 25 μg/l had severe fibrosis (NPV of 89%). Applying the higher cut-off (HA greater than 160 μg/l) to the training set, 10 (NPV of 91%) out of 11 patients with HA greater than 160 μg/l had severe fibrosis, and none of the 13 patients with HA greater than 160 μg/l from the validation set had severe fibrosis (PPV of 100%) (Table
Cirrhosis
Two cut-off values were chosen for identifying absence (less than 50 μg/l) and presence (greater than 237 μg/l) of cirrhosis (F4). Applying the lower cut-off, 100 (NPV of 99%) of the 101 patients with HA lower than 264 μg/l had no cirrhosis. In the validation set, none of the patients with HA lower than 50 μg/l had cirrhosis (NPV of 100%). When applying the higher cut-off (237 μg/l) to the training set, a fair PPV of 71% was obtained for predicting the presence of cirrhosis. The PPV was lower when applying the cut-off to the validation set (PPV of 57%) (Table
Discussion
Combining HA level with other serum markers for assessing liver fibrosis has been considered in some other studies
In the present study, HA level was accurate in predicting significant fibrosis, severe fibrosis, and cirrhosis, with AUCs of 0.75, 0.82, and 0.89, respectively, in the training set; and of 0.73, 0.77, and 0.97, respectively, in the validation set. Using values below the lower cut-off level or above the higher cut-off level, one could predict absence or presence of significant fibrosis, severe fibrosis, and cirrhosis in 31%, 51%, and 78%, respectively, in the validation set patients. Therefore, it is likely that the association of HA level with the degree of hepatic fibrosis represents an indirect one, expressing the functional correlation between fibrosis and both the concomitant capillarization and hepatic hemodynamic changes.
Significant fibrosis can be predicted by a HA level of <16 μg/l for its absence (NPV of 82%) and of >121 μg/l for its presence (PPV of 94%) in the validation set. Severe fibrosis can be predicted by a HA level of ≤ 25 μg/l for its absence (NPV of 89%) and of >160 μg/l for its presence (PPV of 100%). Cirrhosis can be predicted by an HA level of ≤ 50 μg/l for its absence (NPV of 100%) and of > 237 μg/l for its presence (PPV of 57%). Considering a serum HA cut-off of 60 μg/l for absence of cirrhosis diagnosis, our data are in the same range as those of other studies
A cut-off value of 110 μg/l was suggested for separating patients with and without cirrhosis
Our study included a sufficient proportion of patients with significant fibrosis: 47% in the training set and 46% in the validation set; however, the proportion of patients with cirrhosis was low in the two sets: 7% and 5%, respectively. The second limitation of this study is the number of unclassified patients (between 22% and 69%).
Conclusion
This study showed that significant fibrosis, severe fibrosis, and cirrhosis can be predicted by serum HA levels in patients with HCV infection. The notion of routinely measuring a marker that reflects the function of the sinusoidal endothelial cells, rather than the hepatocytes themselves, is an exciting concept. Serum HA would be clinically useful as a non-invasive marker of liver fibrosis or cirrhosis in HCV-infected patients. It suffers from the need to limit, as much as possible, potential confounding variables such as the effects of exercise and eating.
Further studies conducted in a large cohort of cirrhosis patients are needed to corroborate this study, namely because few of our patients had cirrhosis and the cut-off levels must be considered in an independent study. Moreover, a comparison of HA levels with other non-invasive markers and scores of liver fibrosis (FT, APRI, Forns, age-platelets index, platelet count, prothrombin time, etc.) would be of interest.
Methods
Study participants
The cohort included 405 patients (mean age 49 ± 13 years, 53% men) with HCV infection between November 2002 and December 2003, in five centers in southern France [Conception Hospital and Saint-Joseph Hospital (Marseille), Archet Hospital (Nice), Hyères Hospital (Hyères), and Arnault Tzanck Institute (St Laurent du Var)]. HA assessment was evaluated with data from 151 patients (training group) and was validated in the remaining 254 patients (validation group). Only biopsy lengths of 25 mm or more were included in the training group, in accordance with a recent study
Liver biopsies
Liver biopsy examination was performed in each center by evaluating the stage of fibrosis and grade of activity according to the METAVIR scoring system
Hyaluronic acid
HA levels were measured by Alphabio Laboratories, Marseille, with the Corgenix Hyaluronic Acid Test Kit, Corgenix Inc., CO, following the manufacturer's instructions (patients in fasting conditions, no physical effort). Each HA level was measured in duplicate (range 1 to 871 μg/l) and a pool control set was used. All serum samples were obtained on the day of liver biopsy.
Statistical analysis
The association between HA levels and liver biopsy staging was measured with the Kruskal-Wallis multiple comparison test and with the Spearman rank correlation coefficient. A
Authors' contributions
PH and MB conceived and wrote the manuscript. RD, CR, DBF, AT, IP, IA, ARA and DO were responsible for the patient drafting, carried out biochemical analysis, participated in the coordination of the study, and drafted the paper. GP performed the statistical analysis and participated in the writing of the paper. All authors read and approved the final manuscript