Nonfasted Liver Stiffness Correlates with Liver Disease Parameters and Portal Hypertension in Pediatric Cholestatic Liver Disease

In the last decade, transient elastography (TE) has significantly enhanced clinical monitoring of adults with chronic liver disease, changing the role of and need for liver biopsy. This noninvasive technique, which measures liver stiffness, is particularly useful in differentiating advanced fibrosis on liver biopsy from no or minimal fibrosis in adults.⁽¹⁾ TE is not as well studied in pediatrics, although experience is growing. A meta-analysis of transient and shear wave elastography reports in children with chronic liver disease published before 2017 revealed sensitivity 90%, specificity 79%, and receiver operating characteristic 0.92 for varying definitions of portal hypertension.⁽²⁾ One of these studies assessed 249 children with cystic fibrosis, while the pediatric studies in biliary atresia (BA) were single center, including up to 73 children.^{(3, 4)} However, there is a pressing need for high-quality multicenter data for TE in children of varying ages. Specific attention should be accorded to the wide spectrum of liver conditions seen in childhood, which are remarkably different from those seen in adulthood.

While the most common liver conditions in adults are fatty liver disease and chronic hepatitis C infection, infants and children often suffer from a range of congenital cholestatic disorders, including BA, alpha-1 antitrypsin deficiency (A1ATD), and Alagille syndrome (ALGS). BA occurs in one in 8,000 to 18,000 live births and is manifest by uniquely rapid progression to advanced fibrosis and subsequent cirrhosis in the first months to years of life.⁽⁵⁾ BA is also the most common indication for liver transplantation during early childhood. Although most individuals with A1ATD are asymptomatic, 15% may present with neonatal cholestasis or cirrhosis in childhood or adulthood.^{(6, 7)} ALGS is an autosomal-dominant multisystem disorder with paucity of bile ducts and progressive liver disease; 40% to 75% or more require liver transplantation before adulthood.^{(8, 9)} Noninvasive measurements, such as TE, are an unmet need to assess the progression of liver disease in each of these distinct disorders.

Hepatic fibrosis, cirrhosis, and portal hypertension are common final pathways of a number of pediatric liver diseases. Portal hypertension may occur in the first months of life in BA and presents during early childhood in patients with ALGS and A1ATD. While splenomegaly on physical examination and thrombocytopenia are crude measures of portal hypertension, more precise data would be helpful in assessing the progression of pediatric liver disease over time and in predicting the risk of complications of portal hypertension, including variceal hemorrhage and ascites.⁽¹⁰⁾

The National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health-sponsored Childhood Liver Disease Research Network (ChiLDReN) is a multicenter consortium that longitudinally follows infants and children with cholestatic liver diseases, including BA, A1ATD, ALGS, progressive familial intrahepatic cholestasis, bile acid synthetic defects, and mitochondrial disorders. Clinical history, physical examination, and laboratory findings are collected annually on participants in ChiLDReN research protocols. Since 2016, the FibroScan in Pediatric Cholestatic Liver Disease (FORCE) study has investigated the use of TE to assess liver stiffness in children with BA, A1ATD, and ALGS. This study provides a unique opportunity to assess liver stiffness in the context of clinical and laboratory markers of liver disease and portal hypertension in a prospective, longitudinal, multicenter approach in both a variety of cholestatic diseases and in a wide range of pediatric age groups.

Participants and Methods

Results

Participants

Between November 2016 and August 2019, 811 of 1,010 active participants with BA in PROBE or BASIC BA, and with ALGS and A1ATD in LOGIC were potentially eligible for FORCE (Fig. 1). A total of 550 participants consented to the study, 458 of whom had a valid baseline TE measurement (BA, 254; A1ATD, 104; ALGS, 100). Demographics of potentially eligible but not enrolled participants were generally similar to those enrolled in FORCE, although enrollment of participants less than 1 year of age was diminished (Supporting Table S1).

Correlation of LSM with Biochemical Characteristics of Liver Disease

Each parameter was examined for its correlation with LSM in the context of the three diseases. In BA, there was a positive correlation between LSM and TB, INR, AST, ALT, GGT, GPR, PELD score, APRI, and spleen size and a negative correlation with albumin and platelet count (Fig. 2; Supporting Table S4). The negative relationship between platelet count and LSM in BA persisted even with platelet counts in the “normal” range (i.e., >150,000; P = 0.025) (Supporting Fig. S1). Similar correlations existed for A1ATD (except AST, ALT, and albumin) and for ALGS (except for INR, GGT, GPR, and ALT).

Association of LSM with CEPH

The relationship between LSM and portal hypertension was investigated using the recently developed research definition of CEPH.⁽¹⁰⁾ dCEPH was more common in BA (48%) than in A1ATD (8%) or ALGS (15%) (Fig. 3; Supporting Tables S5 and S6). LSM was greater in dCEPH compared to aCEPH for all three diseases (dCEPH vs. aCEPH LSM kPa: mean (+SD) BA, 20.0 (13.6) vs. 10.8 (13.5), P < 0.001; mean A1ATD, 23.8 (12.5) vs. 7.8 (7.7), P < 0.001; mean ALGS, 16.1 (13.6) vs. 9.2 (5.3), P = 0.003). In BA LSM, pCEPH was lower than in dCEPH but not different from aCEPH. In contrast, in ALGS LSM, pCEPH was higher than aCEPH but not different from dCEPH. In A1ATD LSM, pCEPH was lower than dCEPH and higher than aCEPH. Among participants with the same CEPH status, the clinical, biochemical, and LSM varied by disease type. In aCEPH, LSM was higher in BA (Table 2). Participants with ALGS with aCEPH had higher TB, GGT, ALT, and AST levels. Participants with A1ATD with aCEPH had the lowest LSM, GGT, AST, and PELD score. These parameters were similar for BA and A1ATD with pCEPH (Supporting Table S7). Measurements of TB, GGT, AST, ALT, APRI, and PELD score were also higher in participants with ALGS with pCEPH. A complex picture emerged in comparing these parameters for dCEPH (Table 3). Spleen excursion below the costal margin was similar for all three diseases. LSM was higher and platelet count lower in BA and A1ATD compared to ALGS. However, TB, GGT, AST, ALT, and PELD score in ALGS were higher than in BA and A1ATD. The differences in parameters by disease type and CEPH status are summarized in the radar plot in Fig. 4. The number of hours of fasting did not impact the LSM among participants with the same disease type and CEPH status (Supporting Fig. S2; Supporting Table S8).

Table 2. Disease Comparisons for Absent CEPH

Parameter	Mean (SD)			P Value for Differences in Means
	BA (n = 71)	A1ATD (n = 86)	ALGS (n = 61)	Overall Difference	BA vs. A1ATD	BA vs. ALGS	A1ATD vs. ALGS
FibroScan LSM, kPa	10.8 (13.5)	7.8 (7.7)	9.2 (5.3)	0.02	0.01	0.95	0.02
TB, mg/dL	0.6 (0.6)	0.6 (0.7)	1.8 (2.5)	<0.001	0.66	<0.001	<0.001
GGT, mg/dL	149 (250)	57 (105)	418 (423)	<0.001	0.001	<0.001	<0.001
AST, mg/dL	66 (60)	59 (44)	142 (132)	<0.001	0.64	<0.001	<0.001
ALT, mg/dL	72 (89)	73 (59)	161 (140)	<0.001	0.21	<0.001	<0.001
Albumin, g/dL	4.3 (0.5)	4.4 (0.4)	4.3 (0.5)	0.33
INR, g/dL	1.1 (0.1)	1.1 (0.1)	1.0 (0.1)	0.05
GPR, g/dL	1.2 (2.2)	0.4 (0.9)	3.0 (3.1)	<0.001	<0.001	<0.001	<0.001
Platelet count	279 (90)	298 (84)	296 (85)	0.34
APRI	0.6 (0.6)	0.6 (0.6)	1.2 (1.0)	<0.001	0.33	<0.001	<0.001
PELD score	−12.1 (4.8)	−13.1 (4.8)	−7.0 (7.0)	<0.001	0.36	<0.001	<0.001
Spleen size (cm below costal margin)	0.1 (0.3)	0.1 (0.4)	0.0 (0.2)	0.49

Table 3. Disease Comparisons for Definite CEPH

Parameter	Mean (SD)			P Value for Differences in Means
	BA (n = 123)	A1ATD (n = 8)	ALGS (n = 15)	Overall Difference	BA vs. A1ATD	BA vs. ALGS	A1ATD vs. ALGS
FibroScan LSM, kPa	20.0 (13.6)	23.8 (12.5)	16.1 (13.6)	0.14
TB, mg/dL	1.3 (1.5)	0.8 (0.4)	6.4 (8.6)	<0.001	0.59	<0.001	<0.001
GGT, mg/dL	150 (176)	149 (72)	458 (311)	<0.001	0.29	<0.001	0.049
AST, mg/dL	87 (76)	78 (33)	181 (122)	0.001	0.80	<0.001	0.03
ALT, mg/dL	84 (81)	86 (60)	178 (91)	<0.001	0.59	<0.001	0.02
Albumin, g/dL	4.1 (0.5)	4.0 (0.6)	3.9 (0.8)	0.63
Platelet count	96 (55)	88 (42)	149 (70)	0.004	0.67	0.001	0.01
APRI	3.0 (3.2)	2.7 (2.0)	4.0 (3.1)	0.50
PELD score	−6.8 (5.8)	−7.0 (6.0)	0.6 (10.0)	<0.001	0.93	<0.001	0.01
Spleen size (cm below costal margin)	5.3 (4.0)	5.6 (4.5)	5.0 (4.7)	0.93

Discussion

The baseline results of FORCE represent one of the largest prospective studies of TE in children with chronic liver disease. The vast majority of published analyses of TE in children are single-center studies. The largest experiences focus on healthy children, providing key information on technical details of the application of this important technique in pediatrics.^(12-14) The largest TE studies in children with liver disease have examined LSMs in liver transplant recipients (n = 117), cystic fibrosis (n = 249), hepatitis C (n = 223), fatty liver disease (n = 106), and BA (n = 100).^{(3, 15-18)} These studies correlate LSM with a variety of disease parameters, including clinical features of liver disease, manifestations of portal hypertension, and histologic assessment of fibrosis. The FORCE protocol is unique in that it has been developed in the context of three large-scale, prospective, multicenter, observational studies that collected comprehensive and high-quality clinical metadata in three major etiologies of neonatal cholestasis: BA, ALGS, and A1ATD.

One key purpose of FORCE was to confirm the feasibility of measuring LSM in children with chronic liver disease of all ages. Requirements for fasting and sedation were eliminated in FORCE so the findings could be generalized to routine clinical practice. LSM may be higher in the nonfasted state and under general anesthesia.⁽¹³⁾ The difference noted for fasting, while statistically significant, may not be clinically significant especially at the levels generally observed in children with chronic cholestasis. In this study, there did not appear to be a strong effect of fasting on LSM. Valid scans are obtained in ~90% of other TE studies performed in children. It has been consistently reported that these studies are more difficult to perform in children under the age of 2 years, which is not unexpected given behavioral challenges and lack of cooperation with the examination in children at this age. Our findings corroborate this observation. During the conduct of FORCE, two technical issues were identified and corrected; this has led to an increase in the rate of successful scans as the study has moved forward. In real-time, the IQR/median is estimated but may be higher once the examination is finalized. This may lead to studies in which borderline IQR/median levels (25%-30%) during the examination result in final IQR/median levels greater than 30%, leading to an invalid scan. In FORCE, we developed IQR/median targets of <27% to avoid this issue. Care also needs to be taken to select the correct probe (M vs. S) and examination type (S1 vs. S2) based on the thoracic perimeter measurement. The S1 examination is typically only applicable to children <2 years of age. Within our study, FibroScan-based TE measurements are feasible in most children.

The cohort of children who participated in FORCE is representative of patients with liver disease seen in the pediatric outpatient setting. This is not an inception cohort and as such does not necessarily reflect the full range of disease severity associated with these disorders. With an inception cohort, patients with BA and A1ATD who have persistent cholestasis in infancy or complications of advanced liver disease are likely to undergo liver transplantation in the first 2 years of life and will be underrepresented in this type of cohort. Significant ascites precludes an accurate TE examination. The clinical characteristics of participants with BA, A1ATD, and ALGS are distinct (Table 1; Fig. 1). Most had compensated liver disease with very few having significant hypoalbuminemia or coagulopathy. Not surprisingly, PELD scores were generally negative. Importantly, features of portal hypertension were more commonly found in children with BA, of whom nearly 50% had thrombocytopenia and/or CEPH. In contrast, only a minority of participants with ALGS or A1ATD had thrombocytopenia. Interestingly, CEPH was more common in ALGS, even without thrombocytopenia, which raises questions about either the utility of CEPH or the sensitivity of thrombocytopenia as a feature of portal hypertension in this disease. ALGS frequently manifests profound cholestasis, a fact that is supported by higher TB and GGT in enrolled participants with ALGS. It appears that hepatocellular injury or inflammation was commensurately increased in ALGS as both ALT and AST levels were higher in these children.

The purpose of this study was not to use LSM to predict the presence or absence of portal hypertension, which can potentially be assessed using simpler techniques. Rather, the primary goal in FORCE was to determine if LSM was different in children with portal hypertension relative to those without portal hypertension. No attempt was made to correlate LSM with histologic findings of fibrosis, primarily because liver biopsy was not a feasible study procedure and in part due to inherent issues with needle-based biopsy approaches to histologic quantification of fibrosis.⁽¹⁹⁾ Based on prior studies, we postulated that LSMs would be significantly higher in those with portal hypertension.⁽⁴⁾ Portal hypertension is rarely quantified in pediatric liver disease. Some publications have used esophageal varices as a marker of portal hypertension; however, the clinical sites in ChiLDReN do not routinely screen for varices, so this marker of portal hypertension could not be used as has been done in other pediatric studies.⁽²⁰⁾ In light of these issues, the described research definition of CEPH was used as a validation for the potential utility of LSM in pediatric cholestasis.⁽¹⁰⁾

We found statistically and clinically relevant differences in LSMs in all three diseases in children with dCEPH versus aCEPH. In A1ATD, there were clear step-wise increases in LSMs based on progressive CEPH. Outside of the neonatal period, A1ATD may act more like a hepatocellular disease rather than one that is primarily cholestatic, like ALGS. In addition, there are likely a significant number of children with A1ATD-related liver disease who have minimal hepatic fibrosis, distinct from BA and ALGS. Thus, TE in children with A1ATD who had aCEPH resembled normal children, more than the children with BA and ALGS. BA is a profoundly fibrotic disease even in those individuals who are doing well and who are not apparently cholestatic. It is likely that children with BA and aCEPH may still have advanced fibrosis or compensated cirrhosis. This is demonstrated by minimal differences in LSMs in aCEPH versus pCEPH in BA. In contrast, there was a significant increase in LSM in children with dCEPH compared to pCEPH, suggesting that dCEPH is indicative of a more advanced stage of disease in BA. The performance of elastography related to CEPH in ALGS is less certain and more complicated. While significant differences were observed in LSMs between aCEPH and dCEPH among those with ALGS, the magnitude of the difference was less. There is limited longitudinal assessment of hepatic fibrosis in ALGS, and as such, continued investigations of fibrosis in ALGS are warranted.

This is the first study to formally apply CEPH definitions in a rigorous research protocol. The radar plots in Fig. 4 depict the impact of various clinical parameters among the different liver diseases and categories of CEPH. There are many similarities in the clinical parameters between BA and A1ATD, but ALGS is uniquely different. Specific comparisons of liver diseases among the same CEPH status are instructive. In dCEPH, spleen excursion below the costal margin is similar for the three diseases as are LSMs, although LSM values are highest in A1ATD. The clinical characteristics of the ALGS participants with dCEPH were clearly distinctive with higher TB, GGT, ALT, AST, and platelet counts. The latter finding is intriguing in the context of splenomegaly and increased LSMs. The comparisons in aCEPH are also enlightening among ALGS. By definition, splenomegaly and thrombocytopenia are not present in this group. LSM differs amongst the diseases, with the lowest value in A1ATD and similar values for BA and ALGS. This may suggest increased normality of the liver in A1ATD and raises the important issue of the impact of degree of cholestasis on LSM relative to the other two diseases. As with dCEPH, liver tests are more elevated in ALGS.

The correlation of liver biochemistries and LSM demonstrate many consistent relationships but also highlight notable differences among the diseases. There was a strong inverse correlation between TB and LSM in all three diseases. Features of portal hypertension, including spleen excursion below the costal margin and platelet count, also correlated with LSM. Despite obvious limitations of physical examination as an assessment of spleen size, positive correlation with LSM was still present. It is not surprising that LSM was inversely correlated with platelet count. It is, however, surprising that this correlation extended into the normal range of platelet counts. This suggests that the normal range is more statistical rather than biological. Platelet counts in the low-normal range may be significant in chronic liver disease, and trends in platelet counts over time may be relevant in staging disease. There were some inconsistencies in relationships between laboratory parameters and LSM in different diseases. Most notably, GGT and ALT levels did not correlate with LSM in ALGS. This may be a reflection of the highly cholestatic nature of ALGS. The difference of disease type is further reflected in the modeling done in the current analysis. The impacts of TB, GGT, and AST on LSM were each significantly greater in BA compared to ALGS. Differences in impact of these parameters in BA versus A1ATD were infrequent or subtle. Decreased serum albumin levels also had a significant effect on LSM in all three diseases; thus, albumin may be a very important parameter to follow in pediatric chronic liver disease.

The current analysis of FORCE assesses baseline measurements. FORCE is a prospective longitudinal study with proposed follow-up LSM at 1 and 2 years. Future investigations will assess change in LSM over time and the correlation of baseline and change in LSM with clinical events identified in the three on-going prospective longitudinal ChiLDReN studies.

LSM can be performed using a variety of techniques, including TE. The larger area of liver assessed by these techniques relative to needle biopsy enhances the potential accuracy of this approach relative to biopsy. FibroScan-based LSM in children is noninvasive and feasible, although there is a critical need to attend to technical details of the measurements. Its use in unsedated children under the age of 2 years may be limited. These studies have confirmed in a large cohort of children with three distinct types of liver disease that LSM correlates with features of portal hypertension and routine clinical laboratory assessments of liver disease in children. LSM may be influenced by the type of liver disease in ways that are not clear at present, warranting careful interpretation of results and further investigation.