Recent data from the US National Health and Nutrition Examination Survey (NHANES) reported elevated ALT (>30 U/L), a marker of potential NAFLD, is prevalent in 8% of adolescents 12–19 years of age.24
Histology-based autopsy data suggest that 8% of the population aged 2 to 19 years may be affected by NAFLD1
. Progressive disease in even a small fraction represents a significant potential for disease burden. In a retrospective evaluation of screening practices at academic centers, 2% of general pediatricians and 23% of pediatric gastroenterologists performed screening for NAFLD among obese children, suggesting that most cases of pediatric NAFLD are currently undiagnosed7
. Given the potential for NAFLD to progress to cirrhosis, it is imperative that screening tools be developed; ideally, with non-invasive means to identify children at greatest risk for progressive disease. Serum aminotransferase levels would represent the simplest screening test for this purpose, but have not been proven to accurately reflect histologic activity. We analyzed data collected for the NASH CRN, a large, multi-center study, to identify clinical indicators of histological activity.
Assuming that Not NASH, borderline zone 3 pattern and definite NASH represent a range of conditions along a spectrum, we sought to identify clinical features that would distinguish these patterns. AST and GGT were significant predictors of histology pattern in this analysis. While statistically significant, the cumulative odds ratios for AST (OR 1.017 per U/L = 1.18 per 10 U/L = 5.40 per 100 U/L) and GGT (OR 1.017 per U/L = 1.18 per 10 U/L = 5.40 per 100 U/L), demonstrate that these laboratory measures are likely of limited clinical utility in distinguishing children with these histological patterns.
Serum antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA) have been reported in 6–15% of the population and 8–25% of patients with chronic liver disease in whom these antibodies often do not signify autoimmune liver disease.25–27
The largest study of autoantibodies in NAFLD was conducted in 225 patients with biopsy-proven NAFLD (age 13–73 years) from the Mayo Clinic.28
20% had positive titers for ANA, 3% for ASMA and a positive ANA and/or ASMA was associated with higher fibrosis stage and inflammation grade. An Italian study found ANA positive in 21.4%, ASMA positive in 4.7% and anti-mitochondrial antibody (AMA) positive in 2.4% of 84 adult patients with biopsy-proven NAFLD.29
3.6% of patients in this series had overlapping features of autoimmune hepatitis on biopsy. In a Japanese study of 212 adult patients with biopsy-proven NAFLD, 33% had positive ANA titers and 1.4% had both ANA and ASMA titers positive.30
An ANA titer of 1:80 or greater was associated with greater prevalence of severe necroinflammation (25%) compared to those with titers of 1:40 or less (11.3%, P=0.037).
Autoantibody positivity has been reported to occur in apparently healthy children with no personal or family history of autoimmune disease with rates of 3% for ANA, 2.6% for ASMA and 1.1% for AMA.31
The significance of autoantibody positivity in pediatric NAFLD is unknown. None of the children enrolled in our study had positive titers for AMA, but the prevalence of ANA positivity was 18% and ASMA positivity was 32%. Positive titer for ASMA was a significant predictor of increasing NAS (OR 2.8, 95% CI 1.4–5.4). The potential of ASMA as a non-invasive marker of histological severity in pediatric NAFLD is a novel finding that deserves further study.
Borderline zone 1 pattern of NAFLD is a unique histological pattern that appears to predominantly affect children.17
This pattern, previously referred to as type 2 or pediatric-type NASH, has been reported more frequently among boys, to affect younger children, be associated with more severe obesity, and to be the predominant pattern seen among those of Asian or Native American race and Hispanic ethnicity.9
A similar pattern emerged among children enrolled in the NASH CRN, though BMI percentile and percent body fat did not differ between those with borderline zone 1 versus definite NASH. On multiple regression analysis, age, prothrombin time, and insulin resistance were significant predictors of histological pattern. Our findings support the hypothesis that sex hormones and insulin resistance associated with pubertal development may be important variables in the predisposition to and determination of histological subtype of pediatric NAFLD.9, 32
Whether this borderline zone 1 form present in children represents an alternative or distinct pattern of NASH awaits demonstration of potential differences in etiopathogenesis, natural history or treatment response.
Markers of insulin resistance were consistently higher among children with definite NASH compared to other NAFLD patterns and with increasing NAS. In a multiple regression analysis of individual NAS scores, lower Tanner stage was predictive of higher NAS (OR 0.76 per Tanner stage, 95% CI 0.63–0.92) suggesting that hormonal changes associated with pubertal development may influence disease severity. Puberty is associated with a decrease in insulin sensitivity (25–30%) that is compensated for by an increase in insulin secretion. Insulin resistance occurs early in development, typically between Tanner stages I and II, with a nadir in insulin sensitivity at Tanner stage III and recovery by stage V.33, 34
Analysis of children with borderline zone 1 compared to definite NASH support the association of this pattern of developmental insulin resistance with NAFLD expression. Those with borderline zone 1 pattern were younger (median age 11.1 years) with mean Tanner stage 1.8 and had less severe insulin resistance (median HOMA-IR 4.8) compared to those with definite NASH (median age 13.0 years) with mean Tanner stage of 2.7 and greater insulin resistance (median HOMA-IR 7.7, P=0.006). Whether children with borderline zone 1 pattern may evolve into definite NASH and/or children with definite NASH regress to borderline zone 3 or simple steatosis with further developmental maturation is unknown and will require longitudinal data to determine.
Another potential mediator of pubertal development on disease expression in pediatric NAFLD are changes in sex steroid hormones. Sex hormones have been proposed to account for differences in prevalence rates and disease expression among males and females with NAFLD.32
The prevalence of elevated ALT among adolescents aged 12–19 years from NHANES was 12.4% in males compared to 3.5% in females.24
Most published series of pediatric NAFLD have reported males to be more commonly affected.10, 35–37
While our patient population was predominantly male (77%), gender was not predictive of borderline zone 1 pattern versus definite NASH.9
Similarly, while there was a larger percentage of males with definite NASH compared to borderline zone 3 and not NASH, this difference was not significant. We cannot exclude the possibility of type 2 error in determining the role of gender on NAFLD pattern due to the relatively smaller number (n=41) of female subjects.
AST was found to be a significant predictor of NAFLD pattern, fibrosis severity, and NAS. Area under the ROC curve analysis demonstrated AST to be superior to ALT in distinguishing NAFLD pattern and the addition of ALT to AST did not improve discriminate performance. However, AUROC demonstrate that AST value does not have sufficient discriminate power to reliably predict histology. While our results do not support the use of AST in place of liver biopsy, the strong association between AST and meaningful histological features in pediatric NAFLD support current recommendations to use serum aminotransferase levels in screening overweight children.38
It is important to note that the majority of participants in this study were required to have elevated ALT for study inclusion. Baseline ALT values were ≥60 U/L in 50% of Database participants and 99% of TONIC participants. Therefore, the associations that we have reported between serum aminotransferase levels and histology in NAFLD may not be applicable to other patient populations in whom serum transaminase levels may not be elevated to this extent.
Predictors of fibrosis varied depending upon whether all degrees of fibrosis were compared or only those with mild versus moderate fibrosis were considered. Overall predictors of increasing severity of fibrosis were increasing AST and white blood cell count and decreasing hematocrit. Platelet count, frequently included as a non-invasive marker of fibrosis in chronic hepatitis C virus infection, was not associated with fibrosis severity.39, 40
Hispanic ethnicity was predictive of fibrosis severity when comparing those with mild and moderate degrees of fibrosis, likely accounted for by the relatively small percentage (31%) of subjects with moderate zone 3 and periportal fibrosis of Hispanic ethnicity. Children with bridging fibrosis tended to be younger than those with lesser degrees of fibrosis, perhaps indicating that yet unidentified susceptibility genes predispose to a more aggressive course in these children. In contrast to previous studies, BMI was not associated with fibrosis severity, though percent body fat was lower among subjects without fibrosis. The study population may have been too skewed with respect to BMI (97% had BMI ≥95th
percentile) and/or it may be that body fat distribution is a more important determinant of fibrosis than BMI. _Higher insulin levels also were predictive of moderate versus mild fibrosis. While in clinical practice insulin resistance is unlikely to be of use in distinguishing fibrosis stage, this finding supports insulin resistance as an important variable in disease progression.10, 41–43
The association of insulin resistance with portal fibrosis in NASH was recently investigated in context of a histological lesion called ductular reaction.44
In this study, insulin resistance was highly associated with replicative arrest of hepatocytes, an impaired response to necrotic and apoptotic hepatocytes. In the setting of impaired hepatic regeneration, a secondary replicative pathway of hepatic progenitor cells is induced.45
Ductular reaction is a lesion seen at the portal tract interface composed of small biliary ductules, stroma and inflammatory cells that occurs as a by-product of induction of this secondary pathway.46, 47
Ductular reaction was associated with the extent of replicative arrest and the extent of ductular reaction was strongly associated with fibrosis. Further studies are required to determine whether similar processes occur in pediatric NAFLD and whether this provides a mechanistic link between insulin resistance and portal fibrosis.
Because of the expense and risk of liver biopsy, there is enthusiasm for development of noninvasive markers of liver histology. Factors to consider in studies designed to identify such markers include adequacy of biopsy specimens and interpretation of histology by pathologists with appropriate expertise.48
We included biopsy specimen length in all of our analyses and central review by the Pathology Committee of the NASH CRN, composed of pathologists with expertise in the histopathology of NAFLD, was one of the inclusion criteria for our study. More difficult to account for is the possibility of sampling error in NAFLD.49, 50
As recently highlighted, increased size may minimize heterogeneity between biopsy specimens.48
In conclusion, AST, GGT, and positive ASMA titers were the clinical variables most consistently associated with the pattern and severity of NAFLD in this large prospective, multi-center, histology-based study of children. The association between ASMA and pediatric NAFLD is a novel finding deserving of further study. Our results support the hypothesis that insulin resistance associated with pubertal development may be an important determinant of disease expression in pediatric NAFLD. These results also support a role for insulin resistance in the development of fibrosis in pediatric NAFLD. Unfortunately, none of the clinical predictors of histology appear sufficiently powerful to replace liver biopsy as a non-invasive means of staging disease. However, these clinical markers may be employed by pediatric gastroenterologists in evaluating overweight children suspected to have NAFLD.