In the chronically injured liver, fibrogenesis is the complex dynamic interplay among various hepatic cell types and mediators in which the process of perpetuation follows initiation . With the clinical application of magnetic resonance (MR) and ultrasound-based LSM, studies using MR elastography (MRE) , FibroScan, and ARFI elastography have demonstrated significant correlations between liver stiffness and liver fibrosis. However, liver stiffness and liver fibrosis are not equivalent.
In addition to the accumulation of the fibrotic extracellular matrix, other components of chronic liver disease, including cholestasis [31, 32], cardiac congestion , and, particularly, the degree of necroinflammatory activity, can exaggerate ultrasound-based LSM. Patients with CHC usually manifest relatively stable serum ALT levels compared with the abrupt and fluctuating ALT levels in patients with chronic hepatitis B (CHB). Swelling of hepatocytes, interstitial edema, and infiltrates of inflammatory cells may increase liver stiffness in patients with acute hepatitis . After adjusting for other demographic and biological covariates, the results of our study indicate that a 100 IU/L increase in serum ALT levels augmented liver stiffness values by approximately 0.155 m/s (model R2 = 0.630, adjusted R2 = 0.602).
ActiTest is a biomarker of liver necroinflammatory histological activity validated in patients with CHC. The accuracy of ActiTest for grading necroinflammatory activity in HCV-infected patients was significantly higher than serum ALT alone . In our study, the ActiTest A score model (R2 =0.662, adjusted R2 = 0.636) was superior to the models using serum ALT levels (R2 = 0.630, adjusted R2 = 0.602), ALT/ULN categories (R2 =0.661, adjusted R2 = 0.629) and METAVIR A grades (R2 = 0.651, adjusted R2 = 0.620) in explaining the LSM results.
The cutoffs of ARFI LSM in this study (1.55 m/s for F1 versus F2-4; 1.81 m/s for F1-2 versus F3-4) were in part higher than those in the latest study on 139 CHC cases by Rizzo et al. (1.3 m/s for F1 versus F2-4; 1.7 m/s for F1-2 versus F3-4). Necroinflammatory effects explain most of the differences. First, the overall necroinflammatory degree was higher (Student’s t-test, P = .034) in this study (mean ALT, 93.2; SE, 7.2 IU/L; n = 127) than for Rizzo et al. (mean ALT, 77.2; SD, 33.0 IU/L; n = 139). Second, the distribution of necroinflammation tended to be more severe in METAVIR F3 and F2 than F1 stages in this study (Figure 1). Thus, the LSM values were more augmented in F3 and F2 than F1 stages. The analyses of false positivity in patients with F1 to F3 also correlated well with the necroinflammatory effects (Table 4). Third, cirrhotic Taiwanese patients (mean age, 62.7; SE, 1.5 years; n = 18) referred to the unit for SOC tended to be older (Student’s t-test, P = .0083) than those in the study by Rizzo et al. (mean age, 55; SD, 12 years; n = 139) . Despite the advanced countrywide education and health insurance coverage, these patients were characterized by poor patient compliance and late diagnosis. Although age did not affect LSM significantly, as demonstrated in models in the present and previous studies, an older age may have resulted in significant fibrosis progression in these cirrhotic Taiwanese patients despite the relatively compensated liver reserves.
Despite the variation among previous investigations, several recent studies have demonstrated the necroinflammatory effects on liver stiffness by evolving analyses.
In a longitudinal study using FibroScan, Sagir et al.  observed false positivity for cirrhosis (cutoff > 12.5 kPa) in 15 of 20 non-cirrhotic patients with acute liver damage of various etiologies. In 6 patients, the LSMs dropped below 12.5 kPa with normalized ALT levels during follow-up. Using a longitudinal analysis, Arena et al.  demonstrated significant correlations between sequential serum ALT levels and LSM results at different time points. Although these studies showed the need for caution when analyzing LSM in patients with necroinflammatory flares, they did not include regression estimates. Seo et al.  demonstrated that peak ALT levels significantly explained peak LSMs in 31 patients in acute hepatitis A via linear regressions adjusting for age and sex.
A cross-sectional study by Le et al.  showed that LSM using FibroScan in 158 patients was independently associated with histological necroinflammatory grading, but irrespective of serum ALT levels. Fung et al.  reported a suboptimal PPV (as low as 10%) for LSM using FibroScan to diagnose true cirrhosis in 102 patients (median age, 41; range, 18–63 years) with active hepatitis B (median serum ALT, 89; range, 46–501 IU/L). Multiple logistic regressions by Myers et al.  showed that serum ALT levels greater than the optimal cutoff value 60 IU/L from ROC analysis were significantly correlated with the discordance (at least 2 stages between FibroScan and biopsy). Chan et al.  and Kim et al.  proposed distinct sets of cutoff values stratified by distinct ALT profiles.
Tapper et al.  further delineated the positive necroinflammatory effects on LSM using FibroScan through linear regressions in 684 HCV patients with METAVIR F0, F1 and F2. Logistic regressions also showed that false positivity of liver fibrosis staging was associated with both histological and serum hepatic necroinflammatory activity. Using 14.5 kPa as the cutoff of cirrhosis, grade 3 inflammation had an OR of 9.10 (95% CI, 2.49-33.4). Likewise, serum levels of ALT greater than 80 IU/L and 120 IU/L had ORs of 3.84 (95% CI, 2.10-7.00) and 4.10 (95% CI, 2.18-7.69) over references, respectively. Yoon et al.  used ARFI elastography to demonstrate a significant correlation (Pearson's r = 0.431, P < .05) between LSM values and serum ALT levels, and a marked positive effect of histological necroinflammatory activity on LSM. However, this study did not adjust for other relevant essential covariates. ARFI elastography performed potentially better for patients with normal ALT than for those with high ALT (AUCs, 0.88 versus 0.73 for METAVIR F1-2 versus F3-4, 0.92 versus 0.72 for F1-3 versus F4). However, AUCs were not statistically compared. The cutoff values tended to be lower for patients with normal ALT than for those with high ALT levels (ALT > 40 IU/L)(1.09 versus 1.16 m/s for F1-2 versus F3-4, and 1.81 versus 2.23 m/s for F1-3 versus F4, respectively).
In contrast to results indicating positive necroinflammatory effects on liver stiffness, Harata et al.  identified a negative correlation between serum ALT levels and liver stiffness in patients with cholestasis. Cholestasis is a condition in which the release of hydrostatic pressure with synchronous necroinflammatory activity can, paradoxically, reduce the values of LSM using FibroScan. Rizzo et al.  found distinct necroinflammatory effects on LSM between using FibroScan and ARFI elastography. Colombo et al.  demonstrated an insignificant (Spearman’s rank) correlation between necroinflammation and LSM. The serum ALT specific cutoffs of 202 CHB patients in a study by Cardoso et al. did not increase the diagnostic performances using FibroScan for liver fibrosis evaluation .
Although hepatic steatosis is prevalent in patients with CHC, the linear regression analysis in this study did not identify steatosis as a significant independent explanatory factor for the ARFI LSM results. However, larger sample sizes are required to further delineate the effects of more severe forms of steatosis (S3, S4) on ARFI LSM results. Motosugi et al.  also demonstrated the insignificance of different ARFI LSM results among four grades of steatosis (P = .9018). Using MRE in a mouse model, Yin et al.  showed that steatosis did not correlate significantly with liver stiffness at each liver fibrosis stage.
This study shows that BMI significantly and independently explain the results of ARFI LSM (Table 3). Similarly, Roulot et al.  used FibroScan to show that liver stiffness was significantly higher (P = .0005) in obese patients (with BMI > 30 kg/m2) than in overweight or normal patients, after adjusting for age, sex, ALT, aspartate aminotransferase (AST) and ferritin. Baba et al. also demonstrated a significant association between BMI and liver stiffness using FibroScan. Hepatic steatosis, however, was not evaluated in adjusted models . Conversely, Talwalkar et al.  reported an insignificant (linear) correlation between BMI and LSM results using MRE.
Similar to the report by Talwalkar et al. , the effects of age and sex on ARFI LSM were insignificant in this study. Previously demonstrated as a significant factor explaining liver fibrosis evaluation , platelet count was strongly associated with ARFI LSM after adjustment, unlike the inconsistency and insignificance of INR (Table 3).
To minimize histology bias [6, 52], the percutaneous liver biopsy in this study immediately followed ARFI LSM. Specimens were of adequate length, and an expert pathologist interpreted histological findings. Unreliable cases or failed ARFI measurements were primarily derived from obese patients with poor transient apnea maneuvers. Thus, future studies may analyze factors associated with unreliable cases or failure measurements. The present analyses show that the R2 values were modest for the regression models; therefore, other potential explanatory factors, especially direct tissue markers , must be identified to construct an optimal explanatory model (Table 3).
Future analyses would require a larger sample size to develop cutoffs and perform validations that are potentially more reliable and stable than those of this study. These investigations may compare the diagnostic performances of ARFI LSM with another promising diagnostic modality: MRE. Although FibroTest was initially proposed as a similar first-line approach to histology for prediction of 5-year survival in patients with CHC [37, 38], it may also serve as a standard of reference, in addition to METAVIR F staging, for evaluating the diagnostic value of ARFI LSM in baseline or chronological analysis.
The limitations of this study include the lack of stratification of patients with cirrhosis into compensated and decompensated (excluded) groups. The absence of a decompensated group, which may have a high and broad spectrum for LSM, may have caused the insignificant differences in LSM results between METAVIR F3 and F4 (P = .412) before adjusting for other covariates. Further analyses should identify optimal cutoffs for LSM to stratify the broad cirrhosis category and enable risk estimation of end points in chronological analyses . This study does not identify the cutoff for LSM between METAVIR F0 and F1-F4 because of the lack of well-established criteria for recruiting true cases with F0 fibrosis without liver biopsy.
Future noninvasive liver fibrosis evaluation tools should focus on exact staging of fibrosis, rather than classifying patients into categories of milder versus more severe fibrosis stages. In the milder strata of liver fibrosis, necroinflammation can easily overwhelm the fibrotic effects on LSM . In this study, the diagnostic performance of ARFI LSM was potentially limited when distinguishing between METAVIR F1 and F2 categories too. The insignificant regression coefficient in Table 3 reflects the potentially limited sensitivity for ARFI LSM in distinguishing F2 from F1. Therefore, further research is needed prior to the clinical application of ARFI LSM for surveillance of progression, or regression, of liver fibrosis. The real clinical relevance between METAVIR F1 and F2 is still not known with respect to either prognosis or risk of disease progression over the short to intermediate term of 5 to 10 years . Future studies should develop algorithms based on a combination of ARFI LSM and essential serum markers for liver fibrosis evaluation to minimize false positivity of fibrosis staging [24, 27, 54]. A further limitation is that this study did not perform standardization of AUCs based on the prevalence of liver fibrosis stages .