Histopathologic Diagnoses in Validation Non-ALD Controls
The histopathologic diagnoses of validation non-ALD controls were diverse, including metastatic squamous cell carcinoma, hemangioma, inflammatory liver injury, minimal steatosis, and more. Correlation analyses and comparisons related to FGF-21 expression, alcohol-associated liver diseases, and liver disease severity were conducted in different cohorts.
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Supplementary Table 1: Histopathologic diagnoses of validation non-ALD controls Subject Diagnosis 1. Metastatic squamous cell carcinoma 2. Hemangioma 3. Inflammatory liver injury 4. Minimal steatosis 5. Nonspecific portal inflammation 6. Sinusoidal congestion
1A AC p = 0.96 Supplementary Figure 1A | No correlation was found between FGF-21 expression and body mass index in subjects of the discovery cohort, (Spearman s rank correlation of 0.0053, p = 0.96).
1B ALD AC p = 0.336 Supplementary Figure 1B | No correlation was found between FGF-21 expression and body mass index in subjects of the validation cohort. (Spearman s rank correlation of 0.136, p = 0.336).
2A p = 0.39 Supplementary Figure 2A | In the discovery cohort, no clear difference was seen between patients with moderate to severe alcohol-associated hepatitis (AH) or alcohol-related cirrhosis (AC) based on recent consumption of alcohol (EtOH) in the past two weeks, (n = 22). A Kruskal-Wallis test was used given the non- parametric distribution of FGF-21 concentration.
2B Supplementary Figure 2B | No significant difference was observed between FGF-21 expression and alcohol use in subjects with alcohol-related liver disease in the validation cohort (t-test, p = 0.112).
3 AC Supplementary Figures 3 | In the biopsy-proven validation cohort, the only subjects with FGF-21 expression within the 4th interquartile (2124 pg/mL) had alcohol-associated hepatitis.
4 AC Supplementary Figure 4 | Subjects with severe alcohol-associated hepatitis showed the highest FGF-21 within the spectrum of alcohol-related liver disease, including comparison to subjects with non-alcohol associated steatohepatitis (NASH) in the discovery cohort, with Kruskal-Wallis showing significance level of p < 0.001, and pairwise Wilcoxon rank-sum test showing p = 0.31 between control and NASH subgroups. Nonparametric tests used because of FGF-21 expression.
5A 5B AC alone, by MELD p = 0.03 p = ns 8000 8000 FGF21 level (pg/mL) FGF21 level (pg/mL) 6000 6000 4000 4000 2000 2000 0 0 AC 20-30 AC >30 AC (n=5) AH (n = 6) Cirrhosis Alone Supplementary Figures 5A-B | The differences in FGF-21 expression with MELD score did > 30 not reach significance (t-test, p = ns). However, the values for AC were heterogeneous where MELD scores were greater than 30 (n = 5) compared to those with MELD scores between 20-30 (n=5), and were significantly higher.
6A Strata Legend First Quartile Second Quartile Third Quartile Fourth Quartile (Days) Supplementary Figure 6A | Survival analysis of patients diagnosed with alcohol-associated hepatitis (log rank test, p = 0.016), the second quartile (FGF-21 conc. 267-844 pg/mL) still observed with the highest rates of survival without death or orthotopic liver transplantation, but the difference between the first and second quartile now is not significant (log rank test, p = 0.13).
6B Strata Legend First and Second Quartile Third Quartile Fourth Quartile Supplementary Figure 6B | Survival analysis of patients only diagnosed with alcohol-associated hepatitis at 28 days (log-rank test, p = 0.44). This difference is no longer significant despite the first and second quartiles trend towards higher short-term survival rates without death or orthotopic liver transplantation.
6C Strata Legend First and Second Quartile Third Quartile Fourth Quartile Supplementary Figures 6C | Survival analysis of patients only diagnosed with alcohol-associated hepatitis at 90 days (log-rank test, p = 0.44). This difference is no longer significant despite the first and second quartiles trend towards higher short-term survival rates without death or orthotopic liver transplantation.
