Anti-sonic hedgehog immunohistochemistry accurately diagnoses ballooning degeneration in both metabolic dysfunction-associated steatotic liver disease and alcohol-related liver disease

Introduction: Ballooning degeneration is a key histological marker of steatotic liver disease (SLD) severity, but it remains difficult to identify reliably and shows poor inter-observer agreement. Sonic hedgehog (SHH) pathway is involved in ballooning pathogenesis in metabolic dysfunction-associated steatotic liver disease. But data on the expression of SHH ligand in other SLD subtype including alcohol-related liver disease (ALD), and the diagnostic accuracy of anti-SHH immunohistochemistry (IHC) in real-life practice are lacking. Aims: To assess the diagnostic accuracy of anti-SHH IHC for the diagnosis of ballooning degeneration in SLD. Methods: Participants were prospectively recruited from hepatology outpatient clinics and the alcohol withdrawal unit. Prospectively recruited participants with a diagnosis of SLD (MASLD or ALD) on imaging and increased liver stiffness (≥ 7.8 kPa, M probe, Fibroscan®) underwent liver biopsy. Four blinded pathologists scored hematoxylin-eosin stained slides for steatosis and activity including ballooning. Anti-SHH IHC was performed on the same liver samples and quantified manually (positive cells per field at 40-fold magnification) by two untrained observers blinded to the pathology score. SHH positive cells are expressed as mean positive cells between the two observers and divided by total number of examined fields. Results: Eighty-eight participants with SLD (52 with MASLD, 36 with ALD) were prospectively recruited and underwent liver biopsy. Median self-reported alcohol consumption was 80 units per week in the ALD group. Mean BMI was significantly higher in the MASLD group (34.7 kg/m²) compared to the ALD group (27.3 kg/m²; p < 0.0001). 38% of cases were classified by pathologists as fibrosis grade 0-1 (F0-1), 30% as F2, 26% as F3 and 6% as F4 in the pooled SLD group. 46% of MASLD cases were classified as ballooning grade 0 (B0), 27% as B1 and 27% as B2 while 61% of ALD were classified as B0, 14% as B1 and 25% as B2. The count of SHH positive cells strongly correlated between untrained observers (r = 0.89, p < 0.0001) across the whole SLD cohort. SHH positive density positively correlated with ballooning grades in MASLD (r = 0.71, p < 0.0001), ALD (r = 0.75, p < 0.0001) and pooled SLD (r = 0.69, p < 0.0001). In particular, the median SHH density was 0.03 for B0, 0.16 for B1 and 0.96 for B2 in the pooled SLD group (p < 0.0001). SHH density showed a very high diagnostic accuracy for the diagnosis of ballooning in MASLD (AUC = 0.83, p < 0.0001), ALD (AUC = 0.94, p < 0.0001) and pooled SLD (AUC = 0.86, p < 0.0001). Conclusion: Anti-SHH IHC is a highly accurate tool for the diagnosis of ballooning by untrained observers in MASLD and ALD highlighting its potential utility in clinical practice and research in the topic of SLD.