Gut Microbiota as a Biomarker of Hepatocellular Carcinoma in Patients With Cirrhosis

Introduction: Hepatocellular carcinoma (HCC) is often diagnosed at a late stage. Gut dysbiosis – an imbalance of microbial communities – may contribute to the risk of certain malignancies. We investigated the gut microbiota as a potential biomarker of HCC. Methods: This was a nested case-control study of patients with or without HCC in a prospective cohort of liver transplant candidates. Patients with cirrhosis due to hepatitis B (HBV) or C (HCV), alcohol-related liver disease (ALD), or non-alcoholic fatty liver disease (NAFLD) were eligible. Cases with HCC were matched 1:1 to controls based on Childs Turcot Pugh (CTP) class, a known modifier of microbiome composition. One stool sample per patient underwent DNA extraction and 16S rRNA sequencing. Taxonomic differences were assessed using DADA2. Results: We identified 49 cases with HCC and 49 matched controls (Table 1). Compared to controls, cases were older (median 64 vs 60 years, P < 0.01) and more likely to have HCV (78% vs 43%) as opposed to ALD (4% vs 29%) or NAFLD (8% vs 20%) (P < 0.01). Most cases had early-stage HCC: 80% had peak prior tumor burden within Milan criteria and none had prior systemic therapy. Alpha and beta diversity did not differ between groups. Shannon index did not predict HCC in univariate analysis or when adjusted for age, cirrhosis etiology, and antibiotic exposure. However, multiple taxa were differentially abundant by DESeq2. Genus Escherichia-Shigella – which produces pro-inflammatory lipopolysaccharide (LPS), a potential contributor to hepatic fibrosis and carcinogenesis – was overabundant in cases; Prevotella – which produces non-inflammatory LPS – was overabundant in controls (Figure 1A). Because HCC risk differs by liver disease etiology, stratified analyses were performed. Among patients with NAFLD (n = 14), CTP class remained similar between groups. Alpha diversity was higher in cases with NAFLD than controls with NAFLD (P = 0.04; Figure 1B) and there was a trend toward distinct beta diversity (P = 0.06; Figure 1C). Controls with NAFLD had increased abundance of Prevotella and multiple species of Bacteroides, which also produce anti-inflammatory LPS. Conclusion: In cirrhotic liver transplant candidates, presence of HCC was associated with increased abundance of intestinal bacteria that produce pro-inflammatory LPS and reduced abundance of those that produce non-inflammatory LPS. HCC-associated dysbiosis was most pronounced in patients with NAFLD. The gut microbiota may provide a noninvasive biomarker of early-stage HCC. Table 1. - Characteristics of the study population Control (n = 49) HCC (n = 49) P-Value Age (years), median (IQR) 60 (55-65) 64 (60-66) < 0.01 Female sex, no. (%) 14 (29) 13 (27) 1 MELD score, median (IQR) 15 (11-24) 15 (10-19) 0.11 Childs Turcot Pugh class, no. (%) N/A A 20 (41) 20 (41) B 14 (29) 14 (29) C 15 (31) 15 (31) Cirrhosis etiology, no. (%) < 0.01 HCV 21 (43) 38 (78) HBV 4 (8) 5 (10) ARLD 14 (29) 2 (4) NAFLD 10 (20) 4 (8) Figure 1.: (A) Differentially abundant taxa in controls (n = 49) versus patients with HCC (n = 49). (B) In the subset of patients with NAFLD, alpha diversity was higher in patients with HCC and NAFLD (n = 4) than controls with NAFLD (n = 10). (B) Beta diversity in patients with HCC and NAFLD (n = 4) compared to controls with NAFLD (n = 10).