Molecular signatures of long-term hepatocellular carcinoma risk in nonalcoholic fatty liver disease

Abstract

Prediction of hepatocellular carcinoma (HCC) risk is an urgent unmet need in patients with nonalcoholic fatty liver disease (NAFLD). In cohorts of 409 patients with NAFLD from multiple global regions, we defined and validated hepatic transcriptome and serum secretome signatures predictive of long-term HCC risk in patients with NAFLD. A 133-gene signature, prognostic liver signature (PLS)-NAFLD, predicted incident HCC over up to 15 years of longitudinal observation. High-risk PLS-NAFLD was associated with IDO1⁺ dendritic cells and dysfunctional CD8⁺ T cells in fibrotic portal tracts along with impaired metabolic regulators. PLS-NAFLD was validated in independent cohorts of patients with NAFLD who were HCC naïve (HCC incidence rates at 15 years were 22.7 and 0% in high- and low-risk patients, respectively) or HCC experienced (de novo HCC recurrence rates at 5 years were 71.8 and 42.9% in high- and low-risk patients, respectively). PLS-NAFLD was bioinformatically translated into a four-protein secretome signature, PLSec-NAFLD, which was validated in an independent cohort of HCC-naïve patients with NAFLD and cirrhosis (HCC incidence rates at 15 years were 37.6 and 0% in high- and low-risk patients, respectively). Combination of PLSec-NAFLD with our previously defined etiology-agnostic PLSec-AFP yielded improved HCC risk stratification. PLS-NAFLD was modified by bariatric surgery, lipophilic statin, and IDO1 inhibitor, suggesting that the signature can be used for drug discovery and as a surrogate end point in HCC chemoprevention clinical trials. Collectively, PLS/PLSec-NAFLD may enable NAFLD-specific HCC risk prediction and facilitate clinical translation of NAFLD-directed HCC chemoprevention.

Fig. 1.. Study design.

PLS-NAFLD was derived in a cohort of patients with history of prior NAFLD-associated HCC who underwent curative ablation therapies and validated in HCC-naïve and HCC-experienced patients with NAFLD. PLS-NAFLD was translated to serum protein-based PLSec-NAFLD and validated in an independent cohort of HCC-naïve patients with cirrhosis from NAFLD.

Fig. 2.. Derivation of PLS-NAFLD.

(A) Expression of PLS-NAFLD genes and clinico-histological and genetic features in the derivation set. (B) Prognostic association of PLS-NAFLD. (C) Clusters of human liver-derived single cells from a meta-analysis of four scRNA-seq datasets representing healthy to NAFLD-affected livers. (D) Induction of high- and low-risk PLS-NAFLD genes measured by the average relative expression (“score”) across human hepatic single-cell clusters. (E) H&E staining (upper panel) and histological architecture determination over grid-like “spots” for spatial transcriptome profiling (lower panel) of liver tissue from a patient with NAFLD. (F) Induction of high- and low-risk PLS-NAFLD genes measured by the “score” across the four histological architectures. (G) Correlation of high- and low-risk PLS-NAFLD scores with relative abundance of the hepatic single cell clusters across the four histological architectures. (H) Number of inferred cell-cell interactions between the five hepatic single cell clusters co-presenting in the portal tracts and contributing to the high-risk PLS-NAFLD induction. (I) Representative portal tract (outlined by dotted line) with the high-risk PLS-NAFLD induction determined by the spatial transcriptome profiling (right upper panel), where IDO1+ cDCs (left lower panel) and PD-1+ CD8+ T cells (right lower panel) were co-localized in close proximity.

Fig. 3.. Independent validation of PLS-NAFLD in HCC-naïve and HCC-experienced patients with NAFLD.

(A) Overview of tissue sampling and clinical follow-up in the tissue validation set 1. (B) Expression of PLS-NAFLD genes and clinico-histological and genetic features in tissue validation set 1. (C) Prognostic association of PLS-NAFLD with incident HCC in tissue validation set 1. (D) Longitudinal changes in PLS-NAFLD-based HCC risk measured by combined enrichment score (CES) and clinico-histological features and laboratory tests between the serial biopsies. (E) Prognostic association of improved PLS-NAFLD. (F) Time-dependent AUROC of improved PLS-NAFLD and regressed fibrosis (decreased F-stage). (G) Expression of PLS-NAFLD genes with clinico-histological and genetic features in tissue validation set 2. (H) Prognostic association of PLS-NAFLD in tissue validation set 2.

Fig. 4.. Independent validation of serum-based PLSec-NAFLD in HCC-naïve patients with NAFLD cirrhosis.

(A-B) PLSec-NAFLD protein abundance (A) and prognostic association of high-risk PLSec-NAFLD (B) in the serum validation set. (C) Calibration plot of PLSec-NAFLD at 5, 10, and 15 years after blood collection for PLSec-NAFLD assessment. The diagonal dotted line indicates ideal calibration. (D) Prognostic association of etPLSec-NAFLD. (E) Time-dependent AUROC of etiology-agnostic PLSec-AFP, etiology-specific PLSec-NAFLD, and their combination named etPLSec-NAFLD.

Fig. 5.. Therapeutic modulation of PLS-NAFLD.

(A) Study design to assess PLS-NAFLD modulation by bariatric surgery in patients with NAFLD under lifestyle intervention. (B) Proportion of patients with significantly improved PLS-NAFLD with bariatric surgery in patients with NAFLD under lifestyle intervention. (C) Study design to assess association of PLS-NAFLD status with statin use in patients who underwent bariatric surgery. (D) Association of PLS-NAFLD-based HCC risk prediction with lipophilic or hydrophilic statin use. (E) Experimental design to assess the effect of an IDO1 inhibitor, epacadostat, in a PLS-inducible cell culture model (cPLS system). (F) Induction of high-risk pattern of PLS-NAFLD by free fatty acid and its reversal with epacadostat in the cPLS system.