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Benhammou et al. Hepatoma Res 2020;6:35 I http://dx.doi.org/10.20517/2394-5079.2020.16 Page 7 of 15

[91]
95%CI: 0.39-0.61) and chronic liver disease mortality (adjusted relative risk = 0.55, 95%CI: 0.45-0.67) .
In an observational study using the Liver Cancer Pooling Project which consisted of US cohorts from the
National Cancer Institute Cohort Consortium, of the 679 patients who developed HCC, those who were on
aspirin also had a 32% reduction in the risk of HCC (adjusted HR = 0.68, 95%CI: 0.57-0.81) over the follow-
[92]
up period of 11.9 years . Although there was no mention of NAFLD patients (mostly hepatitis B and C),
this early study demonstrated a potential benefit of aspirin in other cirrhosis populations at risk for HCC.
These data led to a more recent study using the Nurses’ Health Study and the Health Professionals Follow-
up Study, where over a median follow-up of 8 years, 133,371 patients were assessed for HCC development.
Regular users of aspirin (≥ 2 doses; 325 mg per week) had a reduced risk of HCC (adjusted HR = 0.51,
[16]
95%CI: 0.34-0.77), which appeared to be dose and time-dependent . Patients who used aspirin for 5 years
or more had the lowest risk of HCC development (adjusted HR = 0.41, 95%CI: 0.21-0.77). Again, the etiology
of HCC and cirrhosis was not evident in the study, although one would expect these data to extrapolate
[16]
to NAFLD-associated HCC . This is further supported by NASH animal models treated with aspirin
demonstrating a decrease in hepatic fibrosis through decreased activation of pro-inflammatory pathways,
[93]
potentially by altering the microbiome . Further studies will be needed to ascertain this however, especially
given the benefit of aspirin in cardiovascular disease, which is one of the leading causes of mortality in that
[94]
patient population .
Microbiome
Advancements in technology, accessibility to sequencing and manipulation of big data has introduced tools
to start understanding how the microbiome contributes to NAFLD, NASH and HCC progression. There
is increasing evidence that gut dysbiosis plays a key role in driving the progression of NAFLD to NASH
[95]
and liver cirrhosis by creating a micro-environment which supports: (1) altered energy absorption ,
[98]
(2) modification of gut permeability [96,97] , (3) promotion of chronic low-level inflammation and, (4)
dysregulation of bile acid signaling [96,99-101] . There is however, a limited understanding of the unique
relationship between intestinal microbiota, dysbiosis and the development of HCC.

Studies have consistently shown that two phyla of bacteria are dominant within the gut flora, Firmicutes
and Bacteroidetes and that their ratios are altered in NAFLD/NASH patients when compared to healthy
controls [100,102-105] . In a recent study using whole-genome shotgun sequencing of DNA extracted from stool
samples, Loomba et al. [106] analyzed the microbiota of subjects with worsening degrees of fibrosis. The authors
discovered that in patients with mild/moderate (F0-F1) fibrosis, the gut flora is dominated by Firmicutes
and Bacterioidetes followed by Proteobacteria and Actinobacteria. In contrast, Proteobacteria levels were
augmented in patients with severe fibrosis (F ≥ 2) while levels of Firmicutes were diminished. In patients
with advanced fibrosis, Bacteroides vulgatus and Escherichia coli (E.coli) were the most abundant organisms.
Given that advanced fibrosis is a risk factor for HCC, it is conceivable that similar differences may be found
in patients with HCC. Indeed, a study performed by Grat et al. [107] comparing the stool composition of
patients with and without HCC (matched by etiology of cirrhosis and MELD scores) discovered that patients
with HCC had significantly higher levels of E. coli in their stools.

Studies have also shown that alcohol espouses gut permeability through the alteration of tight junctions
in gut epithelium [108] . With the liver being a first-pass organ, it is likely that subjects with gut dysbiosis are
predisposed to inappropriate translocation of gut bacteria and their endotoxins, leading to a chronic state
of inflammation. This is supported by data from Ponziani et al. [109] who showed that compared to patients
with NAFLD cirrhosis without HCC, those with HCC have higher levels of fecal calprotectin - a surrogate
measure of gastrointestinal inflammation. Furthermore, Ponziani et al. [109] demonstrated that independent of
HCC, patients with compensated liver cirrhosis have higher levels of plasma lipopolysaccharide (LPS). LPS
is a well-known endotoxin that simulates toll-like (TLR) and nod-like receptors in the intestinal epithelium.
NASH patients have been shown to have higher expression of circulating LPS levels as well as localization to