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Toniutto et al. Hepatoma Res 2020;6:50 I http://dx.doi.org/10.20517/2394-5079.2020.40 Page 9 of 21

Molecular biomarkers
Several studies investigated the usefulness of molecular biomarkers to predict HCC recurrence in liver
[85]
transplanted patients . Regarding DNA alterations/mutations evaluable in liver tissue, the presence of
TP53 mutations, high fractional allelic loss, significant hypo-methylation of 8 tumor suppression genes,
and the absence of CTNNB1 mutations, identified a molecular subclass of aggressive HCC. These features
[86]
were predictive of reduced recurrence-free survival in a small group of 25 liver transplanted patients .
A further interesting approach is to evaluate the impact of gene expression signatures in liver tissue, both
inside and outside of the tumor, in predicting HCC recurrence. Applying this approach in a large cohort of
[87]
132 liver transplanted patients for HCC outside Milan criteria, Miltiadous et al. demonstrated that the
[88]
S2 molecular subclass and progenitor cell markers (CK19 signature ) were independent predictors of
[89]
overall survival and of HCC recurrence after LT, respectively.
The fractional allelic imbalance rate index (FAI), determined from tissue samples, is used to compare the
[90]
acquired mutational load between different tumors . FAI was evaluated in a cohort of 71 liver transplanted
patients with HCC, 18 of whom experienced tumor recurrence. Among the 19 microsatellites evaluated,
[91]
3 loci (D3S2303, D9S251, and D9S254) were found to be predictive of recurrence after LT . If confirmed
in other prospective studies, and in patients outside the Milan criteria, FAI could represent an interesting
tool to identify recipients at higher risk of tumor recurrence.

Enzymes such as histone deacetylases (HDACs) and matrix metalloproteinases (MMPs) have also been
studied in liver transplanted patients. HDACs regulate genes and are involved in tumor cell proliferation,
differentiation, invasion, and metastasis. Liver transplanted patients carrying T alleles in HDAC1 rs1741981
and HDAC3 rs 2547547 single nucleotide polymorphisms have been found to have a low risk of HCC
recurrence . On the contrary, high expression of HDAC3 was related to high risk of HCC recurrence in
[92]
[93]
HBV positive recipients .
MMPs are extracellular matrix-degrading enzymes that can be secreted by tumor cells to enhance tumor
invasiveness and metastasis. Although results among different studies are conflicting , MMP-9 and MMP-2
[42]
[94]
positive staining in stromal tissue adjacent to tumors seems to be associated with HCC recurrence .
While there is a rationale for investigating the role of enzymes in predicting HCC recurrence, the results of
the studies are still too heterogeneous to draw solid conclusions for use in clinical practice.

Besides DNA changes and aberrant gene expression, miRNAs have been evaluated as potential prognostic
biomarkers in HCC. An interesting report proposed a prognostic score incorporating the expression in
liver tissue of miR-214, miR-3187, and the Milan criteria, which improved the accuracy of predicting HCC
[95]
recurrence compared with the Milan criteria alone . Despite several studies reporting a potential role of
[85]
miRNAs in predicting HCC recurrence , none of them were really prospective and adequately powered.
The major limitation in introducing miRNAs in clinical practice is probably the need for a liver tissue
biopsy to evaluate their expression. To overcome this important limitation, liquid biopsy has emerged as a
minimally invasive alternative approach to analyze HCC components without the need of tissue samples.
This method allows the analysis of DNA, RNA in extracellular vescicles such as the exosomes, or circulating
[85]
tumor cells released by the tumor in the bloodstream . Among the few studies that have applied this
[96]
technology, Nakano et al. demonstrated that circulating exosomal miR-92b could have clinical value for
predicting HCC recurrence post-LT. The main limitation of liquid biopsy is the low sensitivity and lack of
[97]
reproducibility when different technologies were applied .
Among the serological markers that can be measured before transplantation, AFP remains the most
clinically useful to date, albeit with the major limitation that it can only be used in protein secreting HCC.