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Page 4 of 12 Armengol et al. Hepatoma Res 2021;7:50 https://dx.doi.org/10.20517/2394-5079.2021.19

The study of DNA methylation has revealed a global genome-wide DNA hypomethylation of HB [33-35] . In
[27]
addition, Carrillo-Reixach et al. reported two epigenomic subtypes of HB called Epi-CA and Epi-CB that
overlapped with C1/C2B and C2-Pure tumors, respectively. The epigenetic differences between Epi-CA and
Epi-CB tumors were found in the degree of DNA hypomethylation and CpG island hypermethylation. In
particular, Epi-CB tumors showed a more pronounced DNA hypomethylation and specific CpG-island
hypermethylation than Epi-CA tumors did. In line with the transcriptomic studies, the differences in the
DNA methylation of the tumors were also associated with tumor histology, specifically the degree of
immaturity of the main epithelial component, and patient clinical outcome. Therefore, patients harboring
tumors with an Epi-CB subtype, which had predominantly a C2 transcriptome, showed a worse prognosis
than patients with tumors of the Epi-CA subtype with a similar C1 or C2B transcriptome. The assessment of
[36]
percentage of unmethylated Alu regions measured using the QUAlu method was proposed as a tool to
discriminate Epi-CA and Epi-CB tumors taking into account the global degree of DNA hypomethylation .
[27]
Later, the finding of two main groups of HBs based on the DNA methylation data, significantly associated
with C1/C2 tumors, was also reported by Sekiguchi et al. who named them F and E from their correlation
[15]
with fetal and embryonic histology, respectively. In this study, the authors found differences in the
methylation level of the HNF4/CEBPA-binding regions in gene bodies, which were more highly
hypermethylated in tumors of the E cluster as compared with tumors of the F cluster.

BIOMARKERS TO IMPROVE FUTURE CLINICAL MANAGEMENT OF HB
Altogether genomic, transcriptomic, and epigenomic studies have offered new clues about HB pathogenesis
and identified biomarkers with strong prognostic predictions. Currently, the treatment of HB patients is
based on the ongoing Pediatric Hepatic International Tumor Trial (PHITT), which uses a highly refined
[37]
risk stratification system of the Children’s Hepatic tumors International Collaboration (CHIC) . This
stratification relies on the main clinical prognostic factors such as the pretreatment extent of disease
(PRETEXT) stage, metastasis, patient age, AFP serum levels, and annotation factors related to vein,
intrahepatic, and extrahepatic tumor dissemination. Up to now, no molecular data have been incorporated
into clinical risk-adapted systems, despite that fact that they have proven effective for prognostic prediction
when combined with CHIC stratification [11,26,27] . First, the 16-gene signature identified to discriminate C1
and C2 tumors was identified as an independent prognostic factor when compared to clinical variables
including PRETEXT, vascular invasion, and metastasis . This signature was widely validated in a recent
[11]
study, using a large cohort of 174 patients, that identified the C2 subtype as the only independent factor
contributing significant additional prognostic information to the clinical parameters compared with three
other biomarkers studied (CTNNB1, NFE2L2, and TERT mutations) . Finally, a recent study proposed a
[26]
first molecular risk stratification of HB by integrating novel transcriptomic and epigenomic biomarkers
such as the C2-Pure subtype, the 14q32-signature of the DLK1/DIO3 locus, and the Epi-CB tumors . In
[27]
this study, the combination of biomarkers performed better when stratifying patients according to their
prognosis than using individual biomarkers did. Therefore, in a similar way to the CHIC clinical
stratification, the integration of multiple molecular prognostic factors has a cumulative effect in terms of
risk prediction. Finally, the combination of clinical and molecular risk-staging systems (CHIC and MRS)
resulted in better patient risk prediction and highlighted the importance of incorporating molecular factors
to the clinical setting [Figure 1].

In summary, the implementation of a combined clinical and molecular risk staging system is key for moving
precision medicine of childhood liver cancer forward and to improve the current patient morbidity and
mortality. The prospective cohort of patients enrolled in the ongoing PHITT (NCT03017326) provides a
unique opportunity to perform a biomarker validation study using a large patient cohort as a pre-step to the
incorporation of a highly validated biomarker panel in the next clinical trials.