DISCUSSION
The scenario of few recurrent driver mutations in HBs represents a
challenge for risk stratification and adjustment of the therapeutic
regimen, and for this reason molecular subclassifications, including
gene signatures, have been proposed 2,29,30.
Expression pattern of 16 genes grouped HBs according to tumor
aggressiveness in two groups, called C1 and C2 signatures where
β-catenin is upregulated in both signatures with genes being related to
hepatocyte markers (GLUL , CYP2E1 , CYP1A1, andAQP9) , cell cycle regulation (E2F5 , BUB1 ,DLG7 ), hepatic stem/progenitor markers (DUSP9 , AFP ,CK19 , and TACSTD1) , and metabolism (APOC4, ALDH2 )29. The differential activation of hepatic
stem/progenitor markers (LIN28B, SALL4 , and AFP ), genes
related to cancer pathways (TERT, TP53 ), metabolism
(NFE2L2 ) and hepatobiliary system (NOCHT1 , HNF )
were also used to stratify HBs in molecular subtypes according to
risk30.
Here, we focused on epigenetic markers (expression of DNMTs and TETs as
well as global DNA methylation) in addition to genes related to the
stages of liver differentiation, in an attempt to perform HB
stratification. Hepatocytes are the major cell type of the liver,
accounting for ~ 70% of the mass of the adult organ.
The anterior portion of the hepatic diverticulum gives rise to the liver
from endoderm cells that differentiate in the bi-potential cells known
as hepatoblasts 31,32, and liver growth and hepatocyte
maturation are processes regulated by genes acting intrinsically in
these cells 33. HB is postulated to develop from
hepatoblasts, the precursor cells of hepatocytes 2;
however, microscopically, HBs are heterogeneous, rarely composed of only
one cell type, often exhibiting combinations of epithelial, stromal,
mesenchymal and/or undifferentiated cells components11.
Genes associated with DNA methylation exhibited in HBs a pattern of
expression directly correlated, which is inverse to the expression
pattern of mature hepatocyte markers, highlighting the central role of
this specific epigenetic pathway in liver differentiation and as well as
in HB stratification. Our data also disclosed a direct correlation ofUHRF1 and AFP expression in tumors, which was previously
described for HCC 34, possibly representing a new
finding to be explored in HBs. Additionally, there is a similar
expression pattern of HFN4A and FOXA2 , which are nuclear
factors that present tissue co-expression and cooperate for hepatic
pathway cell commitment35, emphasizing the key role of
the hepatic differentiation blockage in HB development.
Besides reinforcing the molecular heterogeneity of these embryonal
tumors, we propose a panel of 13 genes for this HB stratification
(TET1, TET2, TET3, DNMT1, DNMT3A, UHRF1, ALB, CYP3A4,
TDO2 , UGT1A1, AFP , HNF4 A, and FOXA2 ). Remarkably,
our data showed that the DNA methylation machinery exerts a key role in
the characterization of HBs, directly reflected in diverse DNA
methylation content. Six out of the eight genes associated with DNA
methylation were determinants for HB stratification, evidencing the
importance of the epigenetic machinery for the biology of this embryonal
liver cancer.
At the beginning of the embryo development, the epigenetic machinery
acts by decreasing the overall level of DNA methylation, allowing the
expression of genes associated with pluripotency 36.
During the maturation of the organ, there is an increase in the DNA
methylation, a process that will culminate in the selective expression
of tissue-specific genes, associated with the promotion of cell
differentiation. The iPSC and definitive endoderm cell lines clustered
separately (Set-4), reinforcing that HBs are composed of cells already
compromised with the hepatocyte differentiation. A small group of
intermediate-risk tumors exhibited high expression of the epigenetic
machinery genes as well as hepatoblast markers (Set-1), a profile
suggestive of a precursor stage of hepatocyte differentiation. This HB
group present the highest expression of TET1 , TET3 , andUHRF1 among all tumors; in a recent work, we proposed an active
demethylation process in HBs, particularly associated with upregulation
of TETs and UHRF1 15 and, accordingly,
Set-1 tumors exhibited marked global DNA hypomethylation. In the other
hand, the Set-3 presented downregulation of the DNA methylation genes
and high expression of mature hepatocyte markers, similarly to
non-tumoral control livers; they were likely derived from cells
compromised with more advanced stages of hepatocyte differentiation, and
their global DNA methylation content is also similar to non-tumoral
livers, corroborating the hypothesis of origin in late stages of
hepatocyte differentiation. Considering that Set-3 comprised HBs from
patients who are deceased and/or developed metastases, as well as one
sample characterized as HB/HCC features, the major markers of hepatocyte
(UGT1A1 , TDO2 , and CYP3A4 ) that are differentially
expressed in this group, deserve further investigation as biomarkers of
worse prognosis. Finally, the Set-2 contains clinically heterogeneous
HBs exhibiting an intermediate expression profile between the Set-1 and
Set-3 clusters, with high expression of the DNA methylation genes and
downregulation of hepatoblast and mature hepatocyte markers. The Set-2
has a global DNA methylation level intermediary between Set-1 and Set-3,
which is in accordance with the hypothesis that these tumor samples have
a molecular profile transitional between hepatoblasts and mature
hepatocytes.
In a recent analysis involving a large group of 113 samples, HBs
presented two epigenetic signatures (Epi-CA and Epi-CB), according to
the degree of sample hypomethylation. Moreover, amplification of the
oncogenic 14q32 DLK1-DIO3 locus in part of the HBs delineated
signatures of moderate or strong expression of genes mapped to this
region. Using both findings, a molecular risk stratification of three
categories was proposed (MRS-HB) 37. Despite using a
smaller cohort of 21 HB samples in our study, we also showed that DNA
methylation is a strong biomarker for HB stratification, pinpointing to
specific DNA methylation genes as important players.
Precision medicine brings
the promise of improvement in diagnosis and matching patients to
personalized targeted therapies using genomics, epigenomics,
metabolomics and proteomics. One of the major barriers to the
implementation of this approach in clinical settings is the intra/inter-
tumor heterogeneity, in addition to the rarity in the case of pediatric
cancer. In particular, HB is a tumor with a genetic background with low
mutational background and few cytogenetic alterations12,30,38–41, in addition to DNA methylation changes13,42.
The present work describes the possibility of molecular stratification
of HBs according to markers of hepatocyte differentiation and DNA
methylation. Tumor heterogeneity could be overcome using molecular
signatures that are linked to clinical data. Here, we evidenced a HB
group (Set-3) with a similarity of late stages of hepatocyte
differentiation and lower DNA methylation genes that present a tendency
to a worse prognosis. Although the other two sets do not have clinical
characteristics that are divided evenly between groups, the association
of Set-3 with a worse prognosis is in concordance with the literature
that have shown metastasis at diagnosis, advanced age of diagnosis27,28,43, mutations at TERT gene30,39 associated with high mortality.
Most drugs based on the mechanisms of anticancer resistance in HB will
act on pathways such as p53, tyrosine kinase, cell cycle control, and
transcriptional and translational events. A specific gene,CYP3A4, upregulated in Set-3, is related to five drugs used in
the treatment of HBs 44, particularly etoposide (a
drug used in neoadjuvant chemotherapy), which causes demethylation in
liver cells 45,46, and Sorafenib, a kinase inhibitor
mainly oxidized by CYP3A4 , with the collaboration ofCYP1A1 and CYP1B1 47. Vincristine,
Cyclophosphamide and Isofosfamide are also metabolized by CYP3A448–51. These findings suggest that changes in this
gene could lead to resistance to treatment, maybe explaining the worse
clinical signs observed in Set-3, with CYP3A4 levels similar to
mature hepatocytes.
Together, data here showed the possibility of HB stratification in three
groups considering the epigenetic machinery and markers of liver
differentiation stages. These groups have levels of DNA methylation
according to their stage of cell differentiation, which indicates that
the clinical heterogeneity widely described for this tumor, also occurs
at the epigenetic level. Therefore, we demonstrate that epigenetics can
be an important tool that must be considered in stratification of HB.