Discussion
HCC patients treated with sorafenib show a highly variable response, and
patients experience resistance and adverse events in approximately 30%
of cases. The molecular mechanisms underlying inter-individual
variability in sorafenib response have yet to be fully elucidated, and a
deeper knowledge of the underlying mechanisms and associated gene
variants would allow us to tailor treatment prescriptions for better
outcomes.
Thus, with this aim, we studied 5 known candidate SNPs in genes
controlling tumor angiogenesis, VEGF-A (rs2010963), VEGF-C(rs4604006), HIF-1α (rs12434438), ANGPT2 (rs55633437), andNOS3 (rs2070744), and genotyped a subgroup of 23 HCC patients
using the DMET plus platform to identify potential prognostic biomarkers
correlated to HCC patient responses to sorafenib treatment. Moreover, in
a subgroup of patients (n = 11), the serum/plasma concentration of
sorafenib was determined. The average steady-state sorafenib
concentration was 4.13 mg/L in responders, and 4.41 mg/L in
non-responders, independent of sorafenib dosages. No significant
correlation was detected between sorafenib concentration and clinical
outcome (data not shown), as already reported in the
literature.35
In our study, the analysis showed that the allele and genotype
frequencies of SNPs in angiogenesis-related genes were significantly
correlated with the response to sorafenib only for the rs2010963 C
allele (p = 0.004) and the CC genotype (p = 0.046) of theVEGF-A gene, in accordance with the results of the retrospective
multicenter study ALICE-2, in which the rs2010963 C allele and CC/CG
genotype were significantly associated with a higher median overall
survival of HCC patients receiving sorafenib.10 It is
likely that VEGF-A -related genetic variants could influence the
level of circulating VEGF,36,37 therefore affecting
sorafenib response. Also, in the SHARP trial,38 it was
reported that a low VEGF-A plasma baseline level, as a prognostic
independent factor, can predict outcomes in patients with advanced HCC,
both in the entire patient population and in the placebo
cohort.37 Consistent with these results, in our GEO
analysis, patients expressing lower levels of VEGF-A mRNA showed a
better response to sorafenib therapy (GSE109211 dataset). Our findings
confirmed the prominent role of the rs2010963 gene variant andVEGF-A expression as significant predictive factors for sorafenib
response in HCC patients.10,39
DMET genotyping showed a statistically significant association of the
sorafenib “non-responder” phenotype with the heterozygous genotypes ofSLC22A14 rs171248, rs149738, and rs183574, and the homozygous
genotypes AA in SULT1A2/CCDC101 rs11401, DPYD rs2297595,FMO2 rs2020863, the TT in DPYD*9 rs1801265, the GG inADH6 rs10008281, and CYP26A1 rs7905939. Instead, the
sorafenib “responder” phenotype was associated with the genotypes CC
in ADH1A rs6811453, the AG in SULT1A2/CCDC101 rs11401,DPYD rs2297595, FMO2 rs2020863, and CT in DPYD*9rs1801265, as well as with the homozygous genotypes of SLC22A14rs171248 (TT), rs149738 (AA) and rs183574 (AA). These results
demonstrate that the ADME genotype is correlated with different
responses to sorafenib, underlying the role of the reference allele or
variant in the effect on treatment response.
Moreover, to verify whether a correlation of SNPs in angiogenesis- and
ADME -related genes might help to discriminate responder/non-responder
patients, we applied a classifier to mine classification rules able to
figure out the principal signatures for discriminating among patients
belonging to the responder/non-responder to sorafenib phenotypes. The
novelty of our study lies in the identification of 10 rules in different
genotype associations for the identification of the non-responder
phenotype, and 3 rules for the responder type. These rules may represent
a genetic signature which could allow the stratification of patients who
are fit for sorafenib treatment. We found that the genetic signature
including 3 ADME-SNPs, SLC22A14 (rs171248), ADH1A
( rs6811453) and CYP26A1 ( rs7905939), and 3 known SNPs in
angiogenesis-related genes, VEGF-A (rs2010963), VEGF-C (rs4604006), and
HIF-1A (rs12434438), was correlated to sorafenib response in our
dataset, allowing the discrimination between responders/non-responders
according to mRECIST criteria. The signature of response, identified by
a decision tree, was also validated by the GRS analysis.
However, while the role of angiogenesis related genes is well known in
HCC patients treated with sorafenib, little information is reported in
the literature regarding the role of selected ADME genes in this
context. SLC22A14 , also known as organic cation transporter-like
2 (OCTL2), is a gene encoding a member of the organic-cation transporter
family and anions (OATs), whose expression is high in the liver. As for
the other SLCs, it is involved in regulating the homeostasis of
metabolites and the uptake of a wide range of molecules and the
disposition of drugs, as well as in promoting cell proliferation,
migration, and invasion in HCC.40 The function of SLC
members in sorafenib resistance is not clear, and only recently have
studies begun to investigate their role in
chemoresistance,41-44 highlighting the role of
aberrant variants or SNPs in organic cation transporters during liver
carcinogenesis, with effects on the ability of HCC to respond to
sorafenib.
The ADH1A gene catalyzes the oxidation of alcohols to aldehydes
and belongs to the superfamily of dehydrogenase enzymes. Several reports
provide a correlation of ADH1A and other ADH s expression
with increased risk of liver cancer, with an impact on the prognosis for
HCC patients.20,45
CYP26A1 , a member of the cytochrome P450 enzyme superfamily, is
mainly involved in retinoic acid metabolism and the synthesis of
cholesterol, steroids, and other lipids, and it contributes to the
development and progression of multiple cancers.46-48Previous studies in HCC have shown that CYP26A1 mRNA is downregulated in
tumor tissue compared to paired-matched non-tumor
tissues,49 but the role of CYP26A1 in HCC is not
entirely clear despite being reported as hypovitaminosis. A, as
potential result from a CYP26A1 depletion, could be correlated to higher
risk of carcinogenesis.50
To support the correlation between ADME- and angiogenesis-related genes,
the network and PEA analysis highlighted the association of 8/12
identified genes in topological key points with a relevant node degree
score in important pathways underlying biological mechanisms implicated
in HCC and sorafenib. In fact, VEGF-A, together with ADH1A ,CYP26A1 , and VEGF-C , showed a common interaction in
“signal transduction pathways” which are known to be dysregulated in
HCC, with consequent uncontrolled cell division and
metastasis,44 alteration of intracellular regulators
or extracellular signals with abnormal epigenetic modification, and gene
expression in the tumor microenvironment. Moreover, all seed genes
identified were involved in multiple pathways, showing their involvement
in significantly affected by biological functions.
Given that the most recent AASLD guidelines on HCC systemic therapy have
approved the use of drugs like bevacizumab+atezolizumab and lenvatinib
as first-line therapies, in addition to sorafenib, the opportunity to
verify a score like the GRS observed in our responder patients or the
genetic signature identified in our retrospective study might help
clinicians to select patients with higher chances to benefit from
sorafenib treatment. In the presence of a favorable GRS, physicians
could treat advanced HCC patients with sorafenib as soon as possible.
Conversely, patients with an unfavorable GRS might not be excellent
candidates for sorafenib.