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Kwee et al. Hepatoma Res 2021;7:8 I http://dx.doi.org/10.20517/2394-5079.2020.124 Page 5 of 13

Consistent with this apparent mechanism of b-catenin mediated immune cell exclusion, clinical
associations between markers of Wnt/b-catenin activation and various molecular signs of immunotherapy
resistance have been revealed through numerous and exhaustive molecular profiling and genotyping
studies of human tumor specimens [10,12,16,17,46] .

Mutated CTNNB1 and other mutated genes known to cause Wnt/b-catenin activation have been associated
with poor clinical response to treatment with ICI antibodies and other targeted agents in HCC [12,16,33,34] .
In one study that included 27 ICI-treated HCC patients who underwent pre-treatment tumor mutation
profiling, 10/10 of HCC patients with tumors bearing CTNNB1 or AXIN1 mutations experienced
progressive disease compared to 5/17 patients with tumors that did not have these b-catenin activating
[16]
mutations (100% vs. 29%, P < 0.001) . The gene AXIN1 codes for a protein involved in b-catenin
destruction, and it is mutated in 5%-19% of HCC [12,30,36,37,42,47] . The specific AXIN1 mutations involved cause
loss-of-function, and they are usually mutually exclusive to CTNNB1 mutations [47,48] . By most estimates,
they are the second most frequent mutation that leads to aberrant b-catenin activation in HCC [12,33,36] . In
addition to experiencing higher rates of progressive disease, ICI-treated HCC patients with CTNNB1 or
AXIN1 mutated tumors also experience significantly shorter progression free survival (median 2.0 months
[16]
vs. 7.4 months, HR = 9.2, 95%CI: 2.9-28.8, P < 0.0001) . These clinical associations imply that the
mutation statuses of these genes may have some value as predictive or prognostic biomarkers for HCC
immunotherapy.

Several other gene mutations that can drive Wnt/b-catenin signaling are relatively uncommon in
HCC [12,28,33,35] . For example, mutations that result in the loss or inactivation of the adenomatous polyposis
coli (APC) gene product are a frequent cause of aberrant b-catenin activation in colon and rectal cancer.
However, while it is estimated that over 80% of colorectal cancers harbor APC mutations, they are relatively
uncommon in HCC with an estimated frequency of 5%-7% [16,49] . With the recent clinical approval of
two cell-free DNA (cfDNA) sequencing panels for mutation profiling with relatively broad (pan-cancer)
coverage, it may now be practical to detect these mutations along with the more prevalent ones through
non-invasive liquid biopsy. In addition to profiling a large set of cancer-associated genes, these panels
also generate blood-based estimates of TMB (bTMB) and MSI-H status, making it conceivable that an
integrative genomic profile of relevance to the HCC immunotherapy space may be discovered. Cohort
studies as well as carefully designed prospective biomarker trials will be necessary to learn, test, and validate
such mutational profiles as predictors of ICI response. Since HCC is a cancer for which tumor tissue is
difficult to obtain clinically, these pan-cancer liquid biopsy panels may eventually prove to be a boon to
the development of more precise targeted therapy for this disease. However, regarding immunotherapy,
mutation status alone may not be able to fully explain the estimated 60%-80% of HCC patients who fail to
exhibit an objective response to ICI therapy.

DOES b-CATENIN ACTIVATED HCC HAVE A METABOLIC PHENOTYPE?
Other molecular mechanisms, including epigenetic alterations , as well as alterations involving
[48]
extracellular pathways [38-40,48] , have also been associated with aberrant Wnt/b-catenin signaling in
HCC. There are also data suggesting that upregulated lipid metabolism in HCC is associated with
b-catenin activation [28,29,50] . This association raises the possibility of inferring tumor b-catenin activation
status based on phenotypic assessments of tumor metabolism. Such assessments are clinically feasible
and can be performed non-invasively using several molecular imaging methods including positron
emission tomography (PET). As a proof of concept with regard to phospholipid metabolism, a recent
series of experiments performed in an adenomatous polyposis coli (APC) knock-out mouse model of
b-catenin activated HCC suggests that tumor uptake of fluorocholine (FCH), a fluorine-18 labeled PET
radiopharmaceutical analog of choline used for in-vivo tracing of tissue phosphatidylcholine synthesis [51,52] ,
is strongly influenced by b-catenin activation . This novel animal model of de-novo HCC can develop
[28]