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Page 6 of 13 Kwee et al. Hepatoma Res 2021;7:8 I http://dx.doi.org/10.20517/2394-5079.2020.124

well, moderately, and poorly differentiated tumors that are capable of recapitulating the salient histological
[53]
and molecular features of b-catenin-driven human HCC . Experimentally, increased tumor FCH uptake
in this system was shown to correlate with increased b-catenin protein expression as well as metabolomic
and transcriptional fingerprints of canonical Wnt/b-catenin activity. Conversely, control animal models
[28]
with non-b-catenin-activated tumors were found to demonstrate low tumor FCH uptake . Providing a
simple mechanistic explanation for how this pathway promotes increased tumor FCH uptake and choline
phospholipid metabolism, it was confirmed that b-catenin activation was able to drive choline membrane
transporter expression. Following a translational route, the team that conducted these experiments also
obtained and sequenced tumor DNA from 13 patients that had underwent clinical FCH PET/CT and found
mutated CTNNB1 in 6/7 patients with FCH-positive tumors and wild-type CTNNB1 in 6/6 patients with
[28]
FCH-negative tumors .

In an attempt to corroborate their results, we examined the tumor genomic profiling results from six HCC
patients who underwent preoperative liver FCH PET/CT imaging prior to liver resection. These patients
were among the participants of a recently completed diagnostic clinical trial of FCH PET/CT in liver
[54]
cancer . To explore whether b-catenin activating mutations found in tumor DNA could also be detected
in cfDNA, we analyzed the pre-treatment plasma samples collected from these patients by performing
targeted mutation profiling of cfDNA using an oncology-specific next generation sequencing panel (56G
Oncology Panel, Swift Biosciences). The results of this liquid biopsy mutation analysis along with the PET
imaging and clinical tumor DNA profiling results are shown in Figure 2.

As a summary of these preliminary findings, mutations associated with Wnt/b-catenin activation were
detected in the tumor DNA of four patients, with three being CTNNB1 mutations and one being a mutation
involving the guanine nucleotide binding protein-alpha stimulating sub-unit (GNAS) gene. These same genes
were also found mutated in the corresponding cfDNA of these patients. Interestingly, mutations involving
GNAS have been reported to be a cause of upregulated Wnt/b-catenin activity and lipid metabolism in
several digestive tract cancers but not yet in HCC [59,60] . Notably, the tumors of all four of these patients
showed high uptake of FCH on PET/CT (image insets on Figure 2). Conversely, no Wnt/b-catenin
activating mutations were identified in the tumor DNA or cfDNA of the remaining 2 patients. The tumors
of both these patients showed low FCH uptake on PET/CT. Interestingly, one of these FCH non-avid
[61]
tumors harbored a SMAD4 gene mutation associated with Wnt/b-catenin pathway downregulation .
While these limited data do not allow us to make any statistical conclusions, they do appear to agree with
the results obtained by the other investigators and, furthermore, provide a demonstration of the potential
capability of liquid biopsy to detect Wnt/b-catenin activating tumor mutations in HCC.

In further corroboration of these findings, we revisited a whole-genome expression dataset generated
[54]
from 41 HCC tumors imaged preoperatively by FCH PET/CT . Using gene set enrichment analysis,
we found that sets of genes associated with b-catenin activation were significantly enriched by FCH-
avid HCC tumors (FDR 0.062) [Figure 3A]. Conversely, we found tumors displaying low FCH uptake
were enriched by a T-cell inflammation signature that has been shown to be strongly predictive of
[15]
clinical response to ICI therapy in several different cancers (FDR 0.116) [Figure 3B] . Furthermore, our
previous published analysis of this radiogenomic dataset reported that tumors displaying high FCH
[54]
uptake disproportionately expressed gene signatures corresponding to distinct molecular classes of HCC,
[62]
[63]
including the S3 class described by Hoshida et al. ; the G5 and G6 classes described by Boyault et al. ;
[64]
and the “CTNNB1-activated” class described by Chiang et al. . All of these classes have more recently
been associated with newly described immunotherapy-relevant HCC sub-types characterized by an
immunosuppressed TME or poor ICI response as well as evidence of abnormal b-catenin activity [10,46,65] .
One recently described immune-suppressed type of HCC shows significant overlap with the Hoshida
S3 class and is notable for its association with a lack of TME infiltration by immune cells as well as a