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Schwertheim et al. Hepatoma Res 2020;6:41 I http://dx.doi.org/10.20517/2394-5079.2020.23 Page 11 of 14

[18]
NI in thyroid carcinomas and pulmonary neuroendocrine tumors [17,18] . Rezk et al. reported that some cases
had β-catenin positive inclusions although they had almost negative cytoplasmic/nuclear staining. This is in
line with our results. Though we found in 11 of 19 cases a correlation of β-catenin immunopositivity within
NI with immunopositivity in the surrounding cytoplasm, there were 8 cases nearly lacking in cytoplasmic
[18]
staining. Rezk et al. suggested that β-catenin might play a role in the development of NI as it is involved
in organizing actin and microtubule polymerization [18,19] . Additionally, Gamachi et al. also observed NI with
immunopositivity for biotin, biotin-binding enzymes and β-catenin in pregnancy-related endometrium
[20]
and morule-associated neoplastic lesions ; they revealed that though biotin and biotin-binding enzymes
were present in neoplastic and non-neoplastic tissues, β-catenin was lacking in non-neoplastic endometrial
lesions. Further, NI with immunoreactivity for PAX8 protein were observed in conventional clear cell renal
[21]
cell carcinoma . The immunopositivity for PAX8 is interesting as it is a transcription factor that is also
involved in the proliferation of tumor cells via the Wnt/β-catenin pathway; overexpression of PAX8 has
[21]
been reported in various carcinomas . These studies did not clarify why NI contained these proteins. In
our current study, to clarify the reason for β-catenin immunopositivity in NI, we examined our HCC cases
on CTNNB1 exon 3 mutations. We differentiated between immunopositivity within the nucleus and in
the intranuclear inclusion and found up to 20 different CTNNB1 mutations, all of which are listed in the
COSMIC catalog and most, reported in the NCBI ClinVar database. A positive mutation status for CTNNB1
correlated significantly with nuclear immunopositivity, which is supported by the literature as CTNNB1
mutations are known to be associated with the translocation of β-catenin proteins from the membrane to the
[22]
nucleus and activation of Wnt/β-catenin signalling . However, we found no association between CTNNB1
mutations and positive β-catenin-immunostaining within the inclusions. Recently, we have shown that NI
[6]
in HCC contained autophagy-associated proteins . Since we could simultaneously detect degraded cell
material and lysosomes in NI by TEM studies, we assumed that biological processes similar to autophagy
[6]
are taking place in NI . These autophagy-associated proteins were partly co-localized within the same NI.
In the current study, we demonstrate that cases with β-catenin immunopostive NI also harbored NI with
immunopositivity for autophagy-associated proteins; this association was significant for p62, cathepsinB/
D, ubiquitin and LC3B. We found that p62 immunoreactivity was almost exclusively located in NI whereby
cytoplasmic staining was diffuse and very weak. We suggest that high p62 positivity in NI could be caused by
[23]
several reasons including alterations in p62 nucleocytoplasmic shuttling as previously reported in our HCC
[6]
study . Intriguingly, we also detected co-localization of β-catenin with p62. The latter strongly suggests that
biological processes with involvement of β-catenin are taking place within NI. We found in 11 of 19 cases
a significant correlation between β-catenin immunopositivity within NI and β-catenin immunoreactivity
in the surrounding cytoplasm, and we suppose that some NI with positive β-catenin immunostaining have
developed by invagination of the cytoplasm into the NI with further closure of the invagination.

To clarify the factors contributing to β-catenin accumulation in NI, we also studied the immunoreactivity of
proteins associated with the Wnt/β-catenin pathway. We detected immunopositivity within NI for glutamine
synthetase, which is a target of β-catenin signalling implicated in the development of HCC [7,24,25] and for
[14]
KDM2A having a role in β-catenin degradation [14,26,27] . Lu et al. reported that lysine demethylase KDM2A
demethylases nuclear β-catenin, which then induces the degradation of β-catenin and consequently, the
downregulation of Wnt/β-catenin target genes. In our study, the presence of NI containing KDM2A protein
was significantly associated with longer survival in HCC patients; this is in line with our previous work in
HCC demonstrating by Kaplan-Meier survival curves that the presence of NI significantly correlated with
[6]
survival . Additionally, HCC patients with KDM2A immunopositivity in the cytoplasm showed a significant
benefit in both, disease-specific OS and recurrence-free survival. Further, we found that even the presence of
immunopositive KDM2A in the cytoplasm of normal tissues adjacent to tumor was associated significantly
with recurrence-free survival. It has been documented by immunohistochemical studies that KDM2A
[28]
positive staining, though predominantly found in the nucleus, can also be detected in the cytoplasm .
[14]
Lu et al. reported methylation by KDM2A in both non-phosphorylated and phosphorylated β-catenin,