Mod Pathol xxx (xxxx) xxx

Journal homepage: https://modernpathology.org/

Research Article

Adenoid Ameloblastoma Shares Clinical, Histologic, and Molecular

Features With Dentinogenic Ghost Cell Tumor: The Histologic Spectrum
of WNT PathwayeAltered Benign Odontogenic Tumors
Kyu-Young Oha,b, Seong-Doo Hongb, Hye-Jung Yoonb,*
a
Department of Oral Pathology, College of Dentistry, Dankook University, Cheonan, Korea; b Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul
National University, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T
Article history:
Received 11 October 2022 An epithelial odontogenic tumor called adenoid ameloblastoma (AA) has recently been included in
Revised 21 October 2022 the new WHO classification. However, AA has considerable overlapping features with a preexisting
Accepted 9 November 2022 entity, dentinogenic ghost cell tumor (DGCT). This study compared the clinical, histologic, and
Available online xxx molecular characteristics of AA and DGCT. Eight cases of odontogenic tumors initially diagnosed as
AA or DGCT were included in this study. Quantitative histologic analysis, b-catenin immunohisto-
Keywords: chemistry, and molecular profiling using next generation sequencing were performed. Additionally,
adenoid ameloblastoma accumulated clinical data of AA and DGCT were statistically analyzed. Nuclear b-catenin accumu-
b-catenin lation was detected in all cases in common, although the tumors studied histologically consisted of
dentinogenic ghost cell tumor varying combinations of the AA-like phenotype, ghost cells, and dentinoid. However, CTNNB1 hot-
next-generation sequencing spot mutations were not found in any case. Instead, loss-of-function mutations in tumor suppressor
WNT pathway genes involved in the WNT pathway, including the APC, SMURF1, and NEDD4L genes, were found
regardless of histologic type. In addition, KRT13 mutations were detected in 2 cases with a high
proportion of ghost cells. Finally, a literature analysis revealed clinical similarities between the
previously reported cases of AA and DGCT. These findings suggest that from a clinical and molecular
point of view, AA and DGCT represent a histologic spectrum of WNT pathwayealtered benign
odontogenic tumors rather than 2 distinct tumors. Moreover, previously unidentified keratin mu-
tations may be associated with ghost cell formation found in specific types of odontogenic lesions.
© 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.

Introduction
Adenoid ameloblastoma (AA) is an epithelial odontogenic tu-
mor newly included in the 5th WHO Classification of Head and
Neck Tumours updated in 2022.1 The key histologic features of AA
are ameloblastoma-like epithelium, characteristic duct-like
structures, cribriform architecture, and cellular condensations
* Corresponding author.
E-mail address: hyejyoon@snu.ac.kr (H.-J. Yoon).
called morules.1,2 Recently, it has been revealed that AA does not
harbor BRAF and KRAS mutations, which are recurrent in histo-
logic mimics of AA including ameloblastoma and adenomatoid
odontogenic tumor.3 Instead, CTNNB1 hotspot mutations have
been detected in AA cases together with nuclear accumulation of
b-catenin.4
Dentinogenic ghost cell tumor (DGCT) is a benign but locally
infiltrative odontogenic tumor characterized by ameloblastoma-
like epithelium with varying amounts of ghost cells and denti-
noid.5 Although there are considerable overlapping histologic
features between AA and DGCT such as ameloblastic epithelium

0893-3952/$ - see front matter © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.modpat.2022.100051
2 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx
and the presence of ghost cells and dentinoid, these 2 tumors have
been distinguished based on the presence (AA) or absence (DGCT)
of adenoid morphology.2,3 However, adenoid/pseudoglandular/
cribriform structures have been described in several DGCT cases
too,6,7 and similarities in demographic and clinical data are found
between AA and DGCT, such as high recurrence rates.7,8 Further-
more, nuclear b-catenin accumulation has been noted in DGCT
cases,9,10 and a CTNNB1 hotspot mutation has been identified in
DGCT.10 Taken together, AA and DGCT have commonalities in
clinical, histologic, and molecular aspects, warranting consider-
ation for their reclassification.
The WNT pathway is one of the main signaling cascades
involved in tumorigenesis, such as colorectal adenocarcinoma and
hepatocellular carcinoma.11 Nuclear translocation of b-catenin is a
hallmark of WNT pathway activation and subsequently promotes
transcription leading to multiple cellular processes.12
WNT-activated cancers harbor mutations not only in the
CTNNB1 gene encoding b-catenin but also in tumor suppressor
genes involved in the WNT pathway such as adenomatous pol-
yposis coli (APC) and AXIN1/2.11,12 Similarly, the absence of CTNNB1
hotspot mutations has been demonstrated in subsets of
WNT-activated odontogenic lesions, such as DGCT,9 in AA,4 and
even in calcifying odontogenic cyst (COC),13,14 which is known for
its high CTNNB1 mutation rate. Considering that loss-of-function
(LOF) mutations in APC have been detected in ghost cell odonto-
genic carcinoma15 and odontogenic carcinoma with dentinoid16
by using next generation sequencing, a detailed genetic analysis
may broaden our understanding of the mutational profile of WNT-
activated odontogenic lesions with wild-type CTNNB1.
In this study, we compared characteristics between AA and
DGCT through quantitative histologic analysis, molecular profiling
using next generation sequencing, and statistical analysis of
accumulated clinical data. In addition, we sought to discover
keratin mutations that may be associated with ghost cell
formation.
Materials and Methods
Tissue Specimens
A retrospective search was performed to identify cases of
odontogenic tumors that were initially diagnosed as AA or DGCT
between January 1, 2000, and December 31, 2020 in the Depart-
ment of Oral Pathology, Seoul National University Dental Hospital.
After reviewing the archived hematoxylin-and-eosinestained
slides of the identified cases based on the WHO Classification of
Head and Neck Tumours1,5 by 2 oral pathologists (K.Y.O., H.J.Y.), 8
cases were included in this study. Clinical features were evaluated
based on medical records. This study was performed in accor-
dance with the tenets of the Declaration of Helsinki and was
granted exemption by the Institutional Review Board of Seoul
National University Dental Hospital (approval No. ERI22030).
Histologic Analysis
According to the essential histologic features of AA proposed in
a previous study,2 the “AA-like phenotype” was defined as a
combination of the following features: (1) ameloblastoma-like
epithelium, (2) duct-like structures, (3) cribriform architecture,
and (4) morules. The proportion of the area showing the AA-like
phenotype to the total tumor area was calculated in each case
and categorized as follows: 0, no; 1%-9%, rare; 10%-49%, focal; and

50%, diffuse. “Ghost cells” were defined as cells with eosinophilic
cytoplasm containing a central hole suggestive of loss of the nu-
cleus. The proportion of ghost cells to the total tumor cells was
calculated in each case. “Dentinoid” was defined as eosinophilic
dentin/osteodentin-like material in proximity to epithelial cells.
Because of the difficulty in its quantification, the relative amount
was calculated for dentinoid and categorized into rare, focal, and
diffuse.
According to the proportions of the AA-like phenotype (no to
rare, AA-negative; focal to diffuse, AA-positive) and ghost cells
(<1%, ghost cellelow;
1%, ghost cellehigh),17 the 8 cases
included in this study were classified into the following histologic
types: (1) typical AA (AA-positive/ghost cellelow); (2) typical
DGCT (AA-negative/ghost cellehigh); (3) mixed type (AA-posi-
tive/ghost cellehigh); and (4) dentinoid-only type (AA-negative/
ghost cellelow).
b-Catenin Immunohistochemistry and CTNNB1 Hotspot Mutation
Analysis
b-Catenin immunohistochemistry was performed using a
monoclonal antibody (Ready-to-Use; clone 17C2; Leica Bio-
systems) and a BOND-MAX autostainer (Leica Biosystems) ac-
cording to the manufacturer’s instructions.
Genomic DNA was extracted from formalin-fixed paraffin-
embedded tissue sections using the QIAamp DNA FFPE Tissue kit
(Qiagen) according to the manufacturer’s instructions. Primers for
PCR and sequencing were designed to detect CTNNB1 hotspot
mutations in exon 3 as follows: F:50 -CCAATCTACTAATGCTAAT
ACTG-30 ; R:50 -CTGCATTCTGACTTTCAGTAAGG-30 .18,19 PCR was
performed as follows: initial denaturation at 95 C for 5 minutes,
45 cycles of amplification (denaturation at 95  C for 30 seconds,
annealing at 52  C for 30 seconds, and extension at 72  C for 60
seconds), and final extension at 72  C for 7 minutes. Sanger
sequencing was performed using an ABI PRISM 3730XL Analyzer
(Applied Biosystems).
Next Generation Sequencing
Five of the 8 cases included in this study had sufficient tumor
tissue for whole-exome sequencing analysis. Exome capture was
performed using the SureSelect Human All Exon V8 (Agilent
Technologies), and libraries were sequenced on an Illumina Hiseq
platform (Illumina, Inc). Reads were mapped to the hg38 human
reference genome,20 and variants were annotated with SnpEff21
using the dbNSFP database.22,23
According to the definition of single nucleotide poly-
morphism,24 variants with a minor allele frequency
0.01 were
excluded from further analysis.25 The remaining variants were
analyzed for their functional effect by the following in silico pre-
diction tools: SIFT, LRT, MutationTaster, MutationAssessor,
FATHMM, PROVEAN, MetaSVM, MetaLR, M-CAP, and fathmm-
MKL.26
Statistical Analysis of Literature Data
Among studies published up to September 2022, the most
recent systematic reviews of reported cases of AA and DGCT,
respectively, were selected for comparative analysis of the
 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx 3

Table 1
Summary of the clinical, histologic, and molecular features of WNT pathwayealtered benign odontogenic tumors included in this study

No Clinical Histologic Immunohistochemical and molecular

Age Sex Site a

FU (y) AA-like (%) GC (%) DN b
Type Nuclear b-cateninc CTNNB1 hotspotsd WES resultse

1 39 M Mx Recur (10) 100 <1 þþ Typical AA þþþ WT APC E789fs

2 47 M Mn NED (4) 100 0 þþ Typical AA þþ WT CTNNB1 Y333fs
3 64 M Mn Recur (13) 40 5 þþ Mixed þþþ WT SMURF1 L424fs, TP53 P278S,
KRT13 Y281H
4 42 M Mn Recur (18) 25 10 þþ Mixed þþþ WT NEDD4L N846fs
5 32 F Mx NED (6) 5 10 þþþ Typical DGCT þþþ WT APC Q1080*, KRT13 M239V
6 42 F Mn Recur (1) 5 20 þ Typical DGCT þþþ WT NA
7 8 M Mnf NED (2) 0 10 þþ Typical DGCT þþþ WT NA
8 28 M Mx Recur (1) 0 0 þþþ DN only þþþ WT NA

AA, adenoid ameloblastoma; DGCT, dentinogenic ghost cell tumor; DN, dentinoid; FU, follow-up; GC, ghost cell; M, male; Mn, mandible; Mx, maxilla; NA, not available;
NED, no evidence of disease; WES, whole-exome sequencing; WT, wild-type.
a
Follow-up period or time from the first surgery to recurrence.
b
þ, rare; þþ, focal; þþþ, diffuse.
c
þþ, focal; þþþ, diffuse.
d
Missense mutations in codons 32, 33, 34, 37, 41, and 45.
e
All frameshift (fs) mutations result in a premature stop codon; all missense mutations are predicted to be damaging or deleterious.
f
Peripheral type.

demographic and clinical features of the 2 entities.7,8 Differences
between categorical data were evaluated using the c2 test.
Results
The clinical, histologic, and molecular features of cases
included in this study are summarized in Table 1 and illustrated in
Figure 1. Nuclear b-catenin and WNT pathway mutations were
identified in all cases analyzed.
Clinical Features of WNT PathwayeAltered Benign Odontogenic
Tumors
The age range of the 8 cases studied was 8-64 years (mean, 37.8
years), and the male-to-female ratio was 3:1. The mandible (5/8;
62.5%), including 1 peripheral case (case 7), was more frequently
affected than the maxilla (3/8; 37.5%). As the first treatment, con-
servative surgery and radical surgery were performed in 7 cases and
1 case, respectively. Recurrence occurred in 5 patients (62.5%)
within 1 to 18 years (mean, 8.6 years) after the first surgery (Table 1).
A comparative analysis of reported demographic and clinical
data between AA and DGCT showed that the mean age was nearly
the same in patients with AA (39.0 years) and central DGCT (38.8
years) despite their wide age ranges. There were no significant
differences in sex distribution, tumor site, and recurrence rate
between AA and central DGCT (Table 2). In addition, cumulative
cases of AA and central DGCT had similar demographic and clinical
characteristics to WNT pathwayealtered benign odontogenic tu-
mors included in this study.
Histologic Spectrum of WNT PathwayeAltered Benign Odontogenic
Tumors
Of the 8 cases studied, 2 (cases 1 and 2) showed a diffuse
distribution of the AA-like phenotype with a low proportion of
ghost cells (typical AA), whereas 3 (cases 5-7) were characterized
by a high proportion of ghost cells but no to rare AA-like pheno-
type (typical DGCT). Of note is that 2 (cases 3 and 4) were clas-
sified as the mixed type because they showed a focal AA-like
phenotype with many ghost cells. By contrast, 1 (case 8) was
accompanied by neither the AA-like phenotype nor ghost cells
and, hence, classified as the dentinoid-only type (Table 1 and
Fig. 1). Representative histologic images of each type are pre-
sented in Figure 2 and Supplementary Figure S1.
In ghost cellehigh cases (cases 3-7), perinuclear eosinophilic
condensation was found within a few tumor cells in close prox-
imity to ghost cells. These distinctive cells had visible nuclei and
were frequently clustered (Fig. 3A, B). All cases studied (cases 1-8)
showed varying amounts of dentinoid with entrapped clear cells
(Fig. 3C, D). Irrespective of histologic type, multiple blood-filled
cavities were found in half of the cases (cases 2, 3, 6, and 8)
(Fig. 3E), and multinucleated giant cells and osteoid were occa-
sionally found in the surrounding fibrous tissue similar to those
observed in aneurysmal bone cyst (Fig. 3F).
Mutational Profile of WNT PathwayeAltered Benign Odontogenic
Tumors
In all but one case, nuclear and cytoplasmic staining of b-cat-
enin was observed in a diffuse pattern, including the AA-like area
(Fig. 4A), tumor cells surrounding ghost cells (Fig. 4B), and clear
cells entrapped in dentinoid (Fig. 4C). The remaining case (case 2)
showed diffuse cytoplasmic but focal nuclear immunoreactivity
for b-catenin, mainly highlighting morules (Fig. 4D). Despite
consistent nuclear b-catenin accumulation, no CTNNB1 hotspot
mutations were found in any case studied (Fig. 4E).
Whole-exome sequencing revealed that all 5 cases analyzed
had a significant mutation in WNT pathway components (Table 1
and Fig. 1A). One typical AA (case 1) and 1 typical DGCT (case 5)
harbored the following LOF mutations in the APC gene: a frame-
shift deletion leading to a premature stop codon (E789fs;
Supplementary Fig. S2) and a nonsense mutation (Q1080*),
respectively. Two mixed types (cases 3 and 4) showed LOF mu-
tations in E3 ubiquitin ligases involved in the WNT pathway,27 a
frameshift insertion leading to premature termination in the
Smad ubiquitination regulatory factor 1 (SMURF1) gene (L424fs)
and the neural precursor cell expressed, developmentally down-
regulated 4-like (NEDD4L) gene (N846fs), respectively. A frame-
shift insertion leading to a premature stop codon was detected in
the CTNNB1 gene (Y333fs) in 1 typical AA (case 2). The effect of the
LOF mutations observed in this study on the WNT pathway is
4 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx

Figure 1.
(A) Clinical, histologic, and molecular data of cases included in this study. (B) Schematic diagram depicting the effect of WNT pathway mutations identified in this study.

schematically depicted in Figure 1B. The schematic structures of
the proteins affected in the present cases are illustrated in
Figure 5.
Mutational Status of Other Signaling Pathways and TP53 in WNT
PathwayeAltered Benign Odontogenic Tumors
None of the cases showed significant mutations in the MAPK
and SHH pathways (Fig. 1A), which have been reported in other
odontogenic tumors such as ameloblastoma, adenomatoid
odontogenic tumor, ameloblastic fibroma, ameloblastic carci-
noma, and ameloblastic fibrosarcoma.28-31
One mixed type (case 3) harbored the TP53 P278S missense
mutation (Table 1 and Fig. 1A), which was predicted to be
damaging/deleterious by 10/10 in silico prediction tools and has
been reported in various types of cancer according to the Cata-
logue of Somatic Mutations in Cancer (COSMIC) database.32
However, the other cases showed no significant mutations in the
TP53 gene, suggesting a low prevalence of TP53 mutations in WNT
pathwayealtered benign odontogenic tumors.
 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx 5

Table 2 both of which showed a high proportion of ghost cells (Fig. 3A, B).
Comparison of demographic and clinical data between adenoid ameloblastoma Although they have not been detected in other solid tumors ac-
and dentinogenic ghost cell tumor based on recently published systematic
cording to the COSMIC database,32 KRT13 Y281H and M239V were
reviews7,8
predicted to be damaging/deleterious by 10/10 and 5/10, respec-
Characteristic Value AA DGCTa P tively, in silico prediction tools. None of the cases showed signif-
Age (y) Mean (range) 39 (15-82) 38.8 (9-80) NA icant mutations in other keratin genes, such as KRT1, KRT4, KRT5,
Sex M:F 1.2:1 (16:13) 1.6:1 (47:29) .533 KRT6A, KRT6B, KRT10, KRT14, KRT16, and KRT17, known to be
Site Mn/(MnþMx) 66.7% (18/27) 61.3% (46/75) .623 altered in genodermatoses associated with perinuclear eosino-
Prognosis Recurrence rate 41.4% (12/29) 30.3% (23/76) .280 philic condensation.33
AA, adenoid ameloblastoma; DGCT, dentinogenic ghost cell tumor; Mn, mandible;
Mx, maxilla; NA, not applicable.
a
Central (intraosseous) DGCT. Discussion

This study comprehensively investigated the histologic and

Keratin Mutations in WNT PathwayeAltered Benign Odontogenic
Tumors
KRT13 missense mutations were found in 1 mixed type (case 3;
Y281H) and 1 typical DGCT (case 5; M239V) (Table 1 and Fig. 1A),
molecular features of odontogenic tumors that can be classified as
AA or DGCT according to the new WHO classification.1,5 As a result,
it was determined that, although these tumors histologically
consist of varying combinations of the AA-like phenotype, ghost
cells, and dentinoid, they all have a WNT pathway mutation in

Figure 2.
Histologic spectrum of WNT pathwayealtered benign odontogenic tumors. AA, adenoid ameloblastoma-like phenotype; GC, ghost cells.
6 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx

Figure 3.
Histologic features observed in WNT pathwayealtered benign odontogenic tumors. (A, B) Perinuclear eosinophilic condensation (arrows) is found within neoplastic epithelial
cells in proximity to ghost cells (asterisks) in 2 ghost cellehigh cases harboring KRT13 mutations. (C, D) Clear cells are found within dentinoid material and occasionally
intermixed with ghost cells (asterisks). (E, F) Blood-filled cavities are found in the stroma, accompanied by multinucleated giant cells and osteoid (arrows) in the surrounding
fibrous tissue.

common. This finding suggests that, from a molecular point of
view, AA and DGCT represent a histologic spectrum of WNT
pathwayealtered benign odontogenic tumors rather than 2
distinct tumors. In addition, literature analysis revealed clinical
similarities between the previously reported cases of AA and
DGCT, further undermining the rationale for distinguishing the 2
entities.
Contrary to the high frequency (
90%) of CTNNB1 hotspot
mutations in COC,13,14 these mutations have been reported in only
1 case of central DGCT to date.10 In addition, CTNNB1 hotspot
mutations are detected in AA at a relatively low frequency
(<50%),4 suggesting that they are not as predominant in WNT
pathwayealtered benign odontogenic tumors as in COC. Instead, it
seems from the results of this study that LOF mutations in tumor
suppressor genes, such as those comprising the b-catenin
destruction complex or encoding E3 ubiquitin ligases, may be
prevalent in WNT pathwayealtered benign odontogenic tumors
as is the case in colorectal adenocarcinoma.11,12 This mutational
landscape characterized by alterations in several different genes in
the same signaling pathway is well recognized in another odon-
togenic tumor, ameloblastoma, which harbors mutations in the
BRAF and 3 RAS genes leading to MAPK activation.31
APC, an essential component of the b-catenin destruction
complex, acts as a tumor suppressor in the WNT pathway and
is the most frequently mutated gene in colorectal cancer.11,12 In
the same manner, APC mutations accounted for the largest
portion (40.0%) of WNT pathway mutations in the 5 cases
analyzed by next generation sequencing in this study. However,
in contrast to the mutation cluster region located between
codons 1286 and 1513 observed in somatic APC mutations in
colorectal cancer34 (Fig. 5), APC LOF mutations occurred before
the mutation cluster region in WNT pathwayealtered benign
odontogenic tumors (codons 789 and 1080). Given that APC
LOF mutations previously reported in ghost cell odontogenic
carcinoma15 and odontogenic carcinoma with dentinoid16 were
located in 123 and 876 codons, respectively, it is assumed that
 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx 7

Figure 4.
b-Catenin immunohistochemistry and CTNNB1 hotspot mutations in WNT pathwayealtered benign odontogenic tumors. (A-C) Diffuse nuclear b-catenin staining is observed in
adenoid structures (A), ghost cellerich area (B), and clear cells entrapped in dentinoid (C). (D) One typical adenoid ameloblastoma with a frameshift mutation in CTNNB1 shows
focal nuclear staining for b-catenin, highlighting morules. (E) All cases studied showed no CTNNB1 hotspot mutations. Codons 33, 37, 41, and 45, which can be phosphorylated by
glycogen synthase kinase-3 (GSK3) and casein kinase-1 (CK1) comprising the b-catenin destruction complex, are included in the hotspot codons.

Figure 5.
Schematic structures of the proteins mutated in WNT pathwayealtered benign odontogenic tumors. aa, amino acids; APC, adenomatous polyposis coli; ARM, armadillo repeats;
C2, N-terminal C2 domain; HECT, homologous to E6AP C-terminus; MCR, mutation cluster region in colorectal cancer (codons 1286-1513); NEDD4L, neural precursor cell
expressed, developmentally downregulated 4-like; SMURF1, Smad ubiquitination regulatory factor 1; WW domains, tryptophan-rich WW domain.
8 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx
the mutation cluster region of APC exists closer to the N-ter-
minus in odontogenic tumors than in colorectal cancer,
resulting in a shorter truncated APC protein.
In addition to APC, other tumor suppressor genes encoding E3
ubiquitin ligases involved in the WNT pathwaydSMURF1 and
NEDD4Ldwere truncated in WNT pathwayealtered benign odon-
togenic tumors. SMURF1 regulates the interaction between Axin
and LRP5/6 by ubiquitinating Axin,27 and NEDD4L degrades
disheveled (Dvl) by polyubiquitination35 and plays a tumor sup-
pressive role in colorectal cancer.36 According to the COSMIC
database,32 nonsense or frameshift mutations in SMURF1 and
NEDD4L have been reported in colorectal adenocarcinoma, endo-
metrial carcinoma, and cutaneous melanoma, which are all known
for high frequencies of WNT pathway mutations.11 In addition, both
SMURF1 (codon 424) and NEDD4L (codon 846) mutations observed
were located within the homologous to E6-AP C-terminus (HECT)
domain that catalyzes polyubiquitin chain packaging37 (Fig. 5),
indicating their implications in a functional defect of the proteins.
Further studies are needed on additional cases to establish the role
of LOF mutations in E3 ubiquitin ligases in WNT-activated tumors.
In this study, WNT pathwayealtered benign odontogenic tu-
mors showed mainly diffuse nuclear b-catenin expression, but
focal immunoreactivity was also noted in a minority. Similarly, b-
catenin immunohistochemistry demonstrated focal to diffuse
nuclear staining in previous studies on AA and COC.4,14 These
findings indicate that b-catenin may show variable expression
levels in WNT-activated odontogenic lesions. In addition, other
tumors characterized by WNT pathway activation, such as ada-
mantinomatous craniopharyngioma,38 salivary basal cell ade-
noma,39 and desmoid fibromatosis,40 display variable extents of
nuclear b-catenin expression. Although the precise mechanism for
these diverse staining patterns remains to be clarified, b-catenin
expression might be affected by the type of mutation given that, in
this study, all 4 cases with an LOF mutation in a tumor suppressor
gene of the WNT pathway showed diffuse nuclear staining.
It has been demonstrated that nuclear b-catenin is present
during tooth development and that canonical WNT activation is
associated with dentin formation,41 which may explain the
frequent presence of dentin-like material called dentinoid in
odontogenic lesions with WNT pathway activation. Among the
cases investigated in this study, 1 WNT pathwayealtered tumor
produced a significant amount of dentinoid but showed neither
the AA-like phenotype nor ghost cells, making it challenging to
classify this case as a specific type of odontogenic tumor according
to the diagnostic criteria of the current WHO classification.1,5
Molecular-based classification, which has recently been intro-
duced in the WHO classification of central nervous system tu-
mors,42 may be considered as a complement to the current
classification system based exclusively on histopathology. One
possible example is a molecularly defined category “WNT
pathwayealtered benign odontogenic tumor.” This newly pro-
posed molecular-based classification can encompass currently
unclassified entities, such as the dentinoid-only type identified in
this study, and may clearly defined tumors that meet the diag-
nostic criteria for more than 1 entity, such as the mixed type
showing the key histologic features of both AA and DGCT. Further
studies are needed to verify the validity of molecular-based clas-
sification in odontogenic tumors.
This study first discovered KRT13 missense mutations in cases
with a high proportion of ghost cells. Germline KRT13 mutations
have been reported in patients with white sponge nevus.43 His-
tologically, perinuclear eosinophilic condensation is noted in the
spinous layer of the oral mucosa in white sponge nevus.33,44
Similar perinuclear eosinophilic bands are observed in other
genodermatoses known to harbor keratin mutations, such as
epidermolytic hyperkeratosis (KRT1 and KRT10),45 epidermolysis
bullosa simplex (KRT5 and KRT14),46 and pachyonychia congenita
(KRT6A, KRT6B, KRT16, and KRT17).47 It is notable that this char-
acteristic histologic feature is also frequently found in odonto-
genic lesions characterized by ghost cells,48 as seen in this study
(Fig. 3A, B). Together, it can be speculated that KRT13 mutations
are associated with perinuclear eosinophilic condensation,
leading to ghost cell formation in specific types of odontogenic
lesions. This hypothesis is further supported by the finding that
keratin 13 is expressed in the odontogenic epithelium but not in
ghost cells in COC.49 Further genetic analysis of additional cases
may clarify the role of somatic keratin mutations in ghost cell
keratinization.
This study demonstrated a histologic continuum of WNT
pathwayealtered benign odontogenic tumors such as AA and
DGCT and identified novel mutations in tumor suppressor genes of
the WNT pathway in these tumors. In addition, we reported
previously unrecognized keratin mutations, which may be asso-
ciated with ghost cells found in odontogenic lesions. In conclu-
sion, we propose that AA and DGCT may represent a histologic
spectrum of WNT pathwayealtered benign odontogenic tumors.
Acknowledgments
Figures 1B and 5 were created with BioRender.com.
Author Contributions
K.Y.O. performed study design, data acquisition, data analysis,
and writing of the paper. S.D.H. performed data interpretation and
review of the paper. H.J.Y. performed study design, data analysis,
and revision of the paper. All authors read and approved the final
version of the paper.
Data availability statement
The data sets used and analyzed during this study are available
from the corresponding author on reasonable request.
Funding
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MSIT)
(2020R1A2C1102907).
Declaration of Competing Interest
The authors declare no competing interests.
Ethics Approval and Consent to Participate
This study was approved by the Institutional Review Board of
Seoul National University Dental Hospital (approval No. ERI22030).
Supplementary Material
The online version contains supplementary material available
at https://doi.org/10.1016/j.modpat.2022.100051
 Kyu-Young Oh et al. / Mod Pathol xxx (xxxx) xxx
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