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\begin{document}

\title{In utero tumor development and identification of CTNNB1 mutation in a
newborn case of ossifying renal tumor of infancy}



\author[1]{Shin Kasai}%
\author[1]{Minori Tamai}%
\author[2]{Eiji Sugihara}%
\author[1]{Naoki Oishi}%
\author[1]{Kyoko Hinata}%
\author[1]{Koshi Akahane}%
\author[1]{Kumiko Goi}%
\author[2]{Yuko Hata}%
\author[1]{Tetsuo Kondo}%
\author[1]{Takahiko Mitsui}%
\author[3]{Mio Tanaka}%
\author[1]{Takeshi Inukai}%
\affil[1]{University of Yamanashi}%
\affil[2]{Cancer Center and Open Facility Center}%
\affil[3]{Kanagawa Children's Medical Center}%


\vspace{-1em}



  
  \date{June 17, 2024}


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\emph{In utero} \textbf{tumor development and identification
of\emph{CTNNB1} mutation in a newborn case of ossifying renal tumor of
infancy}

Shin Kasai\textsuperscript{1}, Minori Tamai\textsuperscript{1}, Eiji
Sugihara\textsuperscript{2}, Naoki Oishi\textsuperscript{3}, Kyoko
Hinata\textsuperscript{1}, Koshi Akahane\textsuperscript{1}, Kumiko
Goi\textsuperscript{1}, Yuko Hata\textsuperscript{2}, Tetsuo
Kondo\textsuperscript{3}, Takahiko Mitsui\textsuperscript{4}, Mio
Tanaka\textsuperscript{5}, and Takeshi Inukai\textsuperscript{1*}

1) Departments of Pediatrics, Faculty of Medicine, University of
Yamanashi, Chuo, Yamanashi, Japan

2) Division of Gene Regulation, Cancer Center and Open Facility Center,
Research Promotion Headquarters, Fujita Health University School of
Medicine, Toyoake, Aichi, Japan

3) Department of Pathology, Faculty of Medicine, University of
Yamanashi, Chuo, Yamanashi, Japan

4) Department of Urology, Faculty of Medicine, University of Yamanashi,
Chuo, Yamanashi, Japan

5) Department of Pathology, Kanagawa Children's Medical Center,
Yokohama, Japan.

*Corresponding author

Takeshi Inukai, Department of Pediatrics, Faculty of Medicine,
University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409--3898,
Japan. Email: tinukai@yamanashi.ac.jp

Word count: 849/850 words

Figures: 2

Supplemental Tables: 2

Supplemental Figures: 5

Running title: \emph{CTNNB1} mutation in ossifying renal tumor of
infancy (44/50 characters)

Key words: \emph{CTNNB1,} Ossifying renal tumor of infancy, \selectlanguage{greek}β-\selectlanguage{english}catenin,
hematuria, calcification, renal tumor

An abbreviations key

ORTI: Ossifying renal tumor of infancy

WES: Whole exome sequencing

EMA: Epithelial membrane antigen

PBMC: Peripheral blood mononuclear cells

CNV: Copy number variation

ILNR: Intralobar nephrogenic rest

CNSET: Calcifying nested stromal-epithelial tumor

\textbf{To the Editor:}

Ossifying renal tumor of infancy (ORTI) is a rare pediatric benign renal
neoplasm first described in 1980.\textsuperscript{1} In the latest
review of 28 cases, whose diagnostic age was
described,\textsuperscript{2} the median age of diagnosis was 4.3 months
old, and 23 cases (82.1\%) were diagnosed within the first year of life.
These observations suggest \emph{in utero} development of ORTI but no
direct confirmation has been made. Here, we reported a newborn girl with
ORTI whose cystic lesion was first noticed at 25 weeks of gestation by
ultrasonography. Furthermore, we identified the \emph{CTNNB1} mutation
in her tumor sample by whole exome sequencing (WES). To our knowledge,
this is the first identification of \emph{CTNNB1} mutation as a genetic
alteration involved in ORTI tumorigenesis.

The patient was transferred to our hospital 10 days after birth due to
intermittent gross hematuria that appeared on the second day of life. In
ultrasonography at 25 weeks of gestation, a cystic lesion was first
noticed in her right kidney, and it was continuously enlarged in
proportion to fetal growth and multiloculated in the follow-up
evaluations (Figure 1A). She was born by scheduled cesarean section at
38 weeks of gestation with a birth weight of 2,672 g. There was no
significant family history. Her general condition was fair, but a soft
mass was palpable on her right abdomen. On admission, ultrasonography
showed a mass lesion with multiple cystic components at the lower pole
of the right kidney (Figure S1). An abdominal computed tomography image
performed 5 days after birth revealed a 4.5 x 4 cm multi-cystic mass
with a small linear calcification in the middle of the solid part
(Figure S2) without any sign of metastasis. Considering the gradually
increased tumor size and continuous hematuria, right nephrectomy was
performed at 23 days old. In the resected kidney, the tumor was
consisted of cystic and solid lesions and was well-demarcated from the
adjacent kidney. Histopathologically, the tumor consisted of three main
components, an osteoid core, osteoblast-like cells, and spindle cells
(Figure 1B), without typical nephrogenic rest. Immunohistochemically,
osteoblast-like cells were positive for cytoplasmic WT1 and epithelial
membrane antigen (EMA) while spindle cells were positive for nuclear WT1
but not EMA (Figure S3). Based on these findings, a final diagnosis of
ORTI was made. She had a good postoperative course and no local
recurrence or metastasis was observed at 9 months of age.

Since genetic alterations involved in the ORTI tumorigenesis have not
been fully elucidated, we conducted WES of the patient's tumor sample
and her peripheral blood mononuclear cells (PBMC) after written informed
consent was obtained from her parents. In copy number variation (CNV)
analysis, several tumor-specific CNVs were identified (Table S1)
including a gain of 7q34 locus containing the \emph{EPHB6} gene that was
reportedly overexpressed in thyroid and breast
cancers,\textsuperscript{3} and a loss of 19p13.3 locus
containing\emph{TLE2} and \emph{TLE6} , which was previously reported in
one sporadic Wilms tumor case.\textsuperscript{4} However, trisomy 4,
which was previously observed in some ORTI cases with a relatively low
prevalence,\textsuperscript{5,6} was undetectable with +1.20 of
threshold. In mutational analysis, c.121A\textgreater{}G (p.T41A)
mutation of the \emph{CTNNB1} gene was confirmed in the tumor tissue
with 0.38 of variant allele frequency (Figures 2A, S4 and Table S1) but
not in her PBSC, suggesting the heterozygous somatic mutation in the
tumor. Threonine 41 is one of the targets for GSK-3\selectlanguage{greek}β-\selectlanguage{english}dependent
phosphorylation and is one of the hotspot mutation sites in diverse
tumors.\textsuperscript{7} Consistently, nuclear accumulations of
\selectlanguage{greek}β-\selectlanguage{english}catenin protein, encoded by \emph{CTNNB1} , were confirmed in both
osteoblast-like and spindle cells of the tumor by immunohistochemistry
(Figure 2B). No further mutations were detected in the tumor sample
including the \emph{WT1} and \emph{WTX} genes, which are frequently
mutated in Wilms tumors.\textsuperscript{8} Loss of heterozygosity
of\emph{WT1} was also unlikely since heterozygous genotypes were
observed in the same single nucleotide polymorphisms in the tumor sample
and her PBSC (Table S2).

Hyperplastic intralobar nephrogenic rest (ILNR), which is known as a
precursor lesion of Wilms tumors, is also considered as possible origin
of ORTI.\textsuperscript{9} In the ILNR-associated Wilms tumors,
co-clustering of \emph{WT1} and \emph{CTNNB1} mutations has been
identified.\textsuperscript{10} In the Wilms tumors with these two
mutations, ILNR lesions had \emph{WT1} mutations but no
\emph{CTNNB1}mutations, indicating that \emph{CTNNB1} mutation is a
later event in tumorigenesis.\textsuperscript{11} These observations
suggest that preceding \emph{CTNNB1} mutation might induce ORTI instead
of Wilms tumor. Of note, the \emph{CTNNB1} mutations in the
GSK-3\selectlanguage{greek}β-\selectlanguage{english}phosphorylation sites have been previously identified in at least
two types of childhood benign tumors characterized by nested
proliferation of spindled and epithelioid cells with calcification or
ossification (Figure 2C). In almost all cases of pilomatricoma, a skin
adnexal tumor in children,\textsuperscript{12} the
\emph{CTNNB1}mutations including threonine 41 were
identified.\textsuperscript{13}Moreover, in all sequenced cases of
calcifying nested stromal-epithelial tumor (CNSET), a rare hepatic tumor
in children and young adults, the\emph{CTNNB1} mutations including
threonine 41 were identified in combination with the \emph{TERT}
promoter mutations,\textsuperscript{14}which were undetectable in our
case (Figure S5).

In summary, our case indicates \emph{in utero} development of ORTI and
involvement of \emph{CTNNB1} mutation in its tumorigenesis. Our
observations warrant further investigation of pathobiological
similarities and differences among pediatric \emph{CTNNB1} -mutated
tumors.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare no conflicts of interest.

\textbf{ACKNOWLEDGMENTS}

The authors would like to thank Keiko Kagami and Chiaki Komatsu
(Pediatrics, University of Yamanashi) for technical supports.

\textbf{REFERENCES}

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2. Banerjee A, Reddy KA, Munghate G, Bodhanwala M, Bendre PS. Ossifying
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3. Pergaris A, Danas E, Goutas D, Sykaras AG, Soranidis A, Theocharis S.
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6. Guan W, Yan Y, He W, et al. Ossifying renal tumor of infancy (ORIT):
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9. Sotelo-Avila C, Beckwith JB, Johnson JE. Ossifying renal tumor of
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10. Maiti S, Alam R, Amos CI, Huff V. Frequent association of \selectlanguage{greek}β-\selectlanguage{english}catenin
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11. Fukuzawa R, Heathcott RW, More HE, Reeve AE. Sequential
\emph{WT1}and \emph{CTNNB1} mutations and alterations of beta-catenin
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10.1136/jcp.2006.043083.

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13. Chan E, Gat U, McNiff J, et al. A common human skin tumour is caused
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14. Papke Jr DJ, Dong F, Zhang X, et al. Calcifying nested
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doi: 10.1038/s41379-021-00822-w.

\textbf{Figure legends}

\textbf{FIGURE 1} (A) Cystic lesion in right kidney observed by
ultrasonography performed at 25, 31, and 35 weeks of gestation.
Diameters of cystic lesion indicated by arrowheads and dotted lines are
shown in lower right panel. (B) Histopathological findings of
osteoblast-like cells with ossification (*) and proliferated spindle
cells (+) in the tumor tissue.

\textbf{FIGURE 2} (A) Tumor-specific heterozygous c.121A\textgreater{}G
(p.T41A) mutation of the \emph{CTNNB1} gene in Sanger sequencing of
genomic PCR products. Lower panel indicates hotspot mutations downloaded
from the MSK-IMPACT pan-cancer study on cBioportal. (B) Nuclear
accumulations of \selectlanguage{greek}β-\selectlanguage{english}catenin protein, encoded by \emph{CTNNB1} , in
osteoblast-like cells (left) and spindle cells (right) of the tumor
tissue by immunohistochemistry. (C) Schematic representation of three
types of childhood benign tumors with \emph{CTNNB1} mutations
characterized by nested proliferation of spindled and epithelioid cells
with calcification or ossification including ossifying renal tumor of
infancy (ORTI) as well as previously described pilomatricoma and
calcifying nested stromal-epithelial tumor (CNSET). Illustration was
created with BioRender.com.

\textbf{Supplemental Tables}

\textbf{TABLE S1} Single nucleotide variation and copy number variation
in the patient's tumor sample compared with her PBSC.

\textbf{TABLE S2} Heterozygous genotypes of \emph{WT1} in the same
single nucleotide polymorphisms in the patient's tumor sample and her
PBSC.

\textbf{Supplemental Figures}

\textbf{FIGURE S1} Abdominal ultrasonography performed on admission (10
days after birth). Size of cystic lesion is indicated.

\textbf{FIGURE S2} Abdominal computed tomography images performed 5 days
after birth. Arrow indicates a small linear calcification in the middle
of the solid part.

\textbf{FIGURE S3} Immunohistochemical findings of osteoblast-like cells
(left panels) and spindle cells (right panels) stained by anti-WT1 (top
panels) and anti-epithelial membrane antigen (EMA) antibodies.

\textbf{FIGURE S4} Whole-exome sequencing (WES) was performed on the
tumor sample and her peripheral blood mononuclear cells (PBMC). The
genomic DNA was sequenced on Illumina NovaSeq6000. WES was performed
using the QIAGEN CLC Genomics Workbench 23.0.4 software. Golden Helix
VarSeq 2.3.0 was used for data assessment. Tumor-specific missense
mutation of \emph{CTNNB1} (c.121A\textgreater{}G, p.T41A) was identified
with 0.38 of variant allele frequency (VAF). Genomic sequences of PBMC
(top) and tumor sample (bottom) are aligned.

\textbf{FIGURE S5} Sanger sequencing of the \emph{TERT} promoter was
performed using genomic PCR products of the patient's tumor sample and
her peripheral blood mononuclear cells (PBMC). Arrows indicate -146C and
-124C, which are reportedly mutated to T in all sequenced cases of
calcifying nested stromal-epithelial tumor (CNSET) \textsuperscript{14}.

\textbf{Hosted file}

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