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\begin{document}

\title{Cerebellopontine angle SHH-activated embryonal tumor without interaction
from the cerebellum in Cowden syndrome: A case report}



\author[1]{Takahiro Ishikawa}%
\author[1]{Kohei Fukuoka}%
\author[1]{Daiju Oba}%
\author[1]{Kayoko Ichimura}%
\author[2]{Yonehiro Kanemura}%
\author[1]{Yutaka Tanami}%
\author[1]{Hirofumi Ohashi}%
\author[1]{Atsuko Nakazawa}%
\author[1]{Jun Kurihara}%
\author[1]{Katsuyoshi Koh}%
\affil[1]{Saitama Kenritsu Shoni Iryo Center}%
\affil[2]{Kokuritsu Byoin Kiko Osaka Iryo Center}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Among intracranial embryonal tumors, genetic/epigenetic analysis can
help unveil the molecular background. Herein, we report a case of a
cerebellar pontine angular embryonic tumor complicated by Cowden
syndrome in an infant. The tumor radiologically lacked evidence of
interaction with the cerebellum and appeared to show continuity with the
pons. Pathological appearance was similar to that of a medulloblastoma
with extensive nodularity. DNA methylation analysis indicated that the
tumor was a ``medulloblastoma, sonic hedgehog-activated'' with a
substantial confidence. Although the diagnosis deviated from the
definition of medulloblastoma, clinical, pathological and molecular data
suggested that it was an ``ectopic'' medulloblastoma.%
\end{abstract}%



\sloppy


\textbf{Introduction}

A recent research using genome-wide DNA methylation analysis by Sturm et
al. highlighted that embryonal tumors of the central nervous system
(CNS), formerly called CNS-primitive neuroectodermal tumors (PNET),
consisted of various types of tumors including a number of novel tumor
entitiessuch as \emph{BCOR} -altered high-grade neuroepithelial
tumors\textsuperscript{1)}. Considering the clinical and molecular
heterogeneities, performing genetic and epigenetic analysis has become
essential to reveal the molecular background of the tumor and establish
a ``true'' diagnosis in patients with CNS embryonal tumors, not
otherwise specified (NOS).

Herein, we report a case of an isolated cerebellar pontine angular
embryonal tumor associated with Cowden syndrome in an infant. This
report highlights the clinical importance of molecular and genetic
diagnostic approaches accompanied by conventional pathological and
radiological techniques for establishing accurate diagnosis to enable
effective treatment of patients with embryonal CNS tumors based on
underlying genetic/epigenetic conditions.

\textbf{Case}

A 1-year-old female infant was referred to our hospital from a local
hospital due to the detection of a tumor in the left cerebellopontine
angle (CPA) on brain magnetic resonance imaging (MRI). Physical
examination and blood tests revealed no notable abnormal findings,
except for an enlarged head circumference (+2.1SD). The patient also
presented with an intermittent squall for a short period of time, with
lacrimation only from the left eye. Radiologically, the tumor did not
directly interact with the cerebellum but appeared to show continuity
with the left border of the pons (Figure 1). The left trigeminal nerve
was compressed by the tumor (Figure 1). The patient underwent a biopsy
with craniotomy, and the postoperative course was uneventful. The tumor
morphologically consisted mainly of large nodular structures with
well-defined borders and low-to-moderate cell densities within the
nodules. Between the nodules, a dense network of cells with a high
nuclear/cytoplasm ratio and round nuclei with hyperchromatic chromatin
was observed. Silver staining revealed numerous reticular fibers in the
dark background surrounding the bright nodules. The MIB-1 index score
was low at the nodules but high (68\%) around the nodules.
Immunohistochemical staining was positive for synaptophysin and NeuN and
negative for GFAP. INI-1 immunoreactivity was retained in all specimens
(Figure 1). Overall, the pathological appearance was very similar to
medulloblastoma with extensive nodularity. Based on the recently-updated
WHO's classification of central nervous system tumors, an initial
diagnosis of ``embryonal tumor, NOS'' was made owing to the unusual
tumor location and the lack of evidence of an interaction with the
cerebellum, which is regarded as the origin of a medulloblastoma.
Molecular analysis was performed to elucidate tumor pathogenesis. Based
on expression analysis using NanoString technology, the case was
included into the SHH subgroup with a high confidence value. Using DNA
methylation analysis with the molecular classifier v12.5, developed by
the German Cancer Center, the tumor was also classified as
``Medulloblastoma, SHH-activated'' with a substantially calibrated score
of 0.766. A mutation panel sequencing of the tumor revealed a pathogenic
variant of \emph{PTEN} (c.493G\textgreater{}T:p. Gly165Ter) with a
variant allele frequency of 84.62\%. Subsequent sequencing of normal
tissue confirmed that the variant was a germline mutation, indicating a
diagnosis of Cowden syndrome (Figure 2A). Additionally, copy number
analysis revealed a loss of heterozygosity at 10q, where the
\emph{PTEN}was located (Figure 2B). Altogether, the molecular analyses
supported the classification of the tumor as medulloblastoma despite its
atypical location and no radiological sign of the relationship with the
cerebellum. She underwent four courses of chemotherapy consisting of
cyclophosphamide, etoposide, cisplatin, vincristine, and intrathecal
methotrexate, followed by high-dose chemotherapy with thiotepa and
melphalan, according to the protocol of the Japanese Pediatric Brain
Tumor Consortium\textsuperscript{2)}. We performed a second-look surgery
for partial resection of the tumor following high-dose chemotherapy due
to minimal reduction in the size of the lesion, as observed on brain
MRI, even after intensive treatment. The pathological specimen obtained
from the second operation revealed disappearance of the high-cellularity
area among the nodules, and the MIB-1 index was reduced to 5\%,
suggesting the cytoreductive effectiveness of chemotherapy. The patient
has remained well without evidence of recurrence for 18 months since the
diagnosis. The periodic squall, initially interpreted as a trigeminal
neuralgia due to nerve compression by the tumor, has persisted with a
mild reduction of the symptoms after administration of carbamazepine.

\textbf{Discussion}

In this report, we presented the case of an infant with SHH-activated
embryonal tumor that developed as an isolated CPA lesion, diagnosed
using both pathological and molecular diagnostics. Molecular
classification using DNA methylation analysis has emerged as a novel
diagnostic tool because most CNS tumors including medulloblastoma may
have distinct methylation profiles. With this analysis, several previous
reports have been documented about ``medulloblastoma'' occurring in
different regions of the cerebellum or fourth ventricle. Liu et al.
reported seven cases of embryonal tumors in the pineal region,
classified as ``Medulloblastoma, WNT-activated (MB-WNT).'' All cases
exhibited missense mutations in exon3 of \emph{CTNNB1} and three cases
exhibited monosomy 6, which is unusual in pineoblastomas but typically
observed in WNT-activated medulloblastomas\textsuperscript{3)}. Arnault
et al. also reported the case of a 5-year-old girl with an embryonal
tumor in the sellar and suprasellar regions, which was initially
diagnosed as CNS-PNET but later classified as a WNT-activated
medulloblastoma based on DNA methylation analysis\textsuperscript{4)}.
In terms of SHH-activated medulloblastoma, Grammel et al. presented
evidence of tumor development outside the cerebellum. In their report,
granule neuron precursors, known to be the cells of origin of SHH
medulloblastoma, were observed in the cochlear nuclei of the brainstem
and was suggested to be the cellular origin of the
tumor\textsuperscript{5)}. The tumor in the present case appeared to
have developed from the ventral side of the pons, which is not
concordant with the location of the cochlear nuclei in the
pons.\emph{PTEN} alteration possibly led to mismigration of granule
cells, leading to tumor occurrence in an unusual location, because the
gene may play a critical role in cell migration during brain
development\textsuperscript{6)}.

In our case, the patient was diagnosed with Cowden syndrome based on
genetic testing of the tumor, which may also support the diagnosis of
medulloblastoma. Cowden syndrome is an autosomal dominant genetic
disease in which multiple hamartomatous lesions. \emph{PTEN} mutations
have been identified as causative genetic alterations in 80\% of the
patients; malignancies occur frequently in young adulthood, and the most
common sites are the breast, thyroid, and uterus\textsuperscript{7)}.
Development of malignant brain tumors is extremely rare in patients with
Cowden Syndrome.

Only six cases of Cowden syndrome with intracranial embryonal tumors
have been reported till date, and all of them were actually
medulloblastomas\textsuperscript{8)9)}. In these reports, all patients
with Cowden syndrome were diagnosed with medulloblastoma at the age of
\textless{}2 years. Among the patients with detailed information
regarding pathology and molecular subgroups, two were diagnosed with
pathologically confirmed desmoplastic/nodular subtype or MBEN, and four
were diagnosed with SHH-activated type medulloblastoma based on
molecular analysis\textsuperscript{8)}. These clinical and molecular
features are consistent with those in our case, except for the site of
tumor development.

\textbf{Conclusion}

Herein, we reported the case of an isolated cerebellar pontine angular
embryonal tumor complicated by Cowden syndrome. Although its location
did not meet the definition of medulloblastoma, the clinical,
histological, and molecular genetic findings strongly suggested its
presence. Therefore, the tumor was considered to be an ``ectopic''
medulloblastoma. Molecular genetic analysis is essential for revealing
the underlying molecular genetic background of CNS embryonal tumors,
NOS, as it can facilitate optimal treatment.

\textbf{Acknowledgement}

This work was supported by JSPS KAKENHI Grant Number 22K15953.

\textbf{References}

\begin{enumerate}
\tightlist
\item
  Sturm D, Orr BA, Toprak UH, et al. New Brain Tumor Entities Emerge
  from Molecular Classification of CNS-PNETs. Cell. 2016 Feb
  25;164(5):1060-1072.
\item
  Yamasaki K, Okada K, Soejima T, et al. Strategy to minimize radiation
  burden in infants and high-risk medulloblastoma using intrathecal
  methotrexate and high-dose chemotherapy: A prospective registry study
  in Japan. Pediatr Blood Cancer. 2020 Jan;67(1):e28012.
\item
  Liu APY, Priesterbach-Ackley LP, Orr BA, et al. WNT-activated
  embryonal tumors of the pineal region: ectopic medulloblastomas or a
  novel pineoblastoma subgroup? Acta Neuropathol. 2020
  Oct;140(4):595-597.
\item
  Tauzi\selectlanguage{ngerman}ède-Espariat A, Simbozel M, Liu APY, et al. A sellar presentation
  of a WNT-activated embryonal tumor: further evidence of an ectopic
  medulloblastoma. Acta Neuropathol Commun. 2023 Apr 3;11(1):58.
\item
  Grammel D, Warmuth-Metz M, von Bueren AO, et al. Sonic
  hedgehog-associated medulloblastoma arising from the cochlear nuclei
  of the brainstem. Acta Neuropathol. 2012 Apr;123(4):601-14.
\item
  Getz SA, DeSpenza T Jr, Li M, et al. Rapamycin prevents, but does not
  reverse, aberrant migration in Pten knockout neurons. Neurobiol Dis.
  2016 Sep;93:12-20.
\item
  Pilarski R, Burt R, Kohlman W, et al. Cowden syndrome and the PTEN
  hamartoma tumor syndrome: systematic review and revised diagnostic
  criteria. J Natl Cancer Inst. 2013 Nov 6;105(21):1607-16.
\item
  Albrecht S, Miedzybrodzki B, Palma L, et al. Medulloblastoma and
  Cowden syndrome: Further evidence of an association. Free Neuropathol.
  2022 Jan 11;3:3-1.
\item
  Caroleo AM, Rotulo S, Agolini E, et al. SHH medulloblastoma and very
  early onset of bowel polyps in a child with PTEN hamartoma tumor
  syndrome. Front Mol Neurosci. 2023 Aug 24;16:1228389.
\end{enumerate}

\textbf{Figure legends:}

\textbf{Figure 1} . Tumor imaging findings

(a--d) A tumor measuring approximately 1.5 cm can be visualized in the
left cerebellopontine angle (red arrow).

(a) Contrast enhancement effect was observed only in the anterior area
on gadolinium-enhanced, T1-weighted images. (b) Diffusion-weighted
images showed diffusion limitation. (c--f) The tumor appeared to be
continuous with the left border of the pons. (d) The right trigeminal
nerve (green arrowhead) is compressed by the tumor.

(g and h) The tumor morphologically consists mainly of large nodular
structures with well-defined borders, and low-to-moderate cell densities
within the nodules. Between the nodules, cells with a high N/C ratio and
round nuclei with densely packed hyperchromatic chromatin are observed.
(i) Silver staining reveals reticular fibers in the dark background
surrounding the bright nodules. (j) The MIB-1 index is low at the
nodules, but high at 68\% around the nodules.

\textbf{Figure 2.} DNA sequencing

(a) DNA sequencing of normal tissue confirms the germline pathogenic
variant of \emph{PTEN} . (b) Copy number analysis of the tumor reveals
copy number neutral loss of heterozygosity of chromosome 10q,
where\emph{PTEN} is located.

\textbf{Hosted file}

\verb`2_Figure1.pptx`  available at \url{https://authorea.com/users/752155/articles/723077-cerebellopontine-angle-shh-activated-embryonal-tumor-without-interaction-from-the-cerebellum-in-cowden-syndrome-a-case-report}

\textbf{Hosted file}

\verb`3_Figure2.pptx`  available at \url{https://authorea.com/users/752155/articles/723077-cerebellopontine-angle-shh-activated-embryonal-tumor-without-interaction-from-the-cerebellum-in-cowden-syndrome-a-case-report}

\selectlanguage{english}
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