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\begin{document}

\title{Evidence for a low-penetrant extended phenotype of RTPS1 from a kindred
with gain of SMARCB1 exon 6}



\author[1]{Uwe Kordes}%
\author[2]{Victor Mautner}%
\author[3]{Florian Oyen}%
\author[1]{Christian Hagel}%
\author[4]{Christian Hartmann}%
\author[5]{Michael Heuser}%
\author[6]{Michael Frühwald}%
\author[7]{Martin Hasselblatt}%
\author[8]{Kathrin Oehl-Huber}%
\author[8]{Reiner Siebert}%
\author[3]{Reinhard Schneppenheim}%
\author[9]{Ulrich Schüller}%
\affil[1]{University Medical Center Hamburg Eppendorf}%
\affil[2]{Universitätsklinikum Hamburg-Eppendorf}%
\affil[3]{University Hospital Medical Center}%
\affil[4]{Institute of Pathology, Section of Neuropathology}%
\affil[5]{Hannover Medical School}%
\affil[6]{University Hospital Augsburg}%
\affil[7]{University Hospital Münster}%
\affil[8]{Ulm University and Ulm University Medical Center}%
\affil[9]{Universitatsklinikum Hamburg Eppendorf Institut fur Neuropathologie}%


\vspace{-1em}



  
  \date{June 07, 2024}


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\selectlanguage{english}
\begin{abstract}
We report on a long-term survivor of an atypical teratoid/rhabdoid tumor
(ATRT-TYR) as an index patient, who carries a SMARCB1 exon 6 gain
inherited from his father. The father was diagnosed with an unusual
sequence of a myxopapillary INI1-negative ependymoma and a relapsing
BRAF V600 wild type hairy-cell leukemia. He has two yet healthy sisters
aged 33 and 38 years carrying the same variant, from which one had lost
an infant to a malignant brain tumor. This family highlights the
existence of RTPS1-associated SMARCB1 germline alterations with reduced
penetrance and extends the spectrum of involved diseases%
\end{abstract}%



\sloppy


\textbf{Introduction}

Pertubations of the SWI/SNF complex are common oncogenic events. The
acquired loss of its core subunit member SMARCB1 drives an expanding
spectrum of intra- and extracranial tumors, some characterized by
rhabdoid features including atypical teratoid/rhabdoid tumor (ATRT), and
others without such features, e. g. cribriform neuroepithelial tumor
(CRINET), poorly differentiated chordoma, desmoplastic myxoid tumor,
sinunasal undifferentiated carcinoma, epithelioid sarcoma, and renal
medullary carcinoma. Distinct heterozygous \emph{SMARCB1} germline
mutations cause Rhabdoid Tumor Predisposition Syndrome Type 1 (RTPS1),
schwannomatosis, or Coffin-Siris syndrome. Although overlap has been
reported, a genotype phenotype correlation exists with certain proximal
and intronic mutations linked to schwannomatosis, distal missense
mutation to Coffin-Siris, and truncating mutations to
RTPS1\textsuperscript{1}.

\textbf{Methods and results}

The index patient \textbf{III.1} (Figure 1A) was diagnosed with a
non-metastatic posterior fossa brain tumor at the age of 6 years.
Histology was consistent with an ATRT. After gross total tumor resection
and treatment with radio-chemotherapy according to the EURHAB
recommendations, the patient has remained in complete remission for 15
years after diagnosis. His father \textbf{II.2} was diagnosed with
a\emph{BRAF} V600 wild-type hairy cell leukemia variant (HCL-v, with
co-expression of CD19, CD103, CD11c, CD25) at the age of 47 years. He
achieved a complete remission after 5 cycles of cladribin, but suffered
from a relapse 9 years later, with a very good partial response to
additional cycles of cladribin. With 54 years he underwent surgery for
an L2-3 spinal ependymoma. At age 57 a hemi-colectomy for
steroid-resistant ulcerative colitis was performed, apparently unrelated
to his other malignoma.

Sanger sequencing of \emph{SMARCB1} did not detect any mutations in
peripheral blood lymphocytes (PBL) of the index patient
\textbf{III.1}(Figure 1B)\textbf{.} However, a pattern suggesting gain
of one copy of exon 6 of \emph{SMARCB1} was found by multiplex
ligation-dependent probe amplification (MLPA). MLPA detected an
apparently identical\emph{SMARCB1} exon 6 gain in PBL of his father
\textbf{II.2} and two of his sisters \textbf{III.2} , \textbf{III.4.}
Targeted next generation sequencing (NGS) confirmed the absence of a
single nucleotide variant or small indel but could not identify the
genomic integration site of the gained exon 6 sequences. \emph{SMARCB1}
mutations were absent in PBL of the index patient's grandmother
\textbf{I.1} , mother \textbf{II.1} , and of another sister
\textbf{III.3} . No material was available from a cousin \textbf{IV.1} ,
who had died from surgical complications of a malignant brain tumor at
the age of 8 months. The index patient's grandfather \textbf{I.2} had
died from non-malignant disease and could not be tested. In the ten
children of the grandfather \textbf{I.2,} a diagnosis of a malignancy
was only known in \textbf{II.2} . No detected carrier had any overt
intellectual impairment, except for a learning disability in
\textbf{III.1} attributed to leukencephalopathy after intensive
multi-modal treatment at young age. High resolution spinal and lower
extremity MRI in \textbf{III.1} and \textbf{III.2} did not detect dorsal
nerve root or peripheral nerve schwannoma.

BAF47 staining was negative in the ATRT of \textbf{III.1} and in the
myxopapillary ependymoma of \textbf{II.1} (Figure 2A-F). Results from
the Infinium Methylation EPIC BeadChip DNA methylation analysis for the
ATRT and the ependymoma did not assign a molecular diagnosis in the
online Heidelberg classifier tool\textsuperscript{2}. However,\emph{t}
-SNE plotting associated the ATRT with the clinically favorable subclass
of ATRT-TYR (Figure 2G) and the ependymoma with the myxopapillary
subclass (Figure 2I). Copy number profiles confirmed a heterozygous loss
of Chr 22 in both tumors (Figure 2H, J). In line MLPA detected large
deletions of \emph{SMARCB1} in both tumors and indicated, that the
allele containing the exon 6 gain was retained in the tumors whereas the
wild-type allele was deleted. No additional \emph{SMARCB1}mutation was
found in the HCL-v of \textbf{II.2} .

\textbf{Discussion}

RTPS1 is characterized by early-onset and even congenital ATRT, and less
commonly extracranial rhabdoid tumors. Synchronous or metachronous tumor
sequences occur. Thus, RTPS1 is a negative prognosticator in ATRT.
Mutations in RTPS1 are usually truncating mutations in distinction to
those in Schwannomatosis or Coffin-Siris syndrome\textsuperscript{1}.
However, overlap between Schwannomatosis and ATRT has been
observed\textsuperscript{3}. We here report a family in which a gain of
SMARCB1 exon 6 segregates with malignant diseases including ATRT. Though
we formally cannot rule out an integration of the gained segment
elsewhere in the genome, the combination of germline and somatic
analyses strongly suggest this change to be pathogenic to one allele
of\emph{SMARCB1,} which is retained in the tumors lacking INI1 staining.
Interestingly, \emph{SMARCB1} exon 6 duplications have previously been
reported in a pedigree with ATRT and schwannoma\textsuperscript{4}.
Another pedigree with a CRINET and incomplete penetrance of
germline\emph{SMARCB1} exon 6 duplication is on
record\textsuperscript{5}.

Our index patient \textbf{III.1} , his sister \textbf{III.2} , and his
father \textbf{II.2} did not present any schwannoma on clinical or MRI
examination. One cousin \textbf{IV.1} died from a malignant brain tumor
in infancy. Her mother \textbf{III.4} is an asymptomatic carrier of
the\emph{SMARCB1} gain, it is possible that she was also afflicted with
an ATRT. Regarding the many siblings and half siblings of the father we
could not accrue more information. Thus, the exact incidence of further
neoplasm was not ascertained and the founding mutation could not be
traced back.

A differential diagnosis of a CRINET was entertained for \textbf{III.1}
, as CRINETs may be similar to ATRT-TYR on methylation profiling,
immunochemistry, and by clinical outcome. However, the absence of any
cribriform features and rosettes combined with an abundance of typical
rhabdoid cells did not support CRINET as a differential diagnosis. No
rhabdoid features were seen in the myxopapillary ependymoma
of\textbf{II.2} . DNA methylation profiling did neither assign a
significant score for the ATRT nor for the myxopapillary ependymoma on
the Heidelberg classifier. While this is a frequent phenomenon for
tumors with germline alterations (unpublished observations), the\emph{t}
-SNE suggested the ATRT to belong to the prognostically favorable TYR
subgroup, in line with the long-term remission observed in our patient.
The spinal tumor showed, both histologically and regarding DNA
methylation high similarity to the subgroup of myxopapillary ependymoma,
a subgroup that is usually occurring in a sporadic setting.

Apart from the myxopapillary ependymoma, the father suffered from a
HCL-v. Classical hairy cell leukemia (HCL-c) is typically associated
with oncogenic \emph{BRAF} V600E mutations. Epigenetic driver mutations
affecting members of the SWI/SNF complex (e.g. \emph{ARID1A} ) have been
detected in some of those HCL-c, but at increased frequency in hairy
cell leukemia variants (HCL-v) that occur without the \emph{BRAF} V600E
mutation.\textsuperscript{6,7} However, both ependymoma and HCL-v have
so far not been observed in the context of \emph{SMARCB1} mutations.

Together, our report highlights the incomplete penetrance of RTPS1
caused by \emph{SMARCB1} gains and the expanding spectrum of
malignancies associated with these mutations.

\textbf{Acknowledgements}

U.S. was supported by the Deutsche Forschungsgemeinschaft and the
F\selectlanguage{ngerman}ördergemeinschaft Kinderkrebszentrum Hamburg. This work was supported
by grants 70112697 and 70114478 from Deutsche Krebshilfe\textbf{.}

Part of this work was a poster presentation at the 2019 Annual Meeting
of the German Society for Neuropathology and Neuroanatomy (DGNN) in
Magdeburg

\textbf{References}

1. Holsten T, Bens S, Oyen F, et al. Germline variants in SMARCB1 and
other members of the BAF chromatin-remodeling complex across human
disease entities: a meta-analysis. \emph{Eur J Hum
Genet.}2018;26(8):1083-1093.

2. Capper D, Jones DTW, Sill M, et al. DNA methylation-based
classification of central nervous system tumours.
\emph{Nature.}2018;555(7697):469-474.

3. Kehrer-Sawatzki H, Kordes U, Seiffert S, et al. Co-occurrence of
schwannomatosis and rhabdoid tumor predisposition syndrome 1. \emph{Mol
Genet Genomic Med.} 2018.

4. Swensen JJ, Keyser J, Coffin CM, Biegel JA, Viskochil DH, Williams
MS. Familial occurrence of schwannomas and malignant rhabdoid tumour
associated with a duplication in SMARCB1. \emph{J Med
Genet.}2009;46(1):68-72.

5. Ibrahim GM, Huang A, Halliday W, et al. Cribriform neuroepithelial
tumour: novel clinicopathological, ultrastructural and cytogenetic
findings. \emph{Acta Neuropathol.} 2011;122(4):511-514.

6. Maitre E, Bertrand P, Maingonnat C, et al. New generation sequencing
of targeted genes in the classical and the variant form of hairy cell
leukemia highlights mutations in epigenetic regulation
genes.\emph{Oncotarget.} 2018;9(48):28866-28876.

7. Swerdlow SH, Campo E, Pileri SA, et al. The 2016 revision of the
World Health Organization classification of lymphoid
neoplasms.\emph{Blood.} 2016;127(20):2375-2390.

\textbf{Legends}

\textbf{Figure 1: Pedigree of described kindred and summary of molecular
results}

Individuals with malignant CNS tumors and leukemia are highlighted
in\textbf{A.} The index patient is marked by an oblique arrow.
Confirmed\emph{SMARCB1} exon 6 gain carriers (gain+) and non-carriers
(gain-) are identified. Carriers \textbf{III.2} and \textbf{III.4} are
unaffected at age 38 and 33 years. Specimens from individual
\textbf{IV.1} were not available. Molecular results from available
samples are summarized in\textbf{B} . MLPA, multiplex ligation-dependent
probe amplification;\emph{t} -SNE, \emph{t} -distributed stochastic
neighbour embedding

\textbf{Figure 2:} \textbf{Analysis of CNS tumors from II.2 and III.1}

Both ATRT (\textbf{A,C} ) and ependymoma (\textbf{B,E} ) display typical
histology with rhabdoid features (\textbf{C} ) or Astra-positive matrix,
respectively, (\textbf{E} ) as hallmark features. Both tumors were
negative for SMARCB1 by immunohistochemistry (\textbf{D, F} ) with
arrows indicating non-neoplastic ependymal cells as an internal positive
control. Analysis of the DNA methylation profiles revealed a classifier
score of 0.48 for the ATRT (0.38 for the TYR-subclass, not shown) and a
score \textless{} 0.3 for the ependymoma (not shown). Using \emph{t}
-SNE together with \textgreater{} 2500 cases from 82 brain tumor classes
from Capper et al.\textsuperscript{1}, the ATRT clustered with the
Tyrosinase subclass (zoom-in shown in \textbf{G} ) and the ependymoma
with the myxopapillary subclass (zoom-in shown in \textbf{I} ). Copy
number plots are shown in (\textbf{H} ) and (\textbf{J} ). Scale bar in
\textbf{A}corresponds to 100 \selectlanguage{greek}µ\selectlanguage{english}m in \textbf{A, B} . Scale bar in C
corresponds to 10 \selectlanguage{greek}µ\selectlanguage{english}m in \textbf{C-F} .

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