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\begin{document}

\title{Clinicopathological characteristics and outcomes in Embryonal tumor with
multilayered rosettes: a decade long experience from a tertiary care
centre}



\author[1]{Kirti Gupta}%
\author[1]{Ridhi Sood}%
\author[1]{Pravin Salunke}%
\author[1]{Debajyoti Chatterjee}%
\author[1]{Renu Madan}%
\author[1]{Chirag  Ahuja }%
\author[2]{Richa Jain}%
\author[3]{Amita Trehan}%
\author[1]{Bishan Radotra}%
\affil[1]{Post Graduate Institute of Medical Education and Research}%
\affil[2]{Postgraduate Institute of Medical Education \& Research}%
\affil[3]{postgraduate Institute of Medical Education \& Research }%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Embryonal tumor with multilayered rosettes (ETMR) are a
heterogenous group clinically, pathologically and topographically. Due
to limited cases, data regarding its molecular genetics, pathology and
prognostic factors is evolving. We retrospectively analysed our cohort
of ETMR over last decade in order to study their clinicopathological
characteristics and outcome. Methods: Our cohort consisted of patients
diagnosed with Embryonal tumor with abundant neuropil and true rosettes
(ETANTR)/Ependymoblastoma (EBL)/ Medulloepithelioma (MEPL) over the past
decade. Clinical details, including outcome and imaging data was
retrieved. Histological analysis including immunohistochemical work-up
was performed. Results: Cohort included 15 patients with age range
between 1-28 years and M:F ratio of 1.5:1. Supratentorial location
predominated in comparison to tumors arising in posterior fossa. ETANTR
and EBL patterns were equally distributed (40\% each), followed by one
case each of mixed pattern (EBL+ETANTR), MEPL and embryonal tumor,
unclassified. All tumors readily expressed LIN 28A and INI-1 was
retained. Recurrence with evidence of glial and rhabdoid differentiation
was noted in a single patient 9 months following resection. Follow-up
period ranged from 1-31 months, with overall median survival of 6.4
months. Eight patients were planned for adjuvant treatment following
surgery, of which only four could complete it. All patients, except for
one, succumbed to the disease. Conclusions: ETMR have a heterogenous
morphology and gathers ETANTR, EBL, MEPL within its spectrum. Following
treatment, the recurrent tumor may feature glial/rhabdoid
differentiation. LIN28A is expressed in all cases, however should be
interpreted in context of histology. Prognosis of ETMR remains dismal
despite multimodal therapy.%
\end{abstract}%



\sloppy


\textbf{Clinicopathological characteristics and outcomes in Embryonal
tumor with multilayered rosettes: a decade long experience from a
tertiary care centre}

\textbf{Authors:}

Kirti Gupta, M.D, Professor\textsuperscript{\textbf{1*}}

Ridhi Sood, M.D., Senior Resident\textsuperscript{1}

Pravin Salunke, M.S., MCh., Additional Professor\textsuperscript{2}

Debajyoti Chatterjee, M.D, D.M., Assistant
Professor\textsuperscript{\textbf{1}}

Renu Madan, M.D, D.M., Assistant Professor\textsuperscript{\textbf{3}}

Chirag Kamal Ahuja, M.D, D.M., Associate Professor\textsuperscript{4}

Richa Jain, M.D, D.M., Assistant Professor\textsuperscript{\textbf{5}}

Amita Trehan, M.D, Professor\textsuperscript{\textbf{5}}

Bishan Dass Radotra, M.D., PhD., Professor\textsuperscript{\textbf{1}}

\textbf{Affiliations} : Departments of
Histopathology\textsuperscript{1}, Neurosurgery\textsuperscript{2},
Radiotherapy\textsuperscript{3}, Radiodiagnosis and
Imaging\textsuperscript{4}, Paediatrics\textsuperscript{5}, Post
Graduate Institute of Medical Education and Research, Chandigarh. India

Keywords: Embryonal tumor with multilayered rosettes, brain tumors,
ependymoblastoma, medulloepithelioma

Running title: Embryonal tumor with multilayered rosettes

\textbf{Word Count:} Abstract=250; Manuscript=2276; Images=6;
References=24

\textsuperscript{\textbf{*}}\textbf{Address for Correspondence} :

Dr. Kirti Gupta, M.D.

Professor, Department of Histopathology,

Postgraduate Institute of Medical Education and Research

(PGIMER), Chandigarh. India

Email: kirtigupta10@yahoo.co.in

Phone: +91-172-2755105

Fax: 0172-2744401

\textbf{Abbreviations}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
ETMR & Embryonal tumor with multilayered rosettes\tabularnewline
ETANTR & embryonal tumor with abundant neuropil and true
rosettes\tabularnewline
EBL & Ependymoblastoma\tabularnewline
MEPL & Medulloepithelioma\tabularnewline
INI-1 & Integrase interactor 1\tabularnewline
CNS-PNET & Central nervous system-primitive neuroectodermal
tumors\tabularnewline
H\&E & Haematoxylin and eosin\tabularnewline
GFAP & Glial fibrillary acidic protein\tabularnewline
NFP & Neurofilament protein\tabularnewline
CSF & Cerebrospinal fluid\tabularnewline
CSI & Cranio-spinal irradiation\tabularnewline
RT & Radiotherapy\tabularnewline
sPNET & supratentorial Primitive Neuroectodermal Tumors\tabularnewline
ATRT & Atypical teratoid rhabdoid tumor\tabularnewline
ASL & Arterial spin labelling\tabularnewline
\bottomrule
\end{longtable}

\textbf{Clinicopathological characteristics and outcomes in Embryonal
tumor with multilayered rosettes: a decade long experience from a
tertiary care centre}

\textbf{Abstract} :

\emph{Background} : Embryonal tumor with multilayered rosettes (ETMR)
are a heterogenous group clinically, pathologically and topographically.
Due to limited cases, data regarding its molecular genetics, pathology
and prognostic factors is evolving. We retrospectively analysed our
cohort of ETMR over last decade in order to study their
clinicopathological characteristics and outcome. \emph{Methods} : Our
cohort consisted of patients diagnosed with Embryonal tumor with
abundant neuropil and true rosettes (ETANTR)/Ependymoblastoma (EBL)/
Medulloepithelioma (MEPL) over the past decade. Clinical details,
including outcome and imaging data was retrieved. Histological analysis
including immunohistochemical work-up was performed. \emph{Results} :
Cohort included 15 patients with age range between 1-28 years and M:F
ratio of 1.5:1. Supratentorial location predominated in comparison to
tumors arising in posterior fossa. ETANTR and EBL patterns were equally
distributed (40\% each), followed by one case each of mixed pattern
(EBL+ETANTR), MEPL and embryonal tumor, unclassified. All tumors readily
expressed LIN 28A and INI-1 was retained. Recurrence with evidence of
glial and rhabdoid differentiation was noted in a single patient 9
months following resection. Follow-up period ranged from 1-31 months,
with overall median survival of 6.4 months. Eight patients were planned
for adjuvant treatment following surgery, of which only four could
complete it. All patients, except for one, succumbed to the disease.
\emph{Conclusions:}ETMR have a heterogenous morphology and gathers
ETANTR, EBL, MEPL within its spectrum. Following treatment, the
recurrent tumor may feature glial/rhabdoid differentiation. LIN28A is
expressed in all cases, however should be interpreted in context of
histology. Prognosis of ETMR remains dismal despite multimodal therapy.

\textbf{Introduction} :

Embryonal tumor with multilayered rosettes (ETMR) are aggressive, WHO
grade 4 tumors which were defined by a distinct histological pattern
about two decades ago.\textsuperscript{1-3} Previously, these tumors
were classified under the broad umbrella of central nervous
system-primitive neuroectodermal tumors
(CNS-PNET).\textsuperscript{4}Pfister and colleagues discovered a novel
miRNA amplification of C19MC at chr19q 13.42 that was later considered
as the genetic hallmark of ETMR.\textsuperscript{5} These tumors are
highly heterogenous in terms of location and histology. The spectrum of
histology described under ETMR includes embryonal tumor with abundant
neuropil and true rosettes (ETANTR), ependymoblastoma (EBL), and
medulloepithelioma (MEPL).\textsuperscript{6,7} The unifying features of
these is amplification of C19MC of which LIN 28A is a surrogate
marker.\textsuperscript{7,8} Recently, DICER mutations have been
identified in C19MC negative tumors.\textsuperscript{9,10} Complete
surgical resection which is attempted for patients with localised
disease is associated with survival advantage similar to other pediatric
brain tumors. These tumors have been conventionally treated with
high-risk medulloblastoma protocols. Due to limited cases and its recent
recognition, no specific treatment strategy using chemotherapy or
adjuvant radiotherapy has been prospectively evaluated in ETMR patients
so far.

We present our experience with this uncommon tumor diagnosed over the
past decade and discuss the morphological features, treatment details
and clinical outcome. Due to limited number of cases reported in the
literature and since the molecular diagnosis is not routinely performed,
the information regarding clinical, pathological features, favourable
prognostic factors, optimal treatment options are still evolving; our
cohort adds observational data to the existing knowledge regarding this
tumor.

Material and methods : Our cohort comprised of 15 cases of ETMR
diagnosed over past 10 years. Demographics, treatment and survival
details were retrieved from neurosurgery and radiotherapy records.
Haematoxylin and eosin (H\&E) stained sections were evaluated and tumors
were further characterized with an immunohistochemical panel consisting
of antibodies to glial fibrillary acidic protein (GFAP), synaptophysin,
neurofilament protein (NFP), Neu N, LIN 28A, INI-1 and Ki67.

Results:

\begin{enumerate}
\tightlist
\item
  \emph{Patients' characteristics} : Age ranged from 1 year to 28 years
  with median age of 4 years and M:F ratio of 1.5:1 (Figure 1a).
  Majority were less than 18 years (80\%) with three patients being more
  than 18 years (Figure 1b) (Table 1).
\item
  \emph{Location} : Tumors arising in the supratentorial compartment was
  seen in majority (n=8 cases, 53.3\%), while in 6 cases, tumor occurred
  in posterior fossa (40\%). A single case of spinal tumor (at L1
  location) was seen (Figure 1c).
\item
  \emph{Imaging} : All the tumors were intra-axial in location having
  large size but were well demarcated. The posterior fossa lesions
  showed compression on peduncles (Figure 1S, \emph{supplementary} ).
  These masses showed no to mild enhancement following contrast
  administration and had mild to moderate perilesional oedema with
  significant mass effect. Diffusion restriction was seen in the solid
  component of the lesions.
\item
  \emph{Management and clinical outcome} : Gross total excision was
  achieved in 11 cases while in 4 cases only a near total resection
  could be achieved owing to the proximity to vital structures.
  Intraoperatively, most tumors were well defined and fleshy. The
  follow-up period ranged from 1-31 months; thirteen patients died with
  overall median survival of 6.4 months. Though the sample size was
  small, we attempted to calculate difference in median survival amongst
  the three patterns (ETANTR versus EBL versus others (including MEPL,
  embryonal tumor, NOS and ETANTR+EBL). The median survival of ETANTR
  was 1.3 months, while for EBL was 9.2 months and for others was 11.4
  months (\emph{p=0.23, not significant} ) (Figure 1d). All patients
  succumbed to the disease with two patients being alive, one month and
  14 months after diagnosis, respectively. Two patients had metastatic
  disease at presentation with cerebrospinal fluid (CSF) being positive.
  Nine patients were found to be eligible for adjuvant treatment
  following surgery. Four patients completed the planned cranio-spinal
  irradiation (CSI) to a dose of 36Gy in 20 fractions followed by tumor
  bed boost of 9-18 Gy in 5-10 fractions and six cycles of
  platinum-based chemotherapy while one patient has recently completed
  first cycle of chemotherapy. Two patients received only radiotherapy
  (CSI and focal radiotherapy in one patient each to a total dose of 54
  Gy in 30 fractions). Rest of the two patients were planned for
  adjuvant chemo-radiotherapy but deteriorated during radiotherapy (RT)
  and thus could not complete the further treatment.
\item
  \emph{Histology} : Diverse histological patterns were noted with
  ETANTR pattern identified in 6 cases; EBL pattern was seen in 6 cases;
  mixed ETANTR and EBL pattern and MEPL was identified in one case each,
  while in a single case the tumor displayed sheets of undifferentiated
  cells with occasional rosettes, the cells were strongly and diffusely
  positive for LIN 28A. The tumor was categorized as embryonal tumors,
  NOS (Table 1). On performing immunohistochemistry, all tumors revealed
  retained nuclear expression for INI-1 (strong and diffuse).
  Synaptophysin, NFP, and Neu N expression was observed more in the
  neuropil in cases of ETANTR. GFAP was largely negative in all cases,
  except for rare areas with positivity. All cases expressed LIN 28A,
  the expression was restricted mainly to rosettes forming cells in
  cases of ETANTR, while in EBL and MEPL it was more widespread
  highlighting the poorly differentiated embryonal cells of EBL and
  tubular and papillary structures of MEPL.
\end{enumerate}

\begin{enumerate}
\tightlist
\item
  ETANTR pattern (n=6): Typical case revealed cellular islands
  interspersed by swathes of neuropil wherein the classic multilayered
  rosettes were abruptly encountered (Figure 2,3). The characteristics
  multilayered rosettes was identified as multilayering of mitotically
  active neuroepithelial cells clustered around a central lumen. The
  central portion of some rosettes were filled with nuclear debris or a
  lumina could be identified. The cellular islands consisted of
  embryonal cells in sheets, cords or as clusters set in a fine
  fibrillar matrix.
\item
  EBL pattern (n=6): The tumors within this pattern featured poorly
  formed ependymoblastic rosettes which were numerous in large portions
  while the neuropil content was relatively less (Figure 4). Mitoses was
  readily identified.
\item
  MEPL pattern (n=1): A single example of this tumor at spinal location
  (L1) was encountered in a teenage girl who presented with low
  backache. On histology, the tumor demonstrated papillary and tubular
  arrangement of neoplastic cells reminiscent of neuroepithelium and
  forming primitive neural tube (Figure 5). Neuropil content was low
  throughout the tumor. Adjacent areas comprised of sheets of embryonal
  cells with presence of rare multilayered ependymoblastic rosettes. No
  other differentiation pattern was observed. Focal expression of
  cytokeratin was noted.
\item
  Undifferentiated embryonal sarcoma, NOS (n=1): The tumor comprised of
  sheets of densely packed, immature cells, with a high nuclear to
  cytoplasmic ratio and round, oval, or angulated hyperchromatic nuclei.
  Occasional rosettes were identified. The cells were variably
  immunopositive for synaptophysin and OLIG2, while GFAP was negative.
  LIN 28A was widely expressed.
\end{enumerate}

All morphological patterns showed numerous mitotic figures and abundant
apoptotic bodies. Ki67 was uniformly high and it brilliantly highlighted
the multilayered rosettes amidst the neuropil islands when central
lumina were poorly defined. Tumor recurrence post-therapy was seen in a
single case 9months following gross-total resection of the primary
tumor. The recurrent tumor consisted of areas of epithelioid and
rhabdoid morphology characterized by large cells with abundant dense
eosinophilic cytoplasm, large pleomorphic vesicular nuclei and
conspicuous nucleoli. GFAP immunostain highlighted areas with glial
differentiation; however, no neuronal differentiation was observed.
Also, INI-1 was widely expressed by the neoplastic cells.

\textbf{Discussion}

Eberhart and colleagues first described this new histologic category of
infant brain tumors and called it embryonal tumors with abundant
neuropil and true rosettes (ETANTR).\textsuperscript{1} Over the years,
this was described with various names/acronyms including embryonal
tumors with multilayered/ ependymoblastic rosettes, MEPL, EBL and
supratentorial Primitive Neuroectodermal Tumors
(sPNET).\textsuperscript{11} Until 2010, when Paulus and Kleihues
proposed ``ETMR'' as a unifying entity to these embryonal
tumors\textsuperscript{12} displaying multilayered rosettes as a common
feature. In 2009, Pfister and colleagues discovered the novel oncogenic
microRNA locus (C19MC) as the diagnostic genetic hallmark of this
tumor.\textsuperscript{5} These studies led to the designation of
ETMR-C19MC altered as a distinct new entity in the 2016 WHO CNS
neoplasms.{[}13{]} C19MC amplification is present in about 90\% of all
ETMR regardless of histology.\textsuperscript{9,14} Transcriptomic
profiling of C19MC-altered tumors revealed enrichment of these tumors
for early neural and pluripotency genes including LIN 28/LIN 28B,
reflecting their primitive nature. In 2013, Spence et al interrogated a
large cohort of 450 paediatric tumors to assess the diagnostic
specificity of C19MC amplification and LIN 28A.\textsuperscript{8} Their
study demonstrated that C19MC alteration is seen in a subset of
CNS-PNETs with high LIN 28 expression, however a small subset of other
tumors such as rhabdoid brain tumors, malignant gliomas, atypical
teratoid rhabdoid tumor (ATRT), germ cells tumors also express LIN 28A.
While LIN 28A immunopositivity is not specific for the diagnosis of
ETMR, we excluded our cohort for ATRT, germ cell tumors, high grade
glioma by performing INI-1, Oct 3/4 and GFAP in all cases. LIN 28A
expression was consistently expressed in all cases in our cohort.
Additionally, to substantiate the above findings, we also performed LIN
28A immunohistochemistry in a case of ATRT (with INI-1 loss) featuring
multilayered true rosettes. LIN 28A expression was seen within these
rosettes as has been reported earlier in
ATRTs.\textsuperscript{15}Although LIN 28A is a surrogate marker for
ETMR, its expression needs to be interpreted in context of histology.
ATRT may often contain multilayered rosettes and presents a diagnostic
challenge; INI-1 is extremely helpful in reliably distinguishing the
two. All the tumors in our cohort readily expressed INI-1. Recently,
using DNA methylation profiling and transcriptomic analysis Lambo et
al,\textsuperscript{9}have demonstrated that approximately 5\% of ETMR
negative for C19MC amplification contain biallelic \emph{DICER}
mutations (ETMR,\emph{DICER-} altered) and these mutations are mutually
exclusive to C19MC amplification.\textsuperscript{10} It is interesting
to note how mutually exclusive \emph{DICER} mutations and C19MC
amplification leads to activation of oncogenic pathways. A common link
is LIN 28A/Let 7 signalling which brings about reprogramming of stem
cells. High expression of LIN 28A leads to depletion of Let-7 miRNA and
subsequent upregulation of Let-7 targets.\textsuperscript{16} ETMR have
low expression of Let-7 miRNA and typically have high expression of LIN
28A.

The tumor predominantly arises in the supratentorial region in 70\% of
cases \textsuperscript{3,7,17} as was also observed in our cohort.
Tumors in infratentorial region comprise 30\% \textsuperscript{3,17,
18}with rare reports of these tumors arising in the
spine.\textsuperscript{17,19} A trend for infratentorial location of
C19MC- altered ETMR has been observed. ETMR has traditionally been
described as a tumor of infants and young children, three patients in
our cohort were more than 18 years of age.

ETMR encompass a wide spectrum of histology from tumors with abundant
neuropil (ETANTR) with tumors with low neuropil content (EBL, MEPL).
Multilayered rosettes are a distinctive feature, recognition of which
amidst sheets of acellular neuropil (Figure 3B) or within cellular
islands is critical. These are best recognized at low magnification,
Ki67 is helpful in highlighting these mitotically active,
undifferentiated cells with high N/C ratio surrounding central lumen
(Figure 3C). In a single case, a continuum of morphological features was
observed with tumor cells exhibiting ependymoblastic rosettes along with
multilayered rosettes. MEPL comprising papillary and tubular structures
reminiscent of embryonic neural tube was identified in the spinal tumor
of adolescence girl. Another case of undifferentiated embryonal tumor
with occasional rosettes was encountered with cells strongly expressing
LIN 28A and retained nuclear expression for INI-1 (thereby excluding
ATRT). ETMR are known to show neuronal and glial differentiation
post-therapy.\textsuperscript{20,21} The tumor recurrence after 9 months
of primary diagnosis in case \#8 demonstrated areas of glial and
rhabdoid differentiation. Interestingly, recurrent tumors on second
surgery are known to transition from one histological variant to
another.\textsuperscript{7}

On imaging, these tumors usually appear large and well demarcated.
Though 75\% of them are supratentorial, posterior fossa lesions are well
described. Large size of the lesions causes significant mass effect on
the adjoining brain parenchyma, which is more marked in the
infratentorial compartment, often with compression on peduncles. These
solid masses are usually hypointense on T1-weighted and hyerintense on
T2-weighted MR images showing diffusion restriction and mild patchy
contrast enhancement. There is usually surrounding oedema and
significant mass effect. The cerebral blood flow has been reported to be
low on arterial spin labelling (ASL) sequence. However, the cerebral
blood volume (CBV) on dynamic susceptibility contrast imaging is usually
high. These features help differentiating ETMR from other childhood
tumors. Pilocytic astrocytoma shows strong contrast enhancement with no
diffusion restriction. Medulloblastomas and ATRT usually enhance well
and have high cerebral blood flow using ASL. Furthermore, ATRT's usually
demonstrate central necrotic areas. Ependymomas often have increased T2
signal like ETMR, but have a variable diffusion restriction
pattern.\textsuperscript{22}

Complete resection undoubtedly translates into better survival; however
certain location particularly brain stem and posterior fossa structures
may not be amenable to complete resection. Possible benefits of second
resections on survival following application of chemotherapy are also on
record.\textsuperscript{21} Consensus guidelines for ideal chemotherapy
regimen and radiotherapy dose- volume (CSI versus focal radiotherapy)
remains poorly defined. Treatment strategies are based on results in CNS
PNETs and other high-risk CNS-embryonal tumors. Various combinations of
chemotherapeutic drugs that have been used so far include lomustine,
vincristine, cyclophosphamide, etoposide and
carboplatin.\textsuperscript{19}

ETMR are aggressive pediatric brain tumors with universally poor
prognosis and a median overall survival (OS) of only 12 months or less
in most of the studies.\textsuperscript{7,8} The 5-year survival rate is
0-30\%.\textsuperscript{14} The overall median survival observed in our
cohort was 6.4 months and was worst for ETANTR (1.3 months) followed by
EBL pattern (9.2 months); however, this did not differ significantly
between the three histological patterns, similar to observations by
Korshunov et al.\textsuperscript{7} As these tumors have recently been
classified, there is limited data to guide prognostication and
treatment.\textsuperscript{23} Cumulative data suggest C19MC patients
who received chemotherapy with radiotherapy had significantly longer
survival compared to untreated patients (median survival of 13 months
versus 0.6 months, respectively).\textsuperscript{8} Studies have
indicated that higher overall survival is seen in completely resected
tumors with patients treated with radiotherapy and high-dose
chemotherapy with autologous stem cell rescue.\textsuperscript{17,24}

In conclusion, our study emphasizes the histological diversity inherent
to ETMR. LIN 28A while being less specific, is a sensitive
immunohistochemical marker. Prognosis of ETMR remains dismal with median
survival of 6.4 months and this does not differ significantly amongst
the three histological patterns.

\textbf{References:}

\begin{enumerate}
\tightlist
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  neurocytic differentiation. J Neurooncol. 2007 Aug;84(1):91-8.
\item
  Gessi M, Giangaspero F, Lauriola L, et al. Embryonal tumors with
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  neuroectodermal tumor. Am J Surg Pathol. 2009 Feb;33(2):211-7.
\item
  Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A,
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  Pfister S, Remke M, Castoldi M, Bai AH, Muckenthaler MU, Kulozik A,
  von Deimling A, Pscherer A, Lichter P, Korshunov A. Novel genomic
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\item
  Korshunov A, McLendon RE, Judkins AR, Embryonal tumour with
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  System, 4th ed., IARC Press, Lyon, 2016, pp. 201--205.
\item
  Korshunov A, Sturm D, Ryzhova M, et al. Embryonal tumor with abundant
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  medulloepithelioma share molecular similarity and comprise a single
  clinicopathological entity. Acta Neuropathol. 2014 Aug;128(2):279-89.
\item
  Spence T, Sin-Chan P, Picard D, et al. CNS-PNETs with C19MC
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\textbf{Figure legends} :

\textbf{Figure 1.} A,B,C: Demographic profile of the cohort; D: Kaplan
Meier curve depicting the overall survival in patients with ETANTR
versus EBL versus others.

\textbf{Figure 1S} (case \#12). A: T1 Sagittal MRI plane showing well
demarcated iso to hypo intense lesion in pons; B: T2 axial MR showing
well defined hyper intense lesion on right side of pons mimicking an
extra axial lesion; C: FLAIR images show ill-defined margins suggesting
an intra axial lesion with possible exophytic component; D: Post
contrast axial MR showing non enhancing lesion expanding the pons on
right side; E: Post contrast sagittal MRI showing non enhancing lesion;
F:3D turbo spin T2 image defining the lesion; G: Diffusion weighted MR
suggesting a cellular tumor; F:T1 coronal MR showing the extent of
lesion.

\textbf{Figure 2: Embryonal tumor with abundant neuropil and true
rosettes.} A: Biphasic histological pattern with cellular islands within
abundant neuropil (H\&E, scale bar, 100\selectlanguage{greek}µ\selectlanguage{english}m); B: Multilayered rosettes
present within sheets of embryonal cells (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); C: Cellular
islands with embryonal cells disposed in sheets, several mitotic figures
and apoptotic debris is noted in the background (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); D:
Multilayered rosettes with a central lumen surrounded by mitotically
active cells (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{Figure 3.} \textbf{Embryonal tumor with abundant neuropil and
true rosettes.} A: High magnification depicting the characteristic
multilayered rosettes with central lumina showing secretions (scale bar,
10\selectlanguage{greek}µ\selectlanguage{english}m); B: Nodular clusters of embryonal cells present within abundant
neuropil (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m);C: Corresponding section showing diffuse
strong immunoreactivity for LIN 28A within the embryonal cells (scale
bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); D: Diffuse strong nuclear expression for INI-1 protein
(scale bar 50\selectlanguage{greek}µ\selectlanguage{english}m); E,F: Focal positivity for synaptophysin (E) and GFAP
(F) within the embryonal cells (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); ; G: Scant positivity
for Vimentin within the embryonal cells (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); H: Ki67
revels a high proliferative index (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); I: Ki67 highlights
the multilayered rosettes composed of mitotically active cells around
the central lumen (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{Figure 4} . \textbf{Ependymoblastoma pattern} . A: Sheets of
embryonal cells with numerous multilayered and perivascular rosettes
with scant neuropil stroma in the background (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); B: High
magnification depicting closely packed multilayered rosettes (scale bar,
20\selectlanguage{greek}µ\selectlanguage{english}m); C: Poorly formed multilayered rosettes (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); D:
Strong and diffuse immunoreactivity for LIN 28A within the embryonal
cells (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); E: Retained widespread nuclear expression for
INI-1 protein (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{Figure 5: Medulloepithelioma pattern.} A: Embryonal cells
disposed in papillary arrangements of neoplastic pseudostratified
neuroepithelium (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); B: Sheets of embryonal cells in zones
away from tubular and papillary structures (scale bar, 50\selectlanguage{greek}µ\selectlanguage{english}m); C, D: High
magnification depicting pseudostratified neuroepithelium around the
vascular cores (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); E: Strong and diffuse immunoreactivity
for LIN 28A within the embryonal cells (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); F: Focal, weak
immunoreactivity for cytokeratin (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); G: GFAP was not
expressed by the tumor cells (scale bar, 20\selectlanguage{greek}µ\selectlanguage{english}m); H: Retained widespread
nuclear expression for INI-1 protein (scale bar, 10\selectlanguage{greek}µ\selectlanguage{english}m).

\textbf{No financial disclosures}

\textbf{No conflicts of interests.}

Data sharing not applicable -- no new data generated, or the article
describes entirely theoretical research\selectlanguage{english}
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\verb`Table 1-Updated.docx`  available at \url{https://authorea.com/users/726898/articles/709087-clinicopathological-characteristics-and-outcomes-in-embryonal-tumor-with-multilayered-rosettes-a-decade-long-experience-from-a-tertiary-care-centre}

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