Pathology of Ewings

sarcoma/PNET: Current
opinion and emerging
concepts
Created by: Saral S. Desai and Nirmala A.
Jambhekar
Tutor:
Letkol CKM dr. Maksum Pandelima, Sp. OT
By: Ria Puji Pangestuti

ABSTRACT
Ewings sarcoma/PNET are small round cell
Ewings sarcoma/PNET are small round cell
tumors showing a varying degree of
tumors showing a varying degree of
neuroectodermal differentiation. They are one
neuroectodermal differentiation. They are one
of the commonest tumors of childhood and
of the commonest tumors of childhood and
occur in bone and within soft tissues.
occur in bone and within soft tissues.
Traditionally, light microscopy with the aid of
Traditionally, light microscopy with the aid of
immunohistochemical stains was suitable for
immunohistochemical stains was suitable for
diagnosis. But now translocation analyses are
diagnosis. But now translocation analyses are
being used not only for the diagnosis and
being used not only for the diagnosis and
classification of small round cell tumors, but to
classification of small round cell tumors, but to
ascertain their prognostic significance, detect
ascertain their prognostic significance, detect
micrometastasis, and monitor minimal residual
micrometastasis, and monitor minimal residual
disease, with potential for targeted therapy
disease, with potential for targeted therapy

PURPOSE
The purpose of
this journal is

to analyzes the
pathology, biology,
and molecular aspects
of Ewings
sarcoma/PNET and
discusses their clinical
and therapeutic
implications

INTRODUCTION
[1918]
[1918]Purdy Stout described a tumor
Arthur
Arthur Purdy
Stout
described
a tumor
composed
of small
round
cells with
rosettes,
composed
of
small
1 round cells with rosettes,
in the ulnar nerve, 1which came to be known
inprimitive
the ulnarneuroectodermal
nerve, which came
to be
known
as
tumor
(PNET)
as primitive neuroectodermal tumor (PNET)

Later, James Ewing described a tumor

Later, James Ewing described a tumor
of long bones composed of
of long bones composed of
undifferentiated cells, which was
undifferentiated cells, which was
radiosensitive (Ewings Sarcoma)
radiosensitive (Ewings Sarcoma)
Over the years, these two tumors
Over the years, these two tumors
were described at various sites as two
were described at various sites as two
distinct entities. IS THAT TRUE?
distinct entities. IS THAT TRUE?

Then...
Angervall
and Enzinger:
described
an
extraskeletal
neoplasm
resembling
Ewings
[1975]
sarcoma

[1984]
Jaffe et al.:
published an
article on
the
neuroectode
rmal tumour
of bone

Now,
we know that
both Ewings
sarcoma and
PNET show
similar
translocations
and are
considered to be
the ends of a
histological
spectrum of
Ewings family of
tumors (EFT)

[1984-Now]
Knowledge about the molecular events
responsible
for
the
development
and
progression
of
EFT
has
increased
dramatically
Numerous technological developments have
contributed to greater understanding of cell
biology
The molecular mechanisms of malignant
transformation has been described.
The analysis of these tumors by various
molecular techniques may allow us not
only to understand the biology of these
lesions better but also to develop
better techniques for their diagnosis
and potential treatment.

EPIDEMIOLOGY
EFT
comprises
510% of
primary
bone
tumors

One of the
most
common
tumor in
childhood

Occurs
predomina
ntly in
children
and young
adults

Shows a
slight
predilectio
n for
males

Sites of involvement and

radiology
1. EFT
usually
arises
from the diaphysis or
metadiaphyseal
region of long bones
2. The less-frequent and
rare locations are the
pelvic
bone,
ribs,
skull
bones,
the
vertebra, the scapula,
and the small bones
of hands and feet

3. The radiological findings are essential for

making a histopathological diagnosis of any
bone tumor
4. Ewings sarcoma involves the diaphysis of
the bones and shows a permeative pattern
of involvement with periosteal reaction

Obtaining Tissue for Pathological

Examination
1. Biopsy of the tumor: the best
1.mode
Biopsy
the tumor:
the best
of of
obtaining
a diagnosis
mode of obtaining a diagnosis
Core biopsy
Open biopsy: if core biopsy have failed (technical
problems, sclerotic bone, and previously treated
case).
Tissue should be fixed in 10% formalin to prevent from
loss of antigens, which renders immunohistochemistry
inconclusive, utolysis and degeneration of DNA,
making the material unsuitable for molecular analysis
2. Frozen section examination:
2.performed
Frozen section
examination:
for adequacy
if the
performed
for
adequacy
if
representativeness of thethe
biopsy is
representativeness
of
the
biopsy
is
an issue
an issue
Reserved for only selective cases: freezing the tissue
can distort the morphology and also lead to loss of
antigen

HISTOLOGY
Ewings sarcoma/PNET is
a prototype of the small
round cell tumor group,
the cytoplasm is scant
EFT
cells
show
membranous expression
of CD99 or MIC2 on
immunohistochemistry
Antibody against FLI1has
been
shown
to
be
specific for EFT

ES/PNET composed of
sheets of small round
blue cells

 Depending on the degree of neuroectodermal

differentiation, the tumor cells may also express
neuron-specific enolase (NSE)
CD99 can also be positive in other tumors,
hence, a panel of immunohistochemical stains is
employed to arrive at a definitive diagnosis.
As stated above, CD99, FLI1, and NSE would be
positive in ES/PNET.

The tumor cells of EFT show membranous expression of

CD99/MIC2 (left), and nuclear positivity for antibodies
against FLI1 (right)

MOLECULAR GENETICS
EFT CASES
85%:
associated with
translocation
1015%:
t(11;22)
(q24;q12). This the translocation
fusion of EWS
t(21;12)(22;12)
gene on 22q12
resulting in
with the FLI1
EWS-ERG (ETSgene on 11q24
related gene)
fusion
results in a
chimeric fusion
transcript EWSFLI1.

1-5%:
translocations,wh
ich involve fusion
of EWS gene and
a member of ETS
family of
transcription
factors. e.g EWS
and ETV1 (Ets
variant 1)
(t(2;22)
(p22;q12))

Involvement of EWS in
Involvement of EWS in
other tumors
other tumors
Translocations involving EWS gene are
Translocations involving EWS gene are
observed in other tumors
observed in other tumors
EWS-ATF1(activating transcription factor
EWS-ATF1(activating transcription factor
1) Fusion: malignant melanoma of soft
1) Fusion: malignant melanoma of soft
parts
parts
EWS-WT1 (Wilms tumor 1) fusion: intra EWS-WT1 (Wilms tumor 1) fusion: intraabdominal DSRCT
abdominal DSRCT
EWS-CHOP Fusion: myxoid liposarcoma
EWS-CHOP Fusion: myxoid liposarcoma
EWS-CHN Fusion: myxoid chondrosarcoma
EWS-CHN Fusion: myxoid chondrosarcoma
EWS-like gene, TLS/FUS, is involved in
EWS-like gene, TLS/FUS, is involved in
tumor-associated gene fusions in myxoid
tumor-associated gene fusions in myxoid
liposarcoma and acute myeloid leukemia
liposarcoma and acute myeloid leukemia

TECHNIQUES FOR DETECTION

OF TRANSLOCATION
1. Chromosomal karyotyping
the classical method for demonstrating
translocations:
Fresh tumor need to be cultured
Required skilled personel to produce and interpret the
karyotype.
2. In situ hybridization fluorescent, i.e.,
FISH, chromogen, or silver based:
Utilizes labeled nucleic acid probes that hybridize to
regions flanking the loci of interest
Can detect aberrant localization of probes
Required multiple probes to detect rarer translocations
Advantage: can be applied easily to touch preparations,
fresh tissue, karyotype preparations, frozen specimens,
and formalin-fixed paraffin-embedded samples.

3. Using polymerase chain reaction (PCR)

Use especially reverse transcriptase PCR
(RT-PCR):
Results in amplification of fusion transcripts encoded by
specific chimeric gene
Can be used with fresh, frozen, or formalin-fixed paraffin
embedded tissue

Post-chemotherapy assessment
1. Excision
specimens
received
after
chemotherapy are examined thoroughly
and the greatest dimension of the tumor is
mapped into grids to assess necrosis
2. The histological response to chemotherapy
is graded semiquantitatively
Grade 1: 50% or less of tumor necrosis
Grade 2: 50-90% of tumor necrosis
Grade 3: 90-99% of tumor necrosis
Grade 4: 100% of tumor necrosis

3. Patients
with
a
good
response
to
chemotherapy (grades 3 and 4) have
superior local recurrence-free survival at 5
years (86% vs 51%, P=0.15).Age and
sex,and the tumor size are thought to
influence the degree of response to
chemotherapy and event-free survival.

FACTORS ASSOCIATED WITH

PROGNOSIS
Stage
Stage

Size
Size

Ages
Ages

Primar
Primar
y
y
tumor
tumor
site
site

Respon
Respon
se to
se to
therap
therap
y
y

THERAPEUTIC TARGETS
The EWS-FLI1 fusion is present only in EFT
cells and does not exist in any normal cell of
the body.
Thus, EFT contains a unique protein
generated by tumor-specific translocation
with a potential for molecular target, but so
far nothing has reached the clinics this
might be due to EWS-FLI1 is poor solubility
As IGF-1 is associated with EFT growth,
monoclonal antibodies against this potential
target are being tried

CONCLUSION
The diagnosis of EFT amalgamates the
The diagnosis of EFT amalgamates the
usual or classical tools such as
usual or classical tools such as
histology and immunohistochemistry
histology and immunohistochemistry
with newer molecular technologies like
with newer molecular technologies like
FISH and PCR
FISH and PCR
The goal of these is to furnish a correct
The goal of these is to furnish a correct
diagnosis and give sufficient
diagnosis and give sufficient
information about the tumor that
information about the tumor that
would aid in better risk assessment,
would aid in better risk assessment,
improve clinical management, and
improve clinical management, and
survival of the patients
survival of the patients