Ewing Sarcoma

Slide 1 · Ewing Sarcoma

Bone and soft-tissue pathology · morphology · fusion biology · diagnosis

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Recognize Ewing sarcoma in its epidemiologic, anatomic, and clinical context.

Explain the EWSR1::ETS fusion-driven pathogenesis and its diagnostic relevance.

Connect imaging, gross findings, and small round cell histology.

Construct an integrated differential and diagnostic confirmation strategy.

Relate systemic treatment and histologic response to clinical outcomes.

Slide 3 · Ewing sarcoma: Defining concept

Aggressive malignant sarcoma of bone or soft tissue.

Classically composed of relatively uniform primitive small round cells.

Molecularly characterized by FET::ETS fusion genes, usually EWSR1::FLI1.

Distinct from other genetically defined undifferentiated round-cell sarcomas.

Slide 4 · Position among primary malignant bone tumors

Ewing sarcoma

Primitive small round cell phenotype

Characteristic gene fusion

Usually no malignant osteoid or cartilage

Matrix-forming bone sarcomas

Osteosarcoma: Malignant osteoid

Chondrosarcoma: Malignant cartilage

Matrix helps establish lineage

Slide 5 · Frequency and rarity

Robbins: Approximately 10% of primary malignant bone tumors.

Second most common bone sarcoma in children, after osteosarcoma.

Roughly 200 new diagnoses per year in the United States.

Denominators matter: These are not population incidence rates.

Slide 6 · Age and sex patterns

Pediatric predominance

Robbins: Approximately 80% of patients are younger than 20 years.

Peak clinical context

Consider Ewing sarcoma in adolescents with an aggressive destructive bone lesion.

Sex distribution

Boys are affected slightly more frequently than girls.

Slide 7 · Common skeletal sites

Diaphysis of long tubular bones is a characteristic location.

Femur is a common long-bone primary site.

Pelvis is an important flat-bone site.

Anatomic site affects symptoms, biopsy planning, and local-control decisions.

Slide 8 · Typical medullary origin and local extension

Schematic anatomy — not a radiograph

Medullary tumor

Cortical breach → soft-tissue extension

Slide 9 · Extraskeletal Ewing sarcoma

Approximately 20% of cases arise outside bone (Robbins).

Molecular entity remains Ewing sarcoma despite an extraosseous primary site.

Clinical differential broadens when no osseous lesion is present.

Tissue morphology and fusion identity become especially important.

Slide 10 · How patients present

Progressively painful and enlarging mass.

Localized swelling, warmth, and tenderness are frequent.

Symptoms reflect rapid bone destruction and surrounding tissue involvement.

Painful symptoms may precede a clearly appreciated mass.

Slide 11 · Ewing sarcoma can resemble infection

Shared findings

Pain, warmth and local tenderness

Fever may occur

ESR elevation, anemia, or leukocytosis

Features requiring oncologic evaluation

Destructive permeative bone lesion

Associated enlarging soft-tissue mass

Persistent symptoms with atypical course

Biopsy needed for specific diagnosis

Slide 12 · Presentation case: Pain, fever, and a bone lesion

Illustrative case

A 15-year-old has worsening thigh pain, swelling, intermittent fever, elevated ESR, and a permeative femoral lesion.

Which clinical findings support inflammation but do not establish infection?

Which radiographic features justify urgent specialist evaluation?

Why must a biopsy be planned as part of definitive care?

Slide 13 · Radiographs: An aggressive lytic pattern

Destructive radiolucent lesion with ill-defined margins.

Permeative or “moth-eaten” pattern indicates marrow-space infiltration.

Tumor can breach cortex and extend beyond bone.

The radiograph can suggest aggressiveness; It cannot specify the fusion.

Slide 14 · Why permeative margins occur

Marrow tumor

Primitive tumor expands within the medullary cavity

Trabecular destruction

Numerous irregular microscopic foci coalesce

Ill-defined image

Radiographic margins appear moth-eaten or permeative

Slide 15 · Onion-skin periosteal reaction

Schematic anatomy — not a radiograph

Reactive periosteal lamellae

Schematic: Successive periosteal layers

Slide 16 · Interpret the periosteal reaction

Successive reactive layers form as tumor advances toward soft tissue.

The laminated “onion-skin” response is a classic clue.

A periosteal reaction is not pathognomonic for Ewing sarcoma.

Integrate the pattern with age, tumor site, imaging, and pathology.

Slide 17 · Imaging has three distinct purposes

Initial detection

Plain radiography characterizes the bone lesion and periosteal response.

Local extent

MRI depicts marrow, soft tissue, joint proximity, and neurovascular relations.

Distant staging

Chest CT and whole-body modalities assess metastatic dissemination.

Slide 18 · Local imaging before tissue sampling

MRI of the primary site is part of formal diagnostic evaluation.

Define intramedullary extent and extraosseous components.

Examine the full involved bone for possible discontinuous tumor deposits.

Plan the biopsy tract with the surgical sarcoma team.

Slide 19 · Clinical staging evaluation

Establish whether disease is localized or metastatic.

Chest CT assesses pulmonary dissemination.

PET and other body imaging are used to evaluate distant spread.

Laboratory studies may include CBC and LDH; No blood test is diagnostic.

Slide 20 · Gross morphology

Typically centered in the medullary cavity.

Soft tan-white tumor tissue replaces marrow and infiltrates cortex.

Extension into periosteum and adjacent soft tissue is common.

Hemorrhage and necrosis frequently contribute to gross heterogeneity.

Slide 21 · Small round cell architecture: Conceptual map

Schematic only — not a photomicrograph

Uniform primitive round cells

Relatively monotonous small round nuclei

Scant, sometimes clear cytoplasm

Tumor lacks its own bone/cartilage matrix

Slide 22 · Microscopic pattern: Sheets of tumor cells

Diffuse sheets of relatively uniform small round cells.

Cells are more cohesive than typical background lymphocytes.

Scant cytoplasm imparts a compact “small blue cell” appearance on H&E.

Stromal fibrous septa may interrupt otherwise solid cellular sheets.

Slide 23 · Nuclear and cytoplasmic detail

Round to oval nuclei and limited cytoplasmic volume characterize the classic phenotype.

Fine dispersed chromatin and inconspicuous nucleoli are often present.

Clear cytoplasm is frequently attributable to abundant glycogen.

Morphology supplies a strong clue—not a stand-alone molecular diagnosis.

Slide 24 · Why glycogen matters

Cytoplasmic clearing to glycogen accumulation.

Periodic acid–Schiff (PAS) staining can demonstrate glycogen.

PAS positivity is supportive but not unique to Ewing sarcoma.

The source notes that tumor cells lack osteoid and cartilage production.

Slide 25 · PAS and diastase: Interpret a supportive stain

H&E clue

Scant / clear cytoplasm within round cell sheets

PAS stain

Cytoplasmic glycogen can become magenta

Diastase digestion

Loss of PAS reactivity supports glycogen

Slide 26 · Homer–Wright rosettes: Schematic

Schematic only — not a photomicrograph

Optional neuronal differentiation

Homer–Wright rosette: Central fibrillary core

May be present; Not obligatory

Cannot establish the diagnosis alone

Slide 27 · Rosettes do not redefine the tumor

Homer–Wright rosettes may be found within Ewing sarcoma.

They form circular cell groupings around a central fibrillary core.

Neuronal differentiation does not imply a separate modern entity when the defining fusion is present.

Absence of rosettes does not argue strongly against Ewing sarcoma.

Slide 28 · Critical morphologic distinction

Ewing sarcoma

Uniform primitive small round cell sheets

Intracytoplasmic glycogen may be present

No neoplastic osteoid

No tumor cartilage

Conventional osteosarcoma

Tumor cells deposit malignant osteoid

Often markedly pleomorphic phenotype

May have other matrix components

Matrix production is diagnostic

Slide 29 · Read a suspected Ewing biopsy systematically

Pattern

Relatively uniform small round cells in diffuse sheets

Cytoplasm

Scant or clear; Glycogen can be present

Architecture

Fibrous septa and occasional rosettes

Matrix

No neoplastic bone or cartilage

Confirmation

Specific FET::ETS fusion in correct context

Slide 30 · The canonical t(11;22) rearrangement

Chr 22: EWSR1

Chr 11: FLI1

EWSR1::FLI1 fusion gene

T(11;22)(q24;q12) • recurrent oncogenic rearrangement

Slide 31 · Chromosomal positions and fusion identity

EWSR1 is located at chromosome 22q12.

FLI1 is an ETS-family transcription factor gene at chromosome 11q24.

T(11;22)(q24;q12) brings together portions of these genes.

The resulting EWSR1::FLI1 is a pathogenic, chimeric fusion gene.

Slide 32 · From fusion gene to dysregulated transcription

Rearrangement

Genetic translocation creates a novel transcriptional regulator

Fusion protein

EWSR1::FLI1 associates with chromatin

Biologic effect

Aberrant transcription drives malignant cellular behavior

Slide 33 · The partner families matter

FET component

EWSR1 is part of the FET RNA-binding protein family.

ETS component

FLI1 contributes an ETS-family DNA-binding regulatory component.

Novel function

The chimeric product changes transcriptional programs.

Slide 34 · Fusion spectrum within Ewing sarcoma

EWSR1::FLI1 is the predominant fusion in pediatric cases (NCI: About 85–90%).

Other ETS partners include ERG, ETV1, ETV4, and FEV.

Rare FUS::ETS fusions can occur.

Fusion naming should identify both gene partners, not merely EWSR1 rearrangement.

Slide 35 · Selected Ewing-associated gene fusions

FET partner

ETS partner

Interpretation

EWSR1

FLI1

Most common canonical fusion

EWSR1

ERG

Important less common fusion

EWSR1

ETV1 / ETV4 / FEV

Uncommon variants

FUS

ERG / FEV

Rare alternative FET partner

Slide 36 · The cell of origin remains uncertain

Mesenchymal stem cells a possible origin.

Primitive neuroectodermal cells have also been proposed.

The fusion-defined molecular identity is more secure than a single lineage-of-origin claim.

Avoid teaching an unproven cell of origin as a settled fact.

Slide 37 · WHO-oriented round-cell sarcoma framework

Molecular class

Defining alteration

Ewing sarcoma

FET::ETS fusion

EWSR1-non-ETS round cell sarcoma

Non-ETS EWSR1-related fusion

CIC-rearranged sarcoma

CIC-related rearrangement

BCOR-altered sarcoma

BCOR genetic alteration

Slide 38 · Older terminology in modern reporting

“Peripheral primitive neuroectodermal tumor” historically emphasized neural features.

“Askin tumor” referred to Ewing sarcoma of the chest wall.

These terms are generally encompassed by Ewing sarcoma when molecular criteria are met.

Do not group genetically distinct “Ewing-like” sarcomas under Ewing sarcoma.

Slide 39 · Supportive IHC: Membrane versus nucleus

CD99: Membranous (supportive)

NKX2.2: Nuclear (supportive)

Neither marker is fully specific; Interpret with morphology and molecular genetics.

Slide 40 · CD99: Diagnostic utility and pitfall

CD99 typically shows strong membranous staining in Ewing sarcoma.

CD99 is a useful screening marker within a small round cell lesion.

It is not specific: Several non-Ewing tumors may express CD99.

A positive CD99 stain does not substitute for genetic confirmation.

Slide 41 · NKX2.2: Additional supporting marker

NKX2.2 is a nuclear antigen commonly assessed in suspected Ewing sarcoma.

Positive staining strengthens the diagnosis in the appropriate context.

NKX2.2 is not perfectly specific.

Its value is highest when integrated with morphology, CD99, and molecular testing.

Slide 42 · Evidence hierarchy for diagnosis

Observation

What it contributes

What it cannot prove

Age, bone site, imaging

Raises clinical suspicion

Tumor identity

Small round cell H&E

Suggests Ewing phenotype

Unique tumor class

CD99 / NKX2.2

Supports the phenotype

Specific gene fusion

Fusion identification

Defines molecular group

Whole clinical stage

Slide 43 · EWSR1 break-apart FISH: Correct use

Test

Detect EWSR1 locus rearrangement

Interpret

Supports EWSR1-related neoplasm

Resolve

Determine fusion partner and WHO class

Slide 44 · FISH alone can misclassify a tumor

EWSR1 break-apart does not reveal the fusion partner.

Other tumor entities harbor EWSR1 rearrangements.

FUS-driven Ewing sarcoma may be negative with an EWSR1 probe.

A small subset of EWSR1::ERG tumors may be missed by break-apart FISH.

Slide 45 · Targeted RNA fusion testing

Fusion-aware assays can identify both partner genes.

NGS fusion panels are frequently used for problematic small round cell tumors.

A partner-aware result helps separate Ewing sarcoma from EWSR1-non-ETS neoplasms.

Interpret negative results alongside specimen quality and assay coverage.

Slide 46 · Integrated diagnostic algorithm

Imaging + clinical context

Aggressive osseous or soft-tissue tumor

Histologic biopsy

Uniform small round cells; Exclude matrix-formers

Immunophenotype

CD99 / NKX2.2 as supportive—not definitive

Molecular test

Confirm FET::ETS fusion; Classify alternatives

Slide 47 · Biopsy adequacy and tissue stewardship

Plan biopsy with a multidisciplinary sarcoma service.

Obtain viable lesional tissue for morphology and ancillary studies.

Extensive necrosis can impair diagnostic assessment.

Preserve suitable material for fusion testing when Ewing sarcoma is considered.

Slide 48 · Differential: Osteosarcoma versus Ewing

Osteosarcoma

Malignant osteoid matrix formation

Often metaphyseal, but site alone insufficient

Histology establishes osteoid-producing tumor

Ewing sarcoma

Primitive small round cells without osteoid

Often diaphyseal or pelvic primary

Specific FET::ETS fusion supports diagnosis

Slide 49 · Differential: Hematolymphoid malignancy

Lymphoma / leukemia

May form small round cell sheets in bone

CD45 / lineage studies may redirect workup

Fusion identity differs from Ewing

Ewing sarcoma

Primitive small round cell phenotype

CD99 and NKX2.2 may support suspicion

EWSR1::ETS or FUS::ETS fusion defines class

Slide 50 · Differential: Rhabdomyosarcoma

Rhabdomyosarcoma

May be primitive and round-cell predominant

Myogenic differentiation assessed by specific markers

Distinct molecular classification

Ewing sarcoma

Often shows uniform round-cell morphology

CD99/NKX2.2 as supportive markers

FET::ETS molecular confirmation

Slide 51 · Differential: Metastatic neuroblastoma

Neuroblastoma

Pediatric small round cell tumor can infiltrate bone

Clinical primary and neuroblastic features guide evaluation

Neuronal marker pattern differs

Ewing sarcoma

Can form Homer–Wright rosettes

Rosettes therefore are not lineage-specific

Genetic confirmation resolves ambiguity

Slide 52 · Practical differential at first biopsy

Candidate

Discriminating question

Ewing sarcoma

Is a specific FET::ETS fusion demonstrable?

Osteosarcoma

Is malignant osteoid formed by tumor cells?

Lymphoma

Are hematolymphoid lineage markers present?

Rhabdomyosarcoma

Is true skeletal muscle differentiation demonstrated?

Metastatic neuroblastoma

Do clinical context and neuroblastic features fit?

Slide 53 · Why EWSR1-non-ETS is not “Ewing”

EWSR1 can participate in fusions outside the ETS transcription factor family.

Examples include EWSR1::NFATC2 and EWSR1::PATZ1 tumors.

Current WHO-oriented taxonomy separates these from Ewing sarcoma.

Partner identity matters more than the shorthand “EWSR1-positive.”

Slide 54 · Two additional WHO round-cell groups

CIC-rearranged sarcoma

A molecularly distinct undifferentiated round cell sarcoma group.

BCOR-altered sarcoma

Genetically defined group that is not conventional Ewing sarcoma.

Diagnostic consequence

Overlapping morphology requires precise fusion/alteration characterization.

Slide 55 · Terminology: Avoid “Ewing-like” as a final diagnosis

Historical umbrella use pooled biologically different tumors.

WHO molecular categories provide more reproducible nomenclature.

A tumor with nonspecific CD99 positivity should not be called Ewing sarcoma.

Use the correct molecular category or a suitably qualified provisional diagnosis.

Slide 56 · Molecular reporting exercise

Illustrative case

A pelvic small round cell sarcoma is CD99-positive. EWSR1 break-apart FISH is positive. RNA testing identifies EWSR1::NFATC2.

Is the correct diagnosis necessarily Ewing sarcoma?

Which finding is decisive for WHO-oriented classification?

How should the initial CD99 and FISH results be described?

Slide 57 · What makes a defensible diagnosis

Clinical setting

Compatible anatomic site and destructive growth

Morphology

Primitive uniform small round cells

Immunophenotype

Supportive but not individually specific

Genetics

FET::ETS fusion; Identify fusion partners

Final label

Avoid collapsing distinct WHO molecular groups

Slide 58 · Localized versus metastatic disease

Local assessment

Size, bone extent, soft-tissue extension

Systemic workup

Chest and whole-body metastasis evaluation

Clinical category

Localized or metastatic guides management

Slide 59 · Why systemic chemotherapy is necessary

Ewing sarcoma is clinically aggressive and may disseminate early.

Apparently localized disease may include occult micrometastases.

Therefore, local surgery alone is not adequate standard treatment.

Multidrug chemotherapy is integrated with local-control strategies.

Slide 60 · Multimodal treatment: Conceptual sequence

Induction therapy

Systemic neoadjuvant chemotherapy

Local control

Surgery and/or radiotherapy

Further therapy

Adjuvant / consolidation chemotherapy

Slide 61 · Surgery and radiotherapy: Complementary roles

Surgical local control

Resection is considered when feasible with acceptable function

Margin status influences local management

Pathology examines viable tumor and treatment effect

Radiotherapy

May contribute to definitive or postoperative local control

Used in selected contexts with difficult anatomy/margins

Treatment decisions require a specialist team

Slide 62 · Histologic response after chemotherapy

Chemotherapy-induced necrosis as a favorable prognostic indicator.

The resection specimen allows estimation of residual viable tumor.

NCI PDQ links minimal viable tumor after induction to better outcomes.

Necrosis should be reported with method and relevant sampling limitations.

Slide 63 · Interpreting prognosis responsibly

Approximately 75% 5-year survival with contemporary multimodal therapy.

It also describes approximately 50% long-term cure rates.

These figures reflect the textbook’s referenced populations and treatment era.

Do not apply the aggregate numbers directly to an individual patient.

Slide 64 · Major prognostic determinants

At presentation

Localized versus metastatic disease

Site and tumor burden

Age and other clinical factors

After therapy begins

Histologic response / remaining viable tumor

Ability to achieve local control

Relapse after initial treatment

Slide 65 · Why survival statistics vary across sources

Outcomes depend on localized versus metastatic case mix.

Endpoint differs: Event-free survival, overall survival, and cure are not interchangeable.

Age, site, disease volume, and treatment regimen influence reported results.

NCI PDQ estimates must not be treated as directly identical to Robbins aggregates.

Slide 66 · Integrated case: A diaphyseal tumor

Illustrative case

A 14-year-old has a painful femoral diaphyseal mass, laminated periosteal reaction, and an H&E biopsy with uniform primitive small round cells.

List two plausible initial diagnoses and the key discriminator.

Identify a supportive immunophenotypic panel and its limits.

What molecular result would confirm the classic Ewing entity?

Slide 67 · Integrated case: Molecular ambiguity

Illustrative case

An undifferentiated soft-tissue sarcoma shows diffuse membranous CD99, nuclear NKX2.2, and an EWSR1 break-apart signal, but fusion-partner testing is pending.

Which evidence favors—but does not establish—Ewing sarcoma?

Why is the FISH result insufficient as a final diagnosis?

How does RNA fusion identification modify the final report?

Slide 68 · Synthesis: Five diagnostic anchors

Who?

Predominantly patients younger than 20 years

Where?

Often long-bone diaphysis, pelvis, or soft tissue

How?

Aggressive permeative lesion ± laminated periosteal reaction

What?

Uniform small round cells, scant glycogen-rich cytoplasm

Why?

Driver FET::ETS fusion; Most often EWSR1::FLI1

Slide 69 · Selected references

Robbins, Cotran & Kumar. Pathologic Basis of Disease. 11th ed. 2026. Chapter 26, Ewing Sarcoma, pp. 1083–1084.

NCI. Ewing Sarcoma and Undifferentiated Small Round Cell Sarcomas of Bone and Soft Tissue Treatment (PDQ®), Health Professional Version.

Dehner CA, Lazar AJ, Chrisinger JSA. Updates on WHO classification for small round cell tumors. Human Pathology. 2024;147:101–113.