Soft Tissue Tumours in Children

Gordan M. Vujanić

School Of Medicine

Cardiff University

Cardiff

United Kingdom

Pathology of Tumours

The Era of Morphology - 1850-1980s

The Era of Immunohistochemistry – 1980 – 2000

The Era of Molecular Diagnosis - 2000 -

Tumours in Children

Challenging to diagnose

- relatively rare

- show challenging morphological features

Different form tumours in adults (carcinomas vs. embryonal)

Rare even in tertiary paediatric referral centres

Multicentre trials resulted in huge improvement (central pathology

review, biology studies, standardized multidisciplinary therapeutic

treatments)

Soft Tissue Tumours in Children - WHO Classification -

1. Fibrous tissue tumours

2. Fibrohistiocytic tumours

3. Lipomatous tumours

4. Smooth muscle tumours

5. Skeletal muscle tumours

6. Endothelial tumours of blood vessels

7. Perivascular tumours

8. Synovial tumours

9. Mesothelial tumours

10. Neural tumours

11. Paraganglion tumours

12. Cartilage and bone tumours

13. Pluripotential mesenchymal tumours

14. Miscellaneous tumours

15. Unclassified tumours

Paediatric Soft Tissue Tumours

Historically, classified according to their degree and type of cellular

differentiation

Skeletal myogenesis = rhabdomyosarcoma

Spindle cell morphology = fibrosarcoma/myofibrosarcoma

Small round cell tumours = undifferentiated sarcoma

Morphological diagnosis of limited value

- Tumours with similar histology – markedly different clinical

behaviour and outcome

Rationale for using molecular techniques

in paediatric tumour pathology

Diagnostic difficulties (especially with needle biopsies)

Immunophenotype often non-diagnostic

CD56 - PNET, RMS, NB, WT

CD99 - PNET, DSRCT, WT

Bcl-2 - PNET, DSRCT, WT, NB, SS

WT1 - PNET, RMS, DSRCT, NB, WT

Important role in diagnostic and prognostic stratification of tumours

Prognosis / treatment / trial protocols

Which ‘molecular’ methods are diagnostically applicable to histopathology samples?

Numerous techniques can be utilised in the molecular genetic

analysis of tumours

Classical cytogenetics

Fluorescence in situ hybridisation (FISH)

PCR and RT-PCR

Paediatric Soft Tissue Tumours

When tumour karyotyping introduced:

STT with recurrent chromosomal translocations

- more uniform cell population

STT with complex karyotypes lacking recurrent translocations

- more likely to be pleomorphic

- RT-PCR and FISH – sorted out many problems ass with classic

cytogenetics

Why molecular diagnostic techniques?

Many paediatric tumours have specific translocations

with associated functional fusion gene products

Tumour Translocation Fusion gene product(s) Prevalence

A-RMS (2;13)(q35;q14) PAX3-FOXO1 ~70%

(2;13)(p36;q14) PAX7-FOXO1 ~10%

ES/PNET (11;22)(q24;q12) EWS-FLI1 ~85%

(21;22)(q22;q12) EWS-ERG ~10%

t(7;22)(q33;q12) EWS-FEV ~1%

DSRCT (11;22)(p13;q12) EWS-WT1 ~93%

Synovial sarcoma (X;18)(p11.2;q12.2) SYS18SSX1; SYS18SSX2; 63%; 37%

SS18-SSX4 rare

IFS (12;15)(p13;q25) ETV6-NTRK3 ~100%

DFSP (17;22)(q22;q13) COL1A1-PDGFB ~92%

AlvSPS (X;17)(p11;q25) ASPL-TFE3 ~100%

FISH vs. PCR in routine practice

PCR

Precise identification of transcripts BUT

Limited to known targets

Reduced sensitivity in FFPE specimens due to lower RNA quality

Usually the housekeeping gene expressed at higher levels than target so

possible false

FISH

Less specific since FISH detects for example any EWS translocation

Translocation partner NOT identified but also not necessarily required to

interpret test !

Role of Molecular Diagnostics in Paediatric Tumours

As a diagnostic tool (Ewing’s sarcoma/PNET, or Synovial Sarcoma)

Tumour Classification (Alveolar RMS vs. Embryonal RMS)

As a prognostic marker (Neuroblastoma, RMS, etc.)

Soft Tissue Tumours in Children

Tumour category Children (%) Adults (%)

Vascular 29 9

Neurogenic 15 9

Myogenic 14 5

Fibroblastic-myofibroblastic 12 7

Fibrohistiocytic 12 17

Lipocytic 6 16

Other 12 38

Rhabdomyosarcoma (RMS)

the most common soft tissue sarcoma in childhood

peak incidence in the first decade

familial form is associated with Li-Faumeni syndrome with increased risk for development

of various neoplasms

most common in soft tissues, but can occur anywhere (cutaneous RMS in association with

epidermal naevus & von Recklinghausen’s disease)

International Classification of RMS

I Superior prognosis Botryoid RMS

Spindle cell RMS

II Intermediate prognosis Embryonal RMS (classical)

III Poor prognosis Alveolar RMS

IV Unknown prognosis RMS with rhabdoid features

Diagnosis of RMS

Morphology

Demonstration of myogenic differentiation

Molecular characterisation

Embryonal RMS

Common in head & neck, genitourinary tract, paraspinal/parameningeal sites

Incidence declines after age 3 yrs

Prognosis is dependant on a number of factors:

Site

Age

Staging

Histology

All are important and form part of an IRS grouping for patient management

Histopathology

Embryonal RMS has a variable pattern

Varying degree of cellularity with alternating dense hypercellular areas and more

loose myxoid areas

A mixture of round cells, spindle cells and more differentiated cells showing

rhabdomyoblastic differentiation

Anaplasia can be present in embyonal and alveolar RMS (?worst prognosis)

Embryonal RMS

Embryonal RMS

Alveolar rhabdomyosarcoma

A-RMS accounts for at least 30-40% of all RMS

Alveolar RMS displays a bimodal age incidence

A peak at 3 years and 15 years of age

Higher incidence in upper and lower limbs

A-RMS - Pathology

Tumour composed of ill defined aggregate/nests of poor differentiated round to oval

cells

Some cells may show rhabdomyoblastic differentiation

The nests usually show central loss of cellular cohesion forming irregular alveolar

spaces

A-RMS - Pathology (continued)

Fibrovascular septa separate the nests of cells, with single cells adherent to these

septa

Mitotic figures are common

Wreath like giant cells are a characteristic and diagnostically useful feature of alveolar

RMS

The alveolar pattern is also seen in metastatic sites

Solid variant of alveolar RMS is an important subtype in which fibrous septa may

be absent or inconspicuous

Alveolar RMS

Alveolar RMS

Wreath like giant cells in A-RMS

Solid variant A-RMS

Reticulin

A-RMS - Differential diagnosis

Any small round cell tumour but in particular pseudo-alveolar pattern in

PNET

alveolar soft part sarcoma

desmoplastic small round cell tumour

Pseudoalveolar pattern in PNET

Recommended panel:

Desmin (in 99%)

Myogenin (in 100%) – most specific

MyoD1 (in 100%)

Muscle Specific Actin (in 94%)

CD99 (weak granular) (in 14%)

CAM5.2

EMA

Synaptophysin

Other useful markers

Myoglobin (in 78%) in more differentiated forms

Sarcomeric actin

Myosin

Immunohistochemistry in RMS

Desmin staining

Myo D1 nuclear staining

CD99 Myogenin

Rhabdomyosarcoma -

immunohistochemistry vs prognosis

Myogenin - >80% ARMS vs >25% ERMS (>50% of tumour cells)

Diffuse myogenin + a marker of poor prognosis (independent of fusion

status and histology)

PAX-5 – 67% ARMS vs 0% ERMS

Rhabdomyosarcoma -

molecular biology

ERMS associated with multiple numeric and structural chromosomal changes (extra

copies of chromosomes 2, 8 and 13; LOH at 11p15 – BW region)

ARMS associated with two specific gene fusions

PAX3-FOXO1 (correlated with t(2;13)(q35;q14) – 3x more common

PAX7-FOXO1 (correlates with t(1;13)(p36;q14) – better prognosis

20-25% ARMS have neither of these translocations – their genetic profile

identical to ERMS - ?prognosis

Synovial Sarcoma

5-10% of STS

42% of paediatric non-RMS STS

85-95% arise in the extremities, then head and neck

Biphasic pattern – spindle cells and epithelial gland components

In children – monophasic SS more common

Focal epithelial differentiation

Cytokeratin staining

SS - Immunoprofile

Cytokeratin

EMA

Vimentin

S100

CD99

BCL2

CD34

Calponin

Desmin

TLE1

+ (some are -)

+

+

+ in 30% of cases

+ in 60% of cases

+ in 75% - 100% of cases

-

+

-

- nearly 100%

Synovial Sarcoma

Characterised by a translocation: t(x;18)(p11.2;q11.2)

(SS18-SSX1); (SS18-SSX2); (SS18-SSX4)

The identification of a specific translocation has resulted in the ability to

diagnose these tumours in unusual sites (viscera)

It is also possible to identify poorly or undifferentiated SS

Is an important tool in differentiating SS from other spindle cell tumours

(congenital fibrosarcoma)

Congenital Infantile Fibrosarcoma

Diagnosed in the 1st year of life (~50% congenital)

Usually occur in distal extremities & head and neck

Histologically simulates adult-type fibrosarcoma

5-year survival of >90%

Recurrence rate 30%

Metastases rare

Chromosomal translocation t(12;15)(p13;q26) (ETV6/NTRK3 genes)

Congenital Fibrosarcoma

Vimentin staining

EWS/PNET

Ewing Family of Tumours

EWS/PNET - a group of tumours with overlapping clinical & pathological

features

peak incidence in childhood & young adolescence

EWS – classically a primary bone neoplasm (rarely extra-osseous)

PNET – usually arises in soft tissues (exceptionally in bone)

PNET - Pathology

i) lobular, ii) diffuse & iii) incohesive growth pattern

foci of haemorrhage but NO calcification

tumour lobules divided by fibro-connective septae

Homer-Wright & perivascular pseudo-rosettes rare

small cells, round-oval nuclei, coarse chromatin, small nucleoli, narrow rim of

eosinophilic cytoplasm

intercellular fibrillary material or ganglion cells exceptionally rare

IHC of EWS/PNET – essential

POSITIVE for:

CD99 ~95%, perimembranous

VIM ~95%, focal or diffuse, dot-like

or perinuclear

NSE, S100, synaptophysin, NFP ~40%

Chromogranin ~20%

GFAP <10%

EMA, Cytokeratin, Desmin <10%

CD99

Ewing’s Family of Tumours –

molecular biology

t(11;22) (involving EWS gene) – 85-90% of ESF

- t(11;22)(q24;q12) (EWSR1-FLI1)

- Type 1 (exon 7 – exon 6) - ?better prognosis

- Type 2 (exon 7 – exon 5)

- t(21;22)(q22;q12) (EWSR1-ERG) – 5-10% of ESF

- 4-9% of ESF – other fusions

Desmoplastic Small Round Cell Tumour

- DSRCT

rare, highly aggressive tumour of uncertain origin

M>F

presents with abdominal distension, pain & mass

at laparotomy – variable sized mass with numerous smaller nodular peritoneal

implants throughout the peritoneal cavity

DSRCT -

Immunohistochemistry

Not specific but distinct co-expression of

i) epithelial (AE1/AE3, CAM5.2)

ii) neural (NSE, PGP9.5; CD99 in 20-35%)

iii) muscle markers (desmin; negative for MyoD1, myoglobin)

DSRCT – Molecular biology

This tumour is defined by a reciprocal translocation

t(11;22)(p13;q12) (EWS-WT1 genes)

This translocation creates a fusion gene that transcribes a chimeric

protein containing portions of WT1 protein & EWS protein

DSRCT - Prognosis

a very aggressive neoplasm

chemotherapy may induce temporary remission

the ultimate outcome remains dismal

Molecular findings are the Gold Standard for diagnosis ?

‘Non-specificity’ of immunomorphology

CD56 - PNET, RMS, NB, WT

CD99 - PNET, DSRCT, WT

Bcl-2 - PNET, DSRCT, WT, NB, SS

WT1 - PNET, RMS, DSRCT, NB, WT

vs. specific diagnosis with molecular techniques

ETV6/NTRK3 in Infantile fibrosarcoma

SS18-SSX1 in Synovial sarcoma

EWS-FLI1 / other translocations in atypical PNET

Histomorphology is the Gold Standard for diagnosis?

‘Non-specificity’ of molecular markers

12;15 ETV6-NTRK3 in CMN/IFS and secretory breast

carcinoma

‘A FISH study of ETV-NTRK3 fusion gene in secretry breats carcinoma’

Marketsov et al. Genes Chrom Cancer 2004; 40: 152-7

X;18 in SS and MPNST ‘Lack of SYT-SSX fusion transcripts in malignant peripheral nerve sheath tumors on RT-

PCR analysis of 34 archival cases’ Tamborini et al. Lab Invest 2002; 82: 609-18

Xp1 RCC and AlvSPS

‘Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a

distinctive entity previously included among renal cell carcinoma of children and

adolescents’

Argani et al. Am J Surg Pathol 2001; 159: 179-92

SUMMARY

Molecular studies invaluable for diagnostic and clinical management of a range of

tumours

In the future, clinical decisions will increasingly be based on a combination of

histological criteria and the molecular identification of genetic abnormalities

Future advances likely to lead to even greater diagnostic and prognostic information,

and identification of new therapeutic targets in treatment

Gold standard is interpretation of molecular findings in the context of the

histopathological features