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Use of Samples in Research - Rhabdomyosarcomas. Janet Shipley Sarcoma Molecular Pathology Team The Institute of Cancer Research Sutton, UK. Childhood Cancers. ~ 1,500 new cases in UK p.a. 1% Liver 3% Germ cell 3% Eye 5% Bone 6% Wilms 6% Soft tissue 7% Neuroblastoma

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Use of samples in research rhabdomyosarcomas

Use of Samples in Research - Rhabdomyosarcomas

Janet Shipley

Sarcoma Molecular Pathology Team

The Institute of Cancer Research

Sutton, UK

Childhood Cancers

~ 1,500 new cases in UK p.a.

1% Liver

3% Germ cell

3% Eye

5% Bone

6% Wilms

6% Soft tissue

7% Neuroblastoma

14% Lymphoma

18% CNS

30% Leukaemia

7% Other

6-8% soft tissue sarcomas (1% in adults)

> 50% rhabdomyosarcoma (RMS)

~ 70 new cases in UK p.a.

Clinical issues:

  • Overall survival rhabdomyosarcomas (RMS) ~70%

  • Certain categories and metastatic disease - dismal

  • Major cause from cancer death in children

  • Toxicity leads to survivorship issues

  • Rhabdomyosarcoma (RMS) histology

  • Small round blue cell tumours

  • Resemble developing skeletal muscle

  • Two main histological subtypes:

    • Embryonal (ERMS)

    • Alveolar (ARMS)

Embryonal RMS (ERMS) (60-80% of RMS cases)

  • Cells resemble embryonic

    skeletal muscle

  • Predominant in younger


  • Generally good prognosis

ERMS genetics

Ploidy changes and aneuploidy (2, 8, 12, 13, 17 and 20)

- chromosomal instability CIN

Abnormalities of 11p:

Increased IGF2 expression through:

- Loss of heterozygosity (LOH)

80% affects IGF2, H19 and CDKN1C (p57KIP2) loci

(duplication of paternal non-silenced locus)

- Loss of imprinting (LOI) – 20%

(loss of maternal IGF2 imprinting)

- RAS mutations including HRAS at 11p

Alveolar RMS (ARMS) (20-40% of RMS cases)

  • Older children

  • Poor prognosis

  • Characteristic translocations

ARMS genetics

  • Ploidy changes, aneuploidy and amplification events

  • TP53 mutations

  • LOI loss of maternal silencing of IGF2 - biallelic expression

    H19 affects IGF2 imprinting

  • Characteristic chromosome translocations present in most, but not all cases



Paired DNA

binding domain




Use of pax foxo1 fusion vs histology in clinical stratification
Use of PAX-FOXO1 Fusion vs Histology in clinical stratification

  • PAX fusion gene status has been used for years as “unofficial” diagnostic aid

  • Current and past treatment stratifications incorporate histology into risk management strategies

  • The definition of Alveolar histology changed in the 90s (from majority to any)

  • Differentiating Alveolar from Embryonal involves finding histological evidence of alveolar spaces (with the exception of solid alveolar)

As 30% of RMS with alveolar histology stratification

do not appear to have fusion gene

Q: Clinical and biological impact of fusion gene status and histology

Williamson et al 2010 JCO

Criteria for inclusion
Criteria for Inclusion stratification

  • Patients with RMS all stages less than 21 years old, both sexes

  • RMS diagnosed or treated in France or UK (through CCLG) between 1989 and 2005 - SIOP protocols

  • Review of histological diagnosis of RMS alveolar and embryonal according to morphology and immunohistochemistry by members of the French/UK panel of pathologists

Analyses stratification

  • Molecular analysis of a representative sample by the Institut Curie or ICR for presence of PAX3/FOXO1, PAX7/FOXO1 or PAX3/NCOA1 by multiplex RT-PCR

  • DNA array CGH profiling

  • Gene expression profiling (Affymetrix Plus 2)

  • Issue of RNA quality critical – rapid snap freezing

Cox regression risk of recurrence
Cox Regression – Risk of Recurrence stratification

  • Fusion gene positive cases greater risk of recurrence

Cox regression risk of death
Cox Regression – Risk of Death stratification

  • Fusion gene positive cases greater risk of death

Frequency of metastases
Frequency of Metastases stratification

Negative matrix factorisation nmf metagenes

ARMSp stratification

This Study



Davicioni et al





Wachtel et al



Laé et al



Negative Matrix Factorisation (NMF) - Metagenes

  • HGU133 plus 2 Our Study

    • 101

      • 69 Alveolar

      • 37 Embryonal

  • HGU133a – Davicioni et al

    • 118

      • 64 Alveolar

      • 55 Embryonal

  • HGU133a – Wachtel et al

    • 30

      • 15 Alveolar

      • 15 Embryonal

  • HGU133a – Laé

    • 38

      • 23 Alveolar

      • 15 Embryonal

Negative matrix factorisation nmf metagenes1
Negative Matrix Factorisation (NMF) - Metagenes stratification






Gain of chromosome 8 is characteristic of fusion negative rms
Gain of Chromosome 8 is Microarray)Characteristic of Fusion Negative RMS


Copy number

Chromosome 8 genes are enriched in metagene f2 linked to fusion neg cases
Chromosome 8 genes are enriched in Metagene F2 linked to fusion neg cases

Highly correlated with F2 metagene

Highly anti-correlated with F2 metagene

Metagenes associated with outcome fusion neg cases

  • Davicioni et al MG34

  • New metagene we derived, less efficient in their dataset

  • - overfitting

  • Heavy association with fusion gene status, PAX3-FOXO1 versus PAX7-FOXO1 cases

  • - COG study, PAX7-FOXO1 better outcome

  • German study, no difference

  • Limited numbers

Pilot data fusion neg cases

N=450 from MMT89, 95 , 98 fusion neg cases

Plus current EpSSG cases

PAX3-FOXO1 fusion neg cases fusion dual-color assay

5’ PAX3 Telomeric Probes (BACs)

3’ FOXO1 Centromeric Probes (BACs)








Chimeric der(13) t(2;13) (q35,q14)



PAX7-FOXO1 fusion neg cases fusion dual color assay

5’ PAX7 Telomeric Probes (BACs)

3’ FOXO1 Centromeric Probes (BACs)








Chimeric der(13) t(1;13) (p36;q14)



Plus RT-PCR analyses where possible

Conclusions 1
Conclusions 1 fusion neg cases

  • PAX fusion negative ARMS clinically and molecularly indistinguishable from ERMS

  • Fusion negative RMS characterised by a distinct and common expression signature including chromosome 8 gain

  • Implications for the ongoing risk stratification strategies in current RMS treatment protocols - under versus over treatment

PLANS: fusion neg cases

  • Prospective study to assess classifier

  • Additional/refinement of potential prognostic markers

  • Identify and validate presence of potential therapeutic targets

Thanks to

Children's Cancer and fusion neg cases

Leukaemia Group

Thanks to…

INSERM U830 Institut Curie

Olivier Delattre

Daniel Williamson

Gaelle Pierron

Benedicte Thuille

Stephanie Reynaud

Départment de Pédiatrie, Institut Curie

Daniel Orbach

Gilles Palenzuela

Pathology Dept. Institut Curie

Paul Fréneaux

Marick Laé

Ligue Nationale Contre le Cancer

Aurélien de Reyniès

Manchester Children’s Hospital

Anna Kelsey

Swiss Bioinformatics Institute

Edoardo Missiaglia


Kathy Pritchard-Jones

Department of Pediatric and Adolescent Oncology,

Institut Gustave Roussy

Odile Oberlin