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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

slide2

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.

slide3

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
slide4

Rhabdomyosarcoma (RMS) histology

  • Small round blue cell tumours
  • Resemble developing skeletal muscle
  • Two main histological subtypes:
    • Embryonal (ERMS)
    • Alveolar (ARMS)
slide5

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

  • Cells resemble embryonic

skeletal muscle

  • Predominant in younger

children

  • Generally good prognosis
slide6

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

slide7

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

  • Older children
  • Poor prognosis
  • Characteristic translocations
slide8

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
slide9

Characteristic translocations present in ~70% of ARMS

    • ~80% of which t(2;13)(q35;q14) PAX3/FOXO1
    • ~20% t(1;13)(p36;q14) PAX7/FOXO1 and rare variants

FOXO1

PAX

Paired DNA

binding domain

Homeodomain

Transactivation

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)
slide12

As 30% of RMS with alveolar histology

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
  • 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
slide14

Analyses

  • 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
  • Fusion gene positive cases greater risk of recurrence
cox regression risk of death
Cox Regression – Risk of Death
  • Fusion gene positive cases greater risk of death
negative matrix factorisation nmf metagenes

ARMSp

This Study

ERMS

ARMSn

Davicioni et al

ARMS

ERMS

ARMSn

ARMSp

Wachtel et al

ERMS

ARMSn

Laé et al

ARMS

ERMS

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
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

slide28

Metagenes associated with outcome

  • 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
slide29

PAX3-FOXO1 versus PAX7-FOXO1 cases

  • Similar gene expression profiles
  • Predictive metagenes linked to PAX3 v PAX7-FOXO1
  • Direct comparison?
  • - COG study, PAX7-FOXO1 better outcome
  • German study, no difference
  • Limited numbers
slide31

N=450 from MMT89, 95 , 98

Plus current EpSSG cases

slide32

PAX3-FOXO1 fusion dual-color assay

5’ PAX3 Telomeric Probes (BACs)

3’ FOXO1 Centromeric Probes (BACs)

RP11-81I18

RP11-452K11

RP11-16P6

RP11-805F18

PAX3-FOXO1

RP11-612G6

RP11-350A18

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

Normal

PAX3-FOXO1

slide33

PAX7-FOXO1 fusion dual color assay

5’ PAX7 Telomeric Probes (BACs)

3’ FOXO1 Centromeric Probes (BACs)

RP11-468N9

RP11-452K11

CTD-2009F7

RP11-805F18

PAX7-FOXO1

RP11-121A23

RP11-350A18

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

Normal

PAX7-FOXO1

Plus RT-PCR analyses where possible

conclusions 1
Conclusions 1
  • 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
slide35

PLANS:

  • 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

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

GOS

Kathy Pritchard-Jones

Department of Pediatric and Adolescent Oncology,

Institut Gustave Roussy

Odile Oberlin

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