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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

    children

  • 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


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)


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

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


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

Training

Test

ARMSp

ARMSn

ERMS





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

Expression

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)

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


PAX7-FOXO1 fusion neg cases 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 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

GOS

Kathy Pritchard-Jones

Department of Pediatric and Adolescent Oncology,

Institut Gustave Roussy

Odile Oberlin


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