Use of samples in research rhabdomyosarcomas
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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

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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


Use of samples in research rhabdomyosarcomas

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.


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

  • Rhabdomyosarcoma (RMS) histology

  • Small round blue cell tumours

  • Resemble developing skeletal muscle

  • Two main histological subtypes:

    • Embryonal (ERMS)

    • Alveolar (ARMS)


Use of samples in research rhabdomyosarcomas

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

  • Cells resemble embryonic

    skeletal muscle

  • Predominant in younger

    children

  • Generally good prognosis


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

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

  • Older children

  • Poor prognosis

  • Characteristic translocations


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

  • 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 samples in research rhabdomyosarcomas

Survival from rhabdomyosarcoma in GB, 1991-2000

Charles Stiller, CCLG data


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)


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

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


Overall and event free survival

Overall and Event Free Survival


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


Frequency of metastases

Frequency of Metastases


Expression profiling of 101 rms

Expression profiling of 101 RMS


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


Negative matrix factorisation nmf metagenes1

Negative Matrix Factorisation (NMF) - Metagenes

Training

Test

ARMSp

ARMSn

ERMS


Supervised analysis support vector machine classification

Supervised Analysis - Support Vector Machine Classification


Supervised analysis sam significance analysis microarray

Supervised Analysis – SAM (Significance Analysis Microarray)


Dna analysis arraycgh 128 rms

DNA analysis - ArrayCGH – 128 RMS


Gain of chromosome 8 is characteristic of fusion negative rms

Gain of Chromosome 8 is 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


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

  • 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


Use of samples in research rhabdomyosarcomas

Pilot data


Use of samples in research rhabdomyosarcomas

N=450 from MMT89, 95 , 98

Plus current EpSSG cases


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

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


Use of samples in research rhabdomyosarcomas

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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