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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

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Use of Samples in Research - Rhabdomyosarcomas

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  1. Use of Samples in Research - Rhabdomyosarcomas Janet Shipley Sarcoma Molecular Pathology Team The Institute of Cancer Research Sutton, UK

  2. 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.

  3. 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

  4. Rhabdomyosarcoma (RMS) histology • Small round blue cell tumours • Resemble developing skeletal muscle • Two main histological subtypes: • Embryonal (ERMS) • Alveolar (ARMS)

  5. Embryonal RMS (ERMS) (60-80% of RMS cases) • Cells resemble embryonic skeletal muscle • Predominant in younger children • Generally good prognosis

  6. 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

  7. Alveolar RMS (ARMS) (20-40% of RMS cases) • Older children • Poor prognosis • Characteristic translocations

  8. 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

  9. 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

  10. Survival from rhabdomyosarcoma in GB, 1991-2000 Charles Stiller, CCLG data

  11. 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)

  12. 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

  13. 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

  14. 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

  15. Overall and Event Free Survival

  16. Cox Regression – Risk of Recurrence • Fusion gene positive cases greater risk of recurrence

  17. Cox Regression – Risk of Death • Fusion gene positive cases greater risk of death

  18. Frequency of Metastases

  19. Expression profiling of 101 RMS

  20. 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

  21. Negative Matrix Factorisation (NMF) - Metagenes Training Test ARMSp ARMSn ERMS

  22. Supervised Analysis - Support Vector Machine Classification

  23. Supervised Analysis – SAM (Significance Analysis Microarray)

  24. DNA analysis - ArrayCGH – 128 RMS

  25. Gain of Chromosome 8 is Characteristic of Fusion Negative RMS Expression Copy number

  26. Chromosome 8 genes are enriched in Metagene F2 linked to fusion neg cases Highly correlated with F2 metagene Highly anti-correlated with F2 metagene

  27. 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

  28. 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

  29. Pilot data

  30. N=450 from MMT89, 95 , 98 Plus current EpSSG cases

  31. 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

  32. 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

  33. 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

  34. PLANS: • Prospective study to assess classifier • Additional/refinement of potential prognostic markers • Identify and validate presence of potential therapeutic targets

  35. 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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