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Genetic Cancer Susceptibility (GCS) Genomics in I4C

Genetic Cancer Susceptibility (GCS) Genomics in I4C. James McKay. GCS aims and focus. Identify and understand genetic susceptibility to cancer applying cutting edge genomics techniques to the large bio-repositories within GEN lung, head and neck, kidney cancers and lymphomas

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Genetic Cancer Susceptibility (GCS) Genomics in I4C

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  1. Genetic Cancer Susceptibility (GCS)Genomics in I4C James McKay

  2. GCS aims and focus • Identify and understand genetic susceptibility to cancer • applying cutting edge genomics techniques to the large bio-repositories within GEN • lung, head and neck, kidney cancers and lymphomas II. Genetics Platform (GSP) • Develop and maintain genotyping / genomic applications • Including the related bioinformatics

  3. GCS studies Cancers prioritized by IARC GWAS – lung, hodgkin’s lymphoma, UADT cancers Pedigree analysis – Exome sequencing in unusual pedigrees NPC cancers in Sarawak, Malaysia, Bladder Cancer in Iran Novel strategies. Use an intregrative approach to maximise our ability to perform meaningful studies within limited sample sets (mutation profiles, loss of second copy of alleles. eQTL)

  4. Bidayuh NPC pedigree Kuching Sarawak Malaysia Pedigree in a loose sense, three isolated villages Discovery Exome sequencing + linkage analysis (snp arrays) Assess for segregation Validation Screening candidate genes in additional cases

  5. Genetics platform Flexibility, suitability to IARC studies and bioropositories • Next generation sequencing LifeTechnology 5500 and PGM • Illuminabead array • High performance computer cluster • Highly dynamic field, what possible this week, needs to be revised next week

  6. Rapid and cheap comprehensive sequencing of many tumours is only now becoming feasible Exome/Genome Sequencing Many genes/Few samples Targeted Resequencing Few genes/Many samples Targeted Resequencing Ion Torrent Experiment Cost (35 amplicons, 150X coverage) 72.8 €/sample X 6 cost through GCS optimization 12.2 €/sample

  7. Genes mutated within HNC • Within 100 (TCGA) head and neck tumours, 95% mutations reported within 10 genes. • It becomes possible to design targeted sequencing assays to cover most mutations, and at low cost. • Mutations can be detected in the DNA from plasma (0.1-1%) Stransky N et al (2011). Science

  8. Highly multiplexed PCR used for enrichment. Targeted combined amplification of genes up to 300-400 amplicons/PCR 5 genes >240,000 amplicons whole exomes Using this to resequence candidate genes. 20 euros/sequence five genes. Applied to circulate tumour DNA (10,000X) to capture rare events Qaigen Generead

  9. I4C genomics applications TEL/AML translocations What is there prevalence at birth (1% or 0.1%?). Dosage? Is presence of TEL/AML translocations predictive of ALL later in life? Is there a relationship between TEL/AML dose/patient and ALL? Is modified by other factors? Birthweight, age of onset (early ALL cases), gender…

  10. TEL/AML translocation assayed using RNA Limitation is the availability of appropriate bio-specimens

  11. DNA assay for TEL/AML translocation Complicated as the breakpoint for the translocations is the variable. A Zelent et al, Oncogene (2004)

  12. Tiled highly multiplexed PCR TEL (ETV6) gene 12 kb N=60 (200bp) AML1 (RUNX1) gene 100 kb N=500 (200bp) Low strigency PCR, coupled with very deep sequencing (10,000) to capture very rare variants

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