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  • Haematologist Main interest pathogen. AML Developing interest in molecular diagnostics ... and I will talk to you about the potential impact of advances in DNA sequencing technologies on the field of cancer Δ , albeit with a slight overrepresentation of examples drawn from haematological cancers
  • 1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
  • 1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
  • As a general indication of how things have been going up until now and as I am sure many of us are aware, the CR-UK str..... recently invited research/Dic centres to apply for funding to offer a service for genotyping a set of mutations deemed to be of clinical relevance. So solid tumours are rapidly going the way of haematological cancers in requiring molecular info as part of their diagnostic work-up.
  • 1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
  • The magnitude of the change is represented here. This represents a >100 trillion-fold increase in sequencing capacity over 10 years
  • And to compare this to something more tangible, the white line here shows the rate at which the cost of transistors for computer circuits has been changing over the last decade (halfs every 2 years) – compared to the real cost of sequencing per megabase of DNA sequence
  • 1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis
  • However, other more selective applications are also being developed All of which need to go through a NGS step
  • The main application of the new technologies is whole matched cancer/constitutional genome sequencing ...
  • The main application of the new technologies is whole matched cancer/constitutional genome sequencing ...
  • There are many examples of PCR based approaches including some multiplex PCRs that tackle lists such as the CR-UK. However, these can only be applied to small substitutions and Indels. An example of such an application is a test we are developing that can be used in AML with NK: In this disease, the genome is stable and numbers of coding mutations small. Approximately 20 recurrent mutations have been identified and for some prognostic implications are know. This tool would enable the determination of the effects of different combinations of mutations on prognosis & response to Rx.
  • 1. 2. Give examples of molecular tests in current use 3. A brief overview of developments in the field of cancer genomics and the technologies underpinning them 4. Talk about novel diagnostic applications 5. Challenges to introducing these and other genomic application to cancer diagnosis

Dr g vassiliou Dr g vassiliou Presentation Transcript

  • The changing face of cancer diagnosis George Vassiliou November 2011
  • Overview
    • Today’s cancer diagnostic lab
    • The era of cancer genomics
    • Novel diagnostic applications
    • Introducing genomics to cancer diagnosis
    • Today’s cancer diagnostic lab
    • The era of cancer genomics
    • Novel diagnostic applications
    • Introducing genomics to cancer diagnosis
    Overview
  • The light microscope remains the central cancer diagnostic tool for 400 years Zacharias and Hans Jansen (ca 1595) Modern microscope (ca 1995)
  • Today’s cancer diagnostic lab
    • Cellular Phenotyping
    • Microscopy (histology/cytology)
    • Immunohistochemistry
    • Flow Cytometry
    • Genetic tests
    • Cytogenetics
    • Molecular Genetics
    • Genotyping for specific mutations (PCR/RT-PCR)
    • Minimal Residual Disease monitoring
    • (CGH and SNP/LOH genotyping)
    • (Gene Expression Profiling)
  • Haemato-oncology lab Microscopy Immunophenotyping Cytogenetics Molecular Genetics Sample Integrated report MICROSCOPY: >80% undifferentiated blasts Morphology of acute lymphoblastic leukaemia Eosinophils, basophils and small megakaryocytes suggest blast phase of chronic myeloid leukaemia IMMUNOPHENOTYPE: Blast cells are CD10, CD19, CD79a, CD34, HLA-DR, TdT positive. Weak CD13. They do not express CD33 or myeloperoxidase. DNA index is 1.0 Phenotype of B lymphoblastic leukaemia or B lymphoblastic transformation of CML CYTOGENETICS: Karyotype: 46,XY,t(9;22)(q34;q11) in 10 of 10 metaphases FISH: BCR/ABL 92% positive MOLECULAR GENETICS: BCR-ABL fusion transcript type: p210 e13a2 by RT-PCR OVERALL CONCLUSION: B lymphoblastic blast crisis of chronic myeloid leukaemia Cytology Histology Immunohistochemistry Diagnostic panels Karyotyping FISH Mutational screening RT-PCR qPCR (MRD)
  • Diagnostic CGH/SNP genotyping
  • n= 241 Diagnostic gene expression profiling MammaPrint – 70 gene signature (NKI) Lymph node positive breast cancer
  • CR-UK stratified medicines initiative Tumour type Gene Mutation Drug Colorectal KRAS Codons 12, 13, 61, 146 Cetuximab/Panitumumab   BRAF V600E/D/K/R/M Sorafenib/Cetuximab   TP53 Exons 2-­‐11     PI3KCA Exons 9 and 20 PI3Kinase inhibitors   UGT1A1 UGT1A1*28 Irinotecan toxicity Breast PI3KCA Exons 9 and 20     TP53 Exons 2-­‐11     PTEN LOH/mutation hotspots mTOR inhibitors   CYP2D6 5 SNPs Response to tamoxifen Prostate PTEN LOH/mutation hotspots mTOR inhibitors   TMPRSS-­‐ERG Junction fragment PCR     TLR4 2 SNPs   Lung EGFR Exons 18-21 Erlotinib/gefitinib   EML4-­‐ALK Fusion product PF02341066 ALK/c-Met inhibitor   XRCC2 5 SNPs Response to platinum agents   ERCC1 mRNA expression     RRMI mRNA expression   Ovary PTEN LOH/mutation hotspots mTOR inhibitors   PI3KCA Exons 9 & 20     BRAF V600E/D/K/R/M Sorafenib/Braf inhibitors Melanoma BRAF V600E/D/K/R/M Sorafenib/Braf inhibitors   CKIT Exons 11,13,17  
    • Today’s cancer diagnostic lab
    • The era of cancer genomics
    • Novel diagnostic applications
    • Introducing genomics to cancer diagnosis
    Overview
  • Advances in DNA sequencing technologies 10 2 10 4 10 6 10 8 10 10 10 12 10 14 10 16 Output kbp / run 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Ion Torrent Capillary (Sanger) Sequencing Next Generation Sequencing (NGS) 454 pyroseq Solexa/ Illumina ABI SOLID Illumina HiSeq Technologies Roche/454 Titanium ABI SOLID 3.0 ABI capillary
  • Rapid reduction in sequencing costs
  • Sanger Institute Total yield by week (Gigabases) 2008 2009 2010 2011
  • How Fast is That?
    • 6000 Gb per week (6 Tb) =
    • 10,000,000 bases per second
    • ½ hour per 6Gb (= 1x Human Genome)
  • Genome Sequencing 2015 ? ~1day ?? $100 Cancer Genomics Sanger (capillary) sequencing 2005 ~3 years ~$ 20million 2010 ~1month $9,500 (Illumina) AML Melanoma Small-cell lung Breast 2008 ~4 months ~$ 1.5million Lung (NSS) 2000 ~10 years ~$ 3.5 billion Myeloma Hepatocellular CLL Mouse AML Next generation sequencing
  • Analysing cancer genomes From Ding et al, Hum Mol Gen, 2010
  • Genomic Circos Plot Circos Plot from Pleasance et al, Nature 2010 Deletions/Insertions Substitution density (het) Substitution density (homo) Coding Substitutions Silent Missense Nonsense Splice site Copy number Regions of LOH Structural rearrangements Intrachromosomal Interchromosomal Genomic coordinates
    • 1000s of individual cancer genomes
    • 100s of recurrent mutations
    • Aetiological links
    • Clinico-pathological correlates
    • Delineation of effects of many mutations
    • Development of new therapies
    • > Increasing use of genomics in cancer diagnosis, prognosis & therapy
    This decade
    • Today’s cancer diagnostic lab
    • The era of cancer genomics
    • Novel diagnostic applications
    • Introducing genomics to cancer diagnosis
    Overview
  • The clinical process in oncology        Pre-clinical phase Presentation Diagnosis Treatment Assessment of response Follow-up Relapse
    • Whole genome sequencing (~6Gb)
    • Exome sequencing (~60Mb)
    • Selected gene/exon DNA sequencing
    • Residual disease monitoring (plasma DNA)
    New diagnostic applications
  • Whole genome sequencing A B C D
  • Diagnostic whole genome sequencing Clinical Report Constitutional genome Cancer genome Compare Other diagnostic data Somatic mutations Subclonal heterogeneity Substitutions Indels Copy number changes Translocations Inherited mutations & polymorphisms
  • Exome sequencing: target enrichment Chr1 NRAS regions covered “ Baits” or PCR amplicons
  • Diagnostic whole exome sequencing Clinical Report Constitutional exome Cancer exome Compare Other diagnostic data Somatic mutations Subclonal heterogeneity Substitutions Indels Copy number changes Translocations Inherited mutations & polymorphisms
  • Selective sequencing example : an AML toolkit
    • ~ 20 genes known to be recurrently mutated
    • Prognostic/treatment implications known for some
    • Target enrichment
    • by “pull down”
    • Can detect:
    • Sequence changes
    • Copy number (UPD/LOH)
    ASXL1 NF1 CBL NPM1 CEBPA NRAS CSF1R RUNX1 DNMT3A TET2 FLT3 WT1 IDH1 EZH2 IDH2 KIT JAK2 KDM6A KRAS TP53 MLL PTPN11 BRAF IKZF1 HPRT1 PAX5 PIK3CA UGT1A1 CYP2D6 TLR4 EGFR XRCC2 PTEN
  • Selective sequencing example : an AML toolkit   Type A (Transcription Factor) Type B (DNA modification) Type C (Signal transduction) PROGNOSIS AML1 NPM1 DNMT3A R882C FLT3-TKD Intermediate AML2   TET2 KRAS K117N Intermediate AML3 CEBPA   NRAS G12D Intermediate AML4 NPM1 IDH1 R132H FLT3-TKD Favourable AML5 ASXL1   KRAS G12D Poor AML6   IDH2 R172K   Poor
  • Plasma DNA Cancer Normal tissues DNA with tumour-specific mutation Slide courtesy of Dr Peter Campbell
  • Tumour-specific rearrangements Individual Breast Cancer Genome 1 st round PCR Nested real-time PCR Chr20 Chr10
  • Relapsing breast cancer Slide courtesy of Dr Peter Campbell 0.1 0.01 1 0.05 0.5 Undiluted patient plasma 1:10 1:100 1:1000 1:10,000 1:100,000 Normal Water 0 5 10 15 20 25 30 35 40 Cycles of real-time PCR Intensity Non-rearranged genomic region 0.1 0.01 1 0.05 0.5 0 5 10 15 20 25 30 35 40 Cycles of real-time PCR Intensity Tumour-specific rearrangement
  • Serial measurements 150 Months after diagnosis Estimated tumour DNA / mL serum (pg) 25 50 75 100 125 Undetectable Detectable at limit of sensitivity 6 7 8 9 10 11 12 13 14 15 16 17 5 Rearrangement 1 Rearrangement 2 First-line chemotherapy Second-line Paclitaxel CT scan: Localised deposits around T9-10 Chemotherapy: CT scan: Widespread soft-tissue metastases Slide courtesy of Dr Peter Campbell
    • Multiple biomarker MRD
    • Methylomics
    • Transcriptomics (RNAseq)
    • Cancer screening / Biomarker assays
    Other applications of NGS
    • Today’s cancer diagnostic lab
    • The era of cancer genomics
    • Novel diagnostic applications
    • Introducing genomics to cancer diagnosis
    Overview
  • Hurdles to the introduction of diagnostic cancer genome sequencing Technology
    • Sample choice/compatibility FFPE, other
    • Cost $10,000/genome
    • Sample to sequence delay 8-10 days
    • Mutation calling Specificity / Sensitivity
    Laboratory
    • Sequencing equipment Choice/Cost
    • New personnel Bioinformaticians
    • Computer storage Petabytes (10 15 )
    • Education/training Pathologists, Clinicians
    Clinic
    • Clinical relevance/utility Evolving
    • Personal genomes/Ethics Being tested
  • Genome Campus Data storage & analysis Sulston Building Morgan Building Research Support Facility Data Centre European Bioinformatics Institute
  • Training of pathologists
    • Core training in genomics
    • New sub-specialty e.g. Molecular Pathology?
    • Impact on other aspects of training
    • How will the training be delivered?
    • Training/role of laboratory scientists
    • Keeping control of the agenda
  • Diagnostic reporting of genomic data
    • Communicating the cancer genome to the clinician
      • Diagnosis
      • Recurrently mutated genes
      • Non-recurrent/private mutations / pathways
      • Prognostic relevance
      • Therapeutic relevance
      • Pharmacogenomics
      • Constitutional genome
      • Mutational signatures
      • Summary / Imagery
  • Implications for cancer classification Cellular origin Morphology Differentiation/grading Mutations: Diagnosis Prognosis Treatment Unified Classification?
  • Acute Myeloid Leukaemia – a paradigm of evolving classification Morphology Single entity Various 1950s         Morphology & cytochemistry M0-M7 FAB 1976         Morphology, AML with recurrent cytogenetic translocations WHO 2002 Immunophenotyping & AML with multilineage dysplasia     Cytogenetics   AML, therapy related       AML not otherwise categorized             Morphology, AML with recurrent genetic abberations WHO 2008 Immunophenotyping , Provisional entity: AML with mutated NPM1     Cytogenetics   & Provisional entity: AML with mutated CEBPA     Genetics  Otherwise as 2002    
  • Will there be a paradigm shift ? ?
  • Summary
    • The advent of cancer genomics is changing cancer medicine
    • Changes will transform cancer diagnosis and the role of pathologists
    • Pathologists need to understand what is coming in order to lead and formulate the future for cancer diagnosis