Clinical implementation of next-generation sequencing for diagnostics - Karin Kassahn

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Advances in next-generation sequencing are enabling the use of genomics in the clinical management of patients. As the clinical applications of next-generation sequencing expand, clinical validation …

Advances in next-generation sequencing are enabling the use of genomics in the clinical management of patients. As the clinical applications of next-generation sequencing expand, clinical validation studies are becoming increasingly important. The challenges and successes of applying these novel technologies in a clinical setting are enormous. Over the past 6 months we have been working towards the implementation of targeted gene panels to stratify cancer patients for targeted therapies and the use of comprehensive gene panels and exome sequencing for the diagnosis of complex, inherited disorders. We are evaluating sample quality from various clinical lung biopsy methods and evaluate their suitability for molecular tumor profiling. We share some of the successes of translating these technologies to diagnostics and outline the remaining challenges. These include the establishment of standardised bioinformatics pipelines for variant calling and annotation, the clinical interpretation and reporting of genomic data, data management within established health IT systems, the ethical implications of offering more comprehensive gene testing than has ever been possible before, and the dynamic relationship between research and diagnostics. We discuss some of the approaches and solutions we have considered. The increasing demand for genomics in health management is challenging current funding models for genetic testing in Australia, but the potential benefits for patients are enormous and are driving implementation of these technologies across the country. In the years to come, genetic diagnosis enabled by NGS will likely become increasingly important in the clinic.

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  • 1. Clinical implementation of next-generation sequencing for diagnostics AMATA 2013 16th October 2013 Dr Karin Kassahn Head, Technology Advancement Unit, Genetic & Molecular Pathology SA Pathology For our patients and our population
  • 2. Tales from the clinical frontline… making personal genomics mainstream AMATA 2013 16th October 2013 Dr Karin Kassahn Head, Technology Advancement Unit, Genetic & Molecular Pathology SA Pathology For our patients and our population
  • 3. 16 May 2013 For our patients and our population
  • 4. Now that the genome is done… June 25, 2000 For our patients and our population
  • 5. Genome Structure 1990-2003 HGP Genome Biology Biology of Disease Advancing Medicine Charting a course for genomic medicine from base pairs to bedside Green, Guyer, NHGRI Nature 2011 2004-2010 2011-2020 Beyond 2020 Improving Healthcare For our patients and our population
  • 6. Personal Genomics on the Rise Intellectual Disability Molecular Oncology Familial Cancer Inherited Disease Pediatric Personal Genomics Cardiomyopathies Pharmacogenomics Personalised Medicine Prenatal Testing Carrier Screening For our patients and our population
  • 7. Outline • Diagnostics today and current trends • What technology (panels vs exomes) • Implementation challenges: Bioinformatics/ IT Clinical annotation/ interpretation For our patients and our population
  • 8. Diagnostic Genetic Testing in Oz  Largely funded at State level with variable patient cocontribution  Limited Medicare-rebate for genetic tests; inconsistent schedule  Specialised referral mechanisms (clinical geneticists)  National Referral Labs for key specialities  NPAAC guidelines – informed by RCPA and HGSA  accreditation via NATA For our patients and our population
  • 9. SA Pathology: Genetic & Molecular Pathology >25,000 genetic tests per annum, >100 genes routinely Sanger sequencing of >50 genes Diversity of methods: • • • • • • • • • • • FISH MLPA Fragment analysis/ AFLP GAP-PCR Long-range PCR qRT-PCR/ fluorescent PCR GC rich PCR Methylation PCR Primer extension assay Sanger sequencing Sequenom For our patients and our population
  • 10. SA Pathology: Genetic & Molecular Pathology >25,000 genetic tests per annum, >100 genes routinely Sanger sequencing of >50 genes Current costs: Sequenom single hotspot: Sanger single amplicon: Sanger full ORF: $50 $100 $250 - $1,700 Send-aways: $40 - $4,000 ($600 avg) For our patients and our population
  • 11. Current trends in diagnostics        Increasing testing demand (20% overall, “Angelina Jolie Effect”) Health budgets under significant pressure New markers of clinical utility (oncology, cardiology, …) “Diagnostic odyssey” (e.g. complex disorders, ID) Emerging Private Market (Wellness Genomes, Direct-to-Consumer) Internationalisation of testing Stronger integration between research and diagnostics For our patients and our population
  • 12. Current trends in diagnostics        Increasing testing demand (20% overall, “Angelina Jolie Effect”) Health budgets under significant pressure New markers of clinical utility (oncology, cardiology, …) NEXT-GENERATION SEQUENCING “Diagnostic odyssey” (e.g. complex disorders, ID) Improve cost-effectiveness EmergingIncreaseMarket (Wellness Genomes, Direct-to-Consumer) Private throughput and automation Improve diagnostic rate Internationalisation of testing Stronger integration between research and diagnostics CLINICAL GENOMICS Faster translation bench to bedside Community panels in diagnostics Every exome a research project Rapidly changing technologies For our patients and our population
  • 13. What technology …. panels vs exomes? For our patients and our population
  • 14. SEQUENOM FLUIDIGM AMPLICON HALOPLEX CAPTURE Mass spectrometry Parallel Amplificat. Highly multiplexed PCR Hybrid (capture + amplificat.) Baits to pull down targets $50 ~$200 $500 $600+ $2500 Hotspots Full Genes Throughput Cost $ incl. seq. For our patients and our population
  • 15. Panels vs Exomes: a diagnostic viewpoint Panels Exomes Cheaper per sample (reagents, labour) Unified laboratory workflow across applications Many clinical applications have few defined targets Complex disorders require broad analysis (ID >600 genes) Compatible with smaller batch sizes Ability to expand analysis to additional targets, if required PANELS Manageable data amount Cost and easeVariants and copy number at of adaptation (storage, processing) once (!AmpliSeq panels) Address current needs Alignment/Variant calling on benchtop sequencer EXOMES (Accreditation!) Complex disorders Whole-genomes … For our patients and our population
  • 16. Off-the-shelf panels Application Company/Product Design Somatic Cancer Sequenom Illumina TruSeq Ion AmpliSeq HaloPlex NimbleGen Mass spectrometry Amplicon Amplicon Capture/Amplicon Capture Familial Cancer Fluidigm Illumina TruSight Amplicon (BRCA1+2,TP53) Capture (94 genes) Inherited Disease Ion AmpliSeq Illumina TruSight Amplicon Capture (552 genes, 2.25Mb) Cardiomyopathy HaloPlex Illumina TruSight Capture/Amplicon Capture (46 genes) For our patients and our population
  • 17. Off-the-shelf panels Application Company/Product Design Somatic Cancer Sequenom Illumina TruSeq Ion AmpliSeq HaloPlex NimbleGen Mass spectrometry Amplicon Amplicon Capture/Amplicon Capture Familial Cancer Fluidigm Illumina TruSight Amplicon (BRCA1+2,TP53) Capture (94 genes) Inherited Disease Ion AmpliSeq Illumina TruSight Amplicon Capture (552 genes, 2.25Mb) Cardiomyopathy HaloPlex Illumina TruSight Capture/Amplicon Capture (46 genes) Custom Sequenom Fluidigm Illumina TruSeq Ion AmpliSeq HaloPlex Mass spectrometry Amplicon Amplicon Amplicon Capture/Amplicon For our patients and our population
  • 18. So …. with all these tools, why is it still hard? For our patients and our population
  • 19. Implementation challenges: Clinical Services perspective: • Integration into existing clinical management workflows • Patient information and consent (esp WES) • Controversy regarding incidental findings (ACMG guidelines) • Developing referral mechanisms (which doctor, which patients, which test) For our patients and our population
  • 20. Implementation challenges: Clinical Services perspective: • Integration into existing clinical management workflows • Patient information and consent (esp WES) • Controversy regarding incidental findings (ACMG guidelines) • Developing referral mechanisms (which doctor, which patients, which test) Strong interest in enhanced testing to support clinical services Some concerns regarding increase in workload For our patients and our population
  • 21. Implementation challenges: Diagnostic Laboratory perspective: • “Commitment issues” (rapidly changing technologies) • Unclear regulatory context • Funding! (R&D, new tests, Medicare?) • Bioinformatics and high performance computing • Defining useful quality metrics • Managing data quantity and VOUS • Standards for clinical annotation, interpretation and reporting • Staff training and change management For our patients and our population
  • 22. Implementation challenges: Diagnostic Laboratory perspective: • “Commitment issues” (rapidly changing technologies) • Unclear regulatory context • Funding! (R&D, new tests, Medicare?) • Bioinformatics and high performance computing • Defining useful quality metrics • Managing data quantity and VOUS • Standards for clinical annotation, interpretation and reporting • Staff training and change management Enthusiasm to adopt new technologies … BUT Concerns about increases in workload due to R&D and data quality Anxiety around change For our patients and our population
  • 23. Implementation challenges: Diagnostic Laboratory perspective: • “Commitment issues” (rapidly changing technologies) • Unclear regulatory context • Funding! (R&D, new tests, Medicare?) • Bioinformatics and high performance computing • Defining useful quality metrics • Managing data quantity and VOUS • Clinical annotation, interpretation and reporting • Staff training and change management For our patients and our population
  • 24. 1. Bioinformatics and HPC For our patients and our population
  • 25. Bioinformatics and HPC • Limited bioinformatics expertise in many diagnostic labs • Training of medical scientists in use of software/ NGS data For our patients and our population
  • 26. Bioinformatics and HPC • Limited bioinformatics expertise in many diagnostic labs • Training of medical scientists in use of software/ NGS data • Identifying commercial vs in-house software solution For our patients and our population
  • 27. Commercial Software Product Secondary Analysis Tertiary Analysis CLC Genomics Workbench x x NextGENe (Softgenetics) x x Geneticist Assistant (Softgenetics) MiSeq Reporter (Illumina) x x Variant Studio (Illumina) Ion Torrent Suite (Life T.) x x Ion Reporter (Life Techn.) SureCall (Agilent) x x Alamut HT Cartagenia BenchLabs NGS DNAStart (Lasergene) BioBase Genome Trax … For our patients and our population (x)
  • 28. Bioinformatics and HPC • • • • Limited bioinformatics expertise in many diagnostic labs Training of medical scientists in use of software/ NGS data Identifying commercial vs in-house software solution Integration with existing health IT infrastructure o Network speed and access o Data storage and management (across sites) o Specialised software requirements (64bit, RAM, dedicated server, …) • IT support for HPC vs outsourcing services • Negotiating service level agreements and compute requirements • Risk aversion (managing sensitive patient data) For our patients and our population
  • 29. 2. Clinical annotation, interpretation, and reporting For our patients and our population
  • 30. Example 1: Clear hit Primary lymphedema Localized fluid retention and tissue swelling caused by a compromised lymphatic system One affected child (affected mother not sequenced) Expect autosomal dominant 492 non-db SNP variants Heterozygous GJC2 mutation Previously described in large independent pedigree For our patients and our population
  • 31. Example 2: Multiple hits Allan-Herndon-Dudley syndrome? global developmental delay, sensorineural hearing loss, dystonic dyskinetic posturing, hypotonia, MRI white matter hypomyelination, hypothyroid with elevated T3. Previous diagnostic testing of MCT8 negative. One affected child (affected mother not sequenced) Expect X-linked 893 non-db SNP variants Homozyg. SLC16A2 mutation (splice site donor in intron) OMIM gene for Allan-Herndon-Dudley syndrome MITF: sensorineural deafness; highly pleiotropic POU1F1:pituitary hormone deficiency - hypothyroidism For our patients and our population
  • 32. Example 3: Further tests Primary recessive microcephaly? Fanconia syndrome? Cytomegalovirus infection, anaemia, alpha thalassaemia, microcephaly, developmental delay, epilepsy, hypertension, moderate conductive hearing loss Single affected child Expect autosomal de novo or recessive Array CGH normal >500 non-db SNP variants Het in FANCA (splice site donor) – pathogenicity? For our patients and our population
  • 33. TP53 mutation? Polymorphic site T>C Homopolymer (6 Cs) For our patients and our population
  • 34. Example 4: Unresolved Lipofuscinosis? Tay-Sachs disease? Ataxia telang.? progressive encephalopathy, scleral telangiectasia, axonal spheroids, and membrane-bound inclusions in skin. Two affected siblings Expect autosomal recessive 479 non-db SNP variants No candidate gene hit Compound het in SLC5A6 (metabolic transporter protein; frameshift + missense muts) Single het in SLC22A1 (spastic paraplegia, neurodegeneration) For our patients and our population
  • 35. Emerging issues Variants of unknown significance (VOUS) time-consuming How to prove causality? How many and which annotation resources constitute “best practise”? Need for automation of variant filtering and prioritization For our patients and our population
  • 36. Emerging issues Variants of unknown significance (VOUS) time-consuming How to prove causality? How many and which annotation resources constitute “best practise”? Standards for annotation, interpretation and Need for automation of variant filtering and prioritization reporting RCPA benchmarking datasets ClinVar International Collaboration for Clinical Genomics (ICCG) HGSA, NIH … For our patients and our population
  • 37. Diagnostics is becoming interdisciplinary and personal … For our patients and our population
  • 38. An interdisciplinary effort Guidelines Accreditation Regulatory bodies Professional societies Clinicians Test requests Clinical management Pharma Drug discovery Clinical trials Diagnostic Labs Testing/ Interpretation Reporting Researchers Guidelines Training/ Registration Biotech companies Software companies IT companies R&D Gene/Variant discovery For our patients and our population Cluster computing Cloud computing Encryption
  • 39. The research/diagnostic feedback: Gene Discovery Disease Management New disease gene Functional studies SOP Assay Diagnostic development Sequencing Patient sequencing Prevalence Screen Clinical Validation Frequency in population Target population Implications for disease management Diagnosis: PPV – NPV Treatment – Prognosis For our patients and our population
  • 40. The research/diagnostic feedback: Disease Management Single-gene sequencing Gene Discovery New disease gene Functional studies STOP Assay Diagnostic development Sequencing Patient sequencing Prevalence Screen Clinical Validation Frequency in population Target population Implications for disease management Diagnosis: PPV – NPV Treatment – Prognosis For our patients and our population
  • 41. The research/diagnostic feedback: Disease Management Panels/ Wholeexomes Gene Discovery Assay Diagnostic development Sequencing Patient sequencing Clinical Validation Build large New disease gene patient cohorts Functional studies Genotype – phenotype r/ships Frequency in Prevalence population Screen Target population Implications for disease management Diagnosis: PPV – NPV Treatment – Prognosis For our patients and our population
  • 42. The research/diagnostic feedback: Disease Management Panels/ Wholeexomes Gene Discovery Assay Diagnostic development Sequencing Patient sequencing Diagnostics is becoming very personal Clinical Validation Build large New disease gene patient cohorts Functional studies Genotype – phenotype r/ships Frequency in Prevalence population Screen Target population Implications for disease management Diagnosis: PPV – NPV Treatment – Prognosis For our patients and our population
  • 43. Conclusions • Diagnostics is changing rapidly • Moving goal posts • Implementation challenges (bioinformatics/ IT; clinical annotation and interpretation) • Clinical validation studies and health economics… • Genetic test requests becoming a consultation • Up-skilling (scientists in medicine and clinicians in technology) • Interdisciplinary effort and case review teams • Motivation is clear: improved pick up rate and personalised medicine For our patients and our population
  • 44. Acknowledgements SA Pathology and Clinical Services Dr Janice Fletcher Prof Hamish Scott Sharon Bain Mark Holloway Karen Ambler Dr Connie Caruso Dr Melanie Hayes Rachel Hall Amanda Tirimacco ACRF SA Cancer Genome Facility Joel Geoghegan Dr Andreas Schreiber Mark Van der Hoek Ming Lin David Lawrence The University of Adelaide Dr Glenice Cheetham Dr Scott Grist Dr Kathie Friend Dr Rosa Katsikeros Dr Chris Hahn Evelyn Douglas Kathie Cox Kristian Brion Prof Jozef Gecz Dr Mark Corbett Prof David Adelson For our patients and our population Prof Michael Brown A/Prof Chris Barnett Prof Eric Hahn Prof A Ruszkiewicz Prof Graeme Suthers Dr Drago Bratkovic Dr Dylan Mordaunt Dr Melody Caramins
  • 45. Thank You! http://www.maths.adelaide.edu.au/biosummer2013/program.html BioInfoSummer 2013 Program Monday, 2 December Introduction to biology and bioinformatics Tuesday, 3 December Evolutionary Biology Wednesday, 4 December Systems Biology Thursday, 5 December Next Generation Sequencing Friday, 6 December Programming for bioinformatics For our patients and our population