For our patients and our population

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

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

16 May 2013

For our patients and our population

Now that the genome is done…

June 25, 2000

For our patients and our population

Genome

Structure

Genome

Biology

Biology of

Disease

Advancing

Medicine

Improving

Healthcare

1990-2003

HGP

2004-2010

2011-2020

Beyond

2020

Charting a course for genomic

medicine from base pairs to bedside

Green, Guyer, NHGRI Nature 2011

For our patients and our population

Pharmaco-

genomics

Personal Genomics on the Rise

Personalised

Medicine

Personal

Genomics

Carrier

Screening

Familial

Cancer

Prenatal

Testing

Inherited

Disease

Cardio-

myopathies

Molecular

Oncology

Pediatric

Intellectual

Disability

For our patients and our population

Outline

• Diagnostics today and current trends

• What technology (panels vs exomes)

• Implementation challenges:

Bioinformatics/ IT

Clinical annotation/ interpretation

For our patients and our population

Diagnostic Genetic Testing in Oz

Largely funded at State level with variable patient co-

contribution

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

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

SA Pathology: Genetic & Molecular Pathology

>25,000 genetic tests per annum, >100 genes routinely

Sanger sequencing of >50 genes

Current costs:

Sequenom single hotspot: $50

Sanger single amplicon: $100

Sanger full ORF: $250 - $1,700

Send-aways: $40 - $4,000 ($600 avg)

For our patients and our population

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

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

NEXT-GENERATION SEQUENCING

Improve cost-effectiveness

Increase throughput and automation

Improve diagnostic rate

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

What technology …. panels vs exomes?

For our patients and our population

SEQUENOM

Mass spectro-metry

Parallel Amplificat.

Highly multiplexed

PCR

Hybrid (capture + amplificat.)

Baits to pull down

targets

Hotspots

Full Genes

Throughput

Cost

$ incl. seq. $50 ~$200 $500 $600+ $2500

AMPLICON CAPTURE FLUIDIGM HALOPLEX

For our patients and our population

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

Manageable data amount (storage, processing)

Variants and copy number at once (!AmpliSeq panels)

Alignment/Variant calling on benchtop sequencer (Accreditation!)

PANELS

Cost and ease of adaptation

Address current needs

EXOMES

Complex disorders

Whole-genomes …

For our patients and our population

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)

Off-the-shelf panels

For our patients and our population

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

Off-the-shelf panels

For our patients and our population

So …. with all these tools, why is it still hard?

For our patients and our population

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

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

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

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

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

1. Bioinformatics and HPC

For our patients and our population

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

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

Commercial Software

Product Secondary Analysis

Tertiary Analysis

CLC Genomics Workbench x x

NextGENe (Softgenetics) x x

Geneticist Assistant (Softgenetics) x

MiSeq Reporter (Illumina) x

Variant Studio (Illumina) x

Ion Torrent Suite (Life T.) x

Ion Reporter (Life Techn.) x

SureCall (Agilent) x (x)

Alamut HT Cartagenia BenchLabs NGS DNAStart (Lasergene) BioBase Genome Trax …

For our patients and our population

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

2. Clinical annotation, interpretation, and reporting

For our patients and our population

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

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

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

TP53 mutation?

Polymorphic site T>C

Homopolymer (6 Cs)

For our patients and our population

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

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

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

Standards for annotation, interpretation and

reporting

RCPA benchmarking datasets

ClinVar

International Collaboration for Clinical Genomics (ICCG)

HGSA, NIH …

For our patients and our population

Diagnostics is becoming interdisciplinary and personal …

For our patients and our population

Biotech

companies Diagnostic

Labs

IT companies

Software

companies

Cluster computing

Cloud computing

Encryption

Clinicians

Researchers

Regulatory

bodies

Guidelines

Accreditation

Professional

societies Guidelines

Training/ Registration

Testing/

Interpretation

Reporting

R&D

Gene/Variant discovery

Test requests

Clinical

management

Pharma

Drug discovery

Clinical trials

An interdisciplinary effort

For our patients and our population

Gene

Discovery

Prevalence

Screen

Diagnostic

Sequencing

Clinical

Validation

New disease gene

Functional studies

Frequency in population

Target population

Implications for disease management

Diagnosis: PPV – NPV

Treatment – Prognosis

Assay development

Patient sequencing

Disease

Management

The research/diagnostic feedback:

SOP

For our patients and our population

Gene

Discovery

Prevalence

Screen

Diagnostic

Sequencing

Clinical

Validation

New disease gene

Functional studies

Frequency in population

Target population

Implications for disease management

Diagnosis: PPV – NPV

Treatment – Prognosis

Assay development

Patient sequencing

Disease

Management

The research/diagnostic feedback:

Single-gene

sequencing

STOP

For our patients and our population

Gene

Discovery

Prevalence

Screen

Diagnostic

Sequencing

Clinical

Validation

New disease gene

Functional studies

Frequency in population

Target population

Implications for disease management

Diagnosis: PPV – NPV

Treatment – Prognosis

Assay development

Patient sequencing

Disease

Management Panels/ Whole-exomes

Build large patient cohorts

Genotype – phenotype r/ships

The research/diagnostic feedback:

For our patients and our population

Gene

Discovery

Prevalence

Screen

Diagnostic

Sequencing

Clinical

Validation

New disease gene

Functional studies

Frequency in population

Target population

Implications for disease management

Diagnosis: PPV – NPV

Treatment – Prognosis

Assay development

Patient sequencing

Disease

Management Panels/ Whole-exomes

Build large patient cohorts

Genotype – phenotype r/ships

The research/diagnostic feedback:

Diagnostics is

becoming

very personal

For our patients and our population

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

Dr Janice Fletcher Dr Glenice Cheetham

Prof Hamish Scott Dr Scott Grist

Sharon Bain Dr Kathie Friend

Mark Holloway Dr Rosa Katsikeros

Karen Ambler Dr Chris Hahn

Dr Connie Caruso Evelyn Douglas

Dr Melanie Hayes Kathie Cox

Rachel Hall Kristian Brion

Amanda Tirimacco

Acknowledgements

SA Pathology and

Clinical Services

ACRF SA Cancer

Genome Facility

Joel Geoghegan

Dr Andreas Schreiber

Mark Van der Hoek

Ming Lin

David Lawrence

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

Prof Jozef Gecz

Dr Mark Corbett

Prof David Adelson

The University of

Adelaide

For our patients and our population

Thank You!

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

BioInfoSummer 2013 Program

http://www.maths.adelaide.edu.au/biosummer2013/program.html