APPLICATIONS OF MOLECULAR DIAGNOSTICS IN CLINICAL CHEMISTRY - PowerPoint Presentation

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APPLICATIONS OF MOLECULAR DIAGNOSTICS IN CLINICAL CHEMISTRY

BY

LT.COL ZUJAJA HINA HAROON

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Molecular diagnostics is the fastest growing segment of the diagnostics industry

~$34 billion world-wide market

6-8% annual growthNew discoveries and technology platforms are leading to the development of more and increasingly sophisticated testsDNA sequencingExpression microarraysArray CGHDetection technology/test platformsMajority of the innovation and discovery takes place in Universities

Molecular Diagnostics is a Rapidly Expanding Field

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Molecular Pathology

A Universal Discipline of Laboratory Medicine

HEMATO

PATHOLOGY

INFECTIOUS

DISEASE

PHARMACO –

GENOMICS

GENETIC

DISEASE

MOLECULAR

ONCOLOGY

Molecular

Pathology

I.D TESTING &

FORENSICS

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Applications of Molecular Diagnostics in Clinical

Chemistry

Oncology – Solid Tumor and HematologicDiagnosisPrognosis

Predict response to therapyMonitor residual disease

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Applications of Molecular Diagnostics in Clinical

Chemistry

Genetics (inherited disease)Diagnosis of: Single gene disorders

Complex polygenic disorders Chromosomal disorders

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Applications of Molecular Diagnostics in Clinical

Chemistry

Identity TestingDetermining familial relationships

Bone marrow engraftment analysisGVHD monitoring

Laboratory specimen identificationForensics

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Applications of Molecular Diagnostics in Clinical

Chemistry

PharmacogenomicsDrug metabolism

Determine drug dosage

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Hematologic Malignancies

Quantitative BCR/ABL

BCR/ABL1 Kinase Mutation AnalysisFLT3 Gene MutationNPM1 MutationCEBPA MutationKIT D816V Mutationt(15;17) PML/RARA Translocationt(14;18) IGH/BCL2 Translocation B Cell (IGH) Gene RearrangementT Cell Gamma (TRG) Gene RearrangementJAK2 V617F Mutation Detection

JAK2 Exon 12 Mutations (March 2010)

Oncology – Solid Tumor and Hematologic

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Solid Tumors

PAX/FOXO1 Translocation, Alveolar Rhabdomyosarcoma

EWSR1/WT1 Translocation, DSRT EWS/FLI1, EWS/ERG Translocations, Ewing Sarcoma SYT/SSX Translocation, Synovial SarcomaEWS/ATF1 Translocation, Clear Cell SarcomaMicrosatellite Instability AnalysisKRAS MutationBRAF V600E MutationKIT Mutation in GISTKIT Mutation in MelanomaHER2 FISH, Breast cancerUroVysion FISH, Bladder cancerOncology – Solid Tumor and Hematologic

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Diagnosis

Prognosis

Predict response to therapyMonitor residual disease

Molecular Diagnostics - Oncology

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Diagnosis – Ewing Sarcoma

cDNA

Reverse

transcription

EWSR1/FLI1

Extract

RNA

~ 1 billion copies of target cDNA

PCR

EWSR1

primer

FLI1

primer

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Detection

PCR products

Capillary electrophoresis

GAPDH control

EWSR1/FLI1 (Type 1)

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Diagnosis

Prognosis

Predict response to therapyMonitor residual disease

Molecular Diagnostics - Oncology

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Prognostic Molecular Testing in AML – The UM Experience

Tests per Month

2004

2005

2006

2007

2008

2009

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Diagnosis

Prognosis

Predict response to therapyMonitor residual disease

Molecular Diagnostics - Oncology

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Predict Response to Therapy: KIT Mutations in Melanoma

Hodi

FS et al.,

2008 J Clin Oncol 26(12):2046

(4 wk)

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DNA Sequencing For KIT Mutation

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Diagnosis

Prognosis

Predict response to therapyMonitor residual disease

Molecular Diagnostics - Oncology

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Cystoscopy - Negative

FISH -

PositiveCase 4History of CIS (bladder), Post Resection

Recurrence of CIS, BCG therapy, Monitoring

Monitoring Residual Disease – UroVysion FISH

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Cystic Fibrosis Carrier Screening

Apolipoprotein E Genotyping

Hereditary Hemochromatosis Mutation Detection Factor V Leiden Mutation DetectionMethylenetetrahydrofolate Reductase C677T MutationProthrombin 20210 MutationUGT1A1 Promoter GenotypingGenetics

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Factor V Leiden Mutation Detection

&

Prothrombin 20210 Mutation

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normal for FII

normal for FVLheterozygous for FIIHomozygous for FVL

FII

FVL

heterozygous for FII

heterozygous for FVL

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Hereditary Hemochromatosis Mutation Detection

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Bone Marrow Transplant Engraftment AnalysisDNA Profiling

Identity Testing

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Bone Marrow Transplant Engraftment Analysis

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DNA Profiling

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The process of Automated DNA profiling involves several stages.

These are:

Item ExaminationTubestarQiagen ExtractionPre-PCRAmplificationPost-PCRCapillary ElectrophoresisInterpretationDNA Profiling

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A DNA Profile

D3

VWAD16

D2

Amelo

D19

D8

D21

D18

THO

FGA

Size Standards

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Pharmacogenomics

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The study of how variations in the human genome affect the response to medicationsTailoring treatments to unique genetic profiles

What is Pharmacogenomics (

PGx)?

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Warfarin Sensitivity Analysis

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Single Nucleotide Polymorphisms (SNPs)

A key to human variability

DNA sequence variation at a single nucleotide that may alter the function of the encoded protein

Functional but

altered

protein

Functional protein

Polymorphisms are common and

contribute to common diseases and influence our response to medications

*

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Pyrosequencing in Dr. Eby’s and McLeod’s Labs:

Thanks to Sharon, Christi, Rhonda

Pyrogram of VKORC1 6853 heterozygote subject. The sequence for nucleotides is: G/C G A G C G.Frequency of VKORC1-6853C allele: 37% in white and 24% in black pts.

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Cytochrome P450 (CYP) 2C9Vitamin K Epoxide Reductase, Complex 1 (VKORC1)

Derivation of pharmacogenetics-based warfarin dosing

Validation of pharmacogenetics-based warfarin dosingOverview

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CYP2C9Metabolizes >90% of active Warfarin

Variant alleles associated with increased sensitivity to Warfarin (CYP2C9*2, *3)

Vitamin K epoxide reductase (VKOR)Inhibited by WarfarinImportant for replenishment of vitamin KVariant alleles of VKORC1 gene associated with altered response to Warfarin Genes important for Warfarin Pharmacogenetics

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Individual Variability in Warfarin Dose

Warfarin maintenance dose (mg/day)

SENSITIVITY

CYP2C9

coding SNPs

RESISTANCE

VKORC1

coding

SNPs

0.5

5

15

Frequency

Common

VKORC1

non-coding SNPs

Adapted from Rettie and Tai, Molecular Interventions 2006

(*3/*3)

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Compared with other laboratory disciplines, the state of the art in quality control (QC) practices for molecular diagnostic tests has fallen behind

Challenges:

new and rapidly evolving technologieshigh expectations of accuracy for once-in-a-lifetime genetic tests lack of quality control materials lack of quantitative test system outputs almost daily appearance of new genetic test targets.QC for Molecular Diagnostics

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Molecular diagnostics VS usual methods

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In other words, we are dealing with a lot of unknowns. We don't have regulatory specifications for quality requirements, Which also means we don't know how well these tests should perform. So it's hard to determine the actual error rate of these tests.

We also have a lot of market forces that work against common control materials. Manufacturers have incentives to create unique testing products, ones that aren't comparable to competitor products. They also have incentives to avoid determining or releasing information on error rates.

QC for Molecular Diagnostics

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Regulations are still catching up with molecular diagnostic testing. While the laboratory director has the same responsibilities (basically, all

the responsibility) for adequate quality of molecular diagnostics, the tools for assessment are primitive.

Quality control for molecular diagnostics is going to grow in importance in the coming years. We hope QC in molecular diagnostics will catch up with the growth in the use of the testing, and before a crisis occurs.QC for Molecular Diagnostics