BY LT.COL ZUJAJA HINA HAROON. Molecular diagnostics is the fastest growing segment of the diagnostics industry ~$34 billion world-wide market 6-8%

APPLICATIONS OF MOLECULAR DIAGNOSTICS

IN CLINICAL CHEMISTRYBY

LT.COL ZUJAJA HINA HAROON

Molecular diagnostics is the fastest growing segment of the diagnostics industry

~$34 billion world-wide market

6-8% annual growth

New discoveries and technology platforms are leading to the development of more and increasingly sophisticated tests◦ DNA sequencing◦ Expression microarrays◦ Array CGH◦ Detection technology/test

platforms

Majority of the innovation and discovery takes place in Universities

Molecular Diagnostics is a Rapidly Expanding Field

Molecular PathologyA Universal Discipline of Laboratory Medicine

HEMATOPATHOLOGY

INFECTIOUSDISEASE

PHARMACO –

GENOMICS

GENETIC

DISEASE

MOLECULARONCOLOGY

MolecularPathology

I.D TESTING &FORENSICS

Applications of Molecular Diagnostics in Clinical Chemistry

Oncology – Solid Tumor and Hematologic- Diagnosis- Prognosis- Predict response to therapy- Monitor residual disease


Genetics (inherited disease)- Diagnosis of:

Single gene disordersComplex polygenic disordersChromosomal disorders


Identity Testing- Determining familial relationships- Bone marrow engraftment analysis- GVHD monitoring- Laboratory specimen identification- Forensics


Pharmacogenomics- Drug metabolism- Determine drug dosage

Hematologic Malignancies Quantitative BCR/ABL BCR/ABL1 Kinase Mutation Analysis FLT3 Gene Mutation NPM1 Mutation CEBPA Mutation KIT D816V Mutation t(15;17) PML/RARA Translocation t(14;18) IGH/BCL2 Translocation B Cell (IGH) Gene Rearrangement T Cell Gamma (TRG) Gene Rearrangement JAK2 V617F Mutation Detection JAK2 Exon 12 Mutations (March 2010)

Oncology – Solid Tumor and Hematologic

Solid Tumors PAX/FOXO1 Translocation, Alveolar Rhabdomyosarcoma EWSR1/WT1 Translocation, DSRT EWS/FLI1, EWS/ERG Translocations, Ewing Sarcoma SYT/SSX Translocation, Synovial Sarcoma EWS/ATF1 Translocation, Clear Cell Sarcoma Microsatellite Instability Analysis KRAS Mutation BRAF V600E Mutation KIT Mutation in GIST KIT Mutation in Melanoma HER2 FISH, Breast cancer UroVysion FISH, Bladder cancer

Oncology – Solid Tumor and Hematologic

Diagnosis

Prognosis

Predict response to therapy

Monitor residual disease

Molecular Diagnostics - Oncology

Diagnosis – Ewing Sarcoma

cDNAReverse

transcription

EWSR1/FLI1

ExtractRNA

~ 1 billion copies of target cDNA

PCR

EWSR1primer

FLI1primer

Detection

PCR products

Capillary electrophoresis

GAPDH control

EWSR1/FLI1 (Type 1)

Diagnosis

Prognosis




0

10

20

30

40

50

60

70

80

90

J an- 04 J an- 05 J an- 06 J an- 07 J an- 08 J an- 09

TotalFLT3KITNPM1CEBPA

Prognostic Molecular Testing in AML – The UM Experience

Test

s per

Month

2004 2005 2006 2007 2008 2009

Diagnosis

Prognosis




Predict Response to Therapy: KIT Mutations in Melanoma

Hodi FS et al., 2008 J Clin Oncol 26(12):2046

(4 wk)

DNA Sequencing For KIT Mutation

Diagnosis

Prognosis




Cystoscopy - Negative FISH - Positive

Case 4History of CIS (bladder), Post ResectionRecurrence of CIS, BCG therapy, Monitoring

Monitoring Residual Disease – UroVysion FISH

Cystic Fibrosis Carrier Screening Apolipoprotein E Genotyping Hereditary Hemochromatosis Mutation

Detection Factor V Leiden Mutation Detection Methylenetetrahydrofolate Reductase

C677T Mutation Prothrombin 20210 Mutation UGT1A1 Promoter Genotyping

Genetics

Factor V Leiden Mutation Detection&Prothrombin 20210 Mutation

normal for FII normal for FVL

heterozygous for FIIHomozygous for FVL

FII FVL

heterozygous for FIIheterozygous for FVL

Hereditary Hemochromatosis Mutation Detection

Bone Marrow Transplant Engraftment Analysis

DNA Profiling

Identity Testing

Bone Marrow Transplant Engraftment Analysis

DNA Profiling

The process of Automated DNA profiling involves several stages.

These are: Item Examination Tubestar Qiagen Extraction Pre-PCR Amplification Post-PCR Capillary Electrophoresis Interpretation

DNA Profiling

A DNA Profile

D3 VWA D16 D2

Amelo

D19

D8 D21 D18

THO FGA

Size Standards

Pharmacogenomics

The study of how variations in the human genome affect the response to medications

Tailoring treatments to unique genetic profiles

What is Pharmacogenomics (PGx)?

Warfarin Sensitivity Analysis

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 proteinFunctional protein

Polymorphisms are common and contribute to common diseases and influence our response to medications

*

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.

Cytochrome P450 (CYP) 2C9 Vitamin K Epoxide Reductase, Complex 1

(VKORC1) Derivation of pharmacogenetics-based

warfarin dosing Validation of pharmacogenetics-based

warfarin dosing

Overview

CYP2C9◦ Metabolizes >90% of active Warfarin ◦ Variant alleles associated with increased

sensitivity to Warfarin (CYP2C9*2, *3) Vitamin K epoxide reductase (VKOR)

◦ Inhibited by Warfarin◦ Important for replenishment of vitamin K◦ Variant alleles of VKORC1 gene associated with

altered response to Warfarin

Genes important for Warfarin Pharmacogenetics

Individual Variability in Warfarin Dose

Warfarin maintenance dose (mg/day)

SENSITIVITY

CYP2C9 coding SNPs

RESISTANCE

VKORC1 coding SNPs

0.5 5 15

Fre

qu

en

cy

Common VKORC1 non-coding SNPs

Adapted from Rettie and Tai, Molecular Interventions 2006

(*3/*3)

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 technologies high 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

http://james.westgard.com/the_westgard_rules/2009/01/qc-for-molecular-diagnostics.html

Molecular diagnostics VS usual methods

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.




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.




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BY LT.COL ZUJAJA HINA HAROON. Molecular diagnostics is the fastest growing segment of the diagnostics industry ~$34 billion world-wide market 6-8%