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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
Applications of Molecular Diagnostics in Clinical Chemistry
Genetics (inherited disease)- Diagnosis of:
Single gene disordersComplex polygenic disordersChromosomal disorders
Applications of Molecular Diagnostics in Clinical Chemistry
Identity Testing- Determining familial relationships- Bone marrow engraftment analysis- GVHD monitoring- Laboratory specimen identification- Forensics
Applications of Molecular Diagnostics in Clinical Chemistry
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
Predict response to therapy
Monitor residual disease
Molecular Diagnostics - Oncology
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
Monitor residual disease
Molecular Diagnostics - Oncology
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
Predict response to therapy
Monitor residual disease
Molecular Diagnostics - Oncology
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
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.
QC for Molecular Diagnostics
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