Download APPLICATIONS OF MOLECULAR DIAGNOSTICS IN CLINICAL

BY
LT.COL ZUJAJA HINA HAROON
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Molecular diagnostics is the
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~$34 billion world-wide market
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6-8% annual growth
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fastest growing segment of the
diagnostics industry
New discoveries and technology
platforms are leading to the
development of more and
increasingly sophisticated tests
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DNA sequencing
Expression microarrays
Array CGH
Detection technology/test
platforms
Majority of the innovation and
discovery takes place in
Universities
Molecular Pathology
A Universal Discipline of Laboratory Medicine
INFECTIOUS
DISEASE
HEMATO
PATHOLOGY
I.D TESTING &
FORENSICS
Molecular
Pathology
PHARMACO –
GENOMICS
MOLECULAR
ONCOLOGY
GENETIC
DISEASE
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 disorders
Complex polygenic
disorders
Chromosomal 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
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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)
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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 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
Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease
Diagnosis – Ewing Sarcoma
Extract
RNA
EWSR1/FLI1
Reverse
transcription
cDNA
EWSR1
primer
FLI1
primer
PCR
~ 1 billion copies of target cDNA
PCR products
Detection
Capillary electrophoresis
EWSR1/FLI1 (Type 1)
GAPDH control
Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease
Prognostic Molecular Testing in AML – The UM
Experience
90
80
Tests per Month
70
60
Total
FLT3
KIT
NPM1
CEBPA
50
40
30
20
10
0
Jan-04
2004 Jan-052005 Jan-062006
2007 Jan-082008
Jan-07
2009
Jan-09
Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease
Predict Response to Therapy: KIT Mutations in
Melanoma
(4 wk)
Hodi FS et al., 2008 J Clin Oncol 26(12):2046
Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease
Monitoring Residual Disease – UroVysion FISH
Case 4
History of CIS (bladder), Post Resection
Recurrence of CIS, BCG therapy, Monitoring
Cystoscopy - Negative
FISH - Positive
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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
FII
FVL
normal for FII
normal for FVL
heterozygous for FII
Homozygous for FVL
heterozygous for FII
heterozygous for FVL
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Bone Marrow Transplant Engraftment Analysis
DNA Profiling
The process of Automated DNA profiling involves
several stages.
These are:
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Item Examination
Tubestar
Qiagen Extraction
Pre-PCR
Amplification
Post-PCR
Capillary Electrophoresis
Interpretation
D3
VWA
D8
Amelo
D19
D16
D21
THO
Size Standards
D2
D18
FGA
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The study of how variations in the human
genome affect the response to medications
Tailoring treatments to unique genetic
profiles
DNA sequence variation at a single nucleotide that
may alter the function of the encoded protein
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Functional protein
Functional but altered protein
Polymorphisms are common and contribute to common diseases
and influence our response to medications
Frequency of VKORC1-6853C allele:
37% in white and 24% in black pts.
Pyrogram of VKORC1 6853 heterozygote subject.
The sequence for nucleotides is: G/C G A G C G.
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Cytochrome P450 (CYP) 2C9
Vitamin K Epoxide Reductase, Complex 1
(VKORC1)
Derivation of pharmacogenetics-based
warfarin dosing
Validation of pharmacogenetics-based
warfarin dosing
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CYP2C9
◦ Metabolizes >90% of active Warfarin
◦ Variant alleles associated with increased sensitivity
to Warfarin (CYP2C9*2, *3)
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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
Frequency
Individual Variability in
Warfarin Dose
SENSITIVITY
RESISTANCE
CYP2C9 coding
VKORC1 coding
SNPs
Common VKORC1
non-coding SNPs
SNPs
(*3/*3)
0.5
5
15
Warfarin maintenance dose (mg/day)
Adapted from Rettie and Tai, Molecular Interventions 2006
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-alifetime genetic tests
 lack of quality control materials
 lack of quantitative test system outputs
 almost daily appearance of new genetic test
targets.
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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.
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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.