1. APPLICATIONS OF MOLECULAR DIAGNOSTICS IN CLINICAL CHEMISTRYBY
LT.COL ZUJAJA HINA HAROON

2. Molecular Diagnostics is a Rapidly Expanding Field
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

3. A Universal Discipline of Laboratory Medicine
Molecular Pathology
A Universal Discipline of Laboratory Medicine
INFECTIOUS
DISEASE
I.D TESTING &
FORENSICS
HEMATO
PATHOLOGY
Molecular
Pathology
PHARMACO –
GENOMICS
MOLECULAR
ONCOLOGY
GENETIC
DISEASE

4. Applications of Molecular Diagnostics in Clinical Chemistry
Oncology – Solid Tumor and Hematologic
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease

5. Applications of Molecular Diagnostics in Clinical Chemistry
Genetics (inherited disease)
Diagnosis of:
Single gene disorders
Complex polygenic disorders
Chromosomal disorders

6. Applications of Molecular Diagnostics in Clinical Chemistry
Identity Testing
Determining familial relationships
Bone marrow engraftment analysis
GVHD monitoring
Laboratory specimen identification
Forensics

7. Applications of Molecular Diagnostics in Clinical Chemistry
Pharmacogenomics
Drug metabolism
Determine drug dosage

8. Oncology – Solid Tumor and Hematologic
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)

9. 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

10. Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease

11. Diagnosis – Ewing Sarcoma
Extract
RNA
cDNA
Reverse
transcription
EWSR1/FLI1
EWSR1
primer
FLI1
PCR
~ 1 billion copies of target cDNA

12. Capillary electrophoresis
PCR products
Detection
Capillary electrophoresis
EWSR1/FLI1 (Type 1)
GAPDH control

13. Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease

14. Prognostic Molecular Testing in AML – The UM Experience
Tests per Month
2004
2005
2006
2007
2008
2009

15. Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease

16. Predict Response to Therapy: KIT Mutations in Melanoma
(4 wk)
Hodi FS et al., 2008 J Clin Oncol 26(12):2046

17. DNA Sequencing For KIT Mutation

18. Molecular Diagnostics - Oncology
Diagnosis
Prognosis
Predict response to therapy
Monitor residual disease

19. Monitoring Residual Disease – UroVysion FISH
Case 4
History of CIS (bladder), Post Resection
Recurrence of CIS, BCG therapy, Monitoring
Cystoscopy - Negative
FISH - Positive

20. Genetics Cystic Fibrosis Carrier Screening Apolipoprotein E Genotyping
Hereditary Hemochromatosis Mutation Detection
Factor V Leiden Mutation Detection
Methylenetetrahydrofolate Reductase C677T Mutation
Prothrombin Mutation
UGT1A1 Promoter Genotyping

21. Factor V Leiden Mutation Detection & Prothrombin 20210 Mutation

22. FII FVL
normal for FII
normal for FVL
heterozygous for FII
Homozygous for FVL
heterozygous for FII
heterozygous for FVL

23. Hereditary Hemochromatosis Mutation Detection

25. Identity Testing Bone Marrow Transplant Engraftment Analysis
DNA Profiling

26. Bone Marrow Transplant Engraftment Analysis

28. DNA Profiling

29. DNA Profiling Item Examination Tubestar Qiagen Extraction Pre-PCR
The process of Automated DNA profiling involves several stages.
These are:
Item Examination
Tubestar
Qiagen Extraction
Pre-PCR
Amplification
Post-PCR
Capillary Electrophoresis
Interpretation

30. A DNA Profile D3 VWA D16 D2 D8 D21 D18 Amelo D19 THO FGA
Size Standards

31. Pharmacogenomics

32. What is Pharmacogenomics (PGx)?
The study of how variations in the human genome affect the response to medications
Tailoring treatments to unique genetic profiles

33. Warfarin Sensitivity Analysis

34. 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 protein
Functional but altered protein *
Single nucleotide variation==Ex)10 nucleotide sequence of DNA fragment; two Fragments might differ b/c they may have a C or a T
Polymorphisms are common and contribute to common diseases and influence our response to medications

35. Pyrosequencing in Dr. Eby’s and McLeod’s Labs: Thanks to Sharon, Christi, Rhonda
Frequency of VKORC1-6853C allele: 37% in white and 24% in black pts.
Pyrogram of VKORC heterozygote subject.
The sequence for nucleotides is: G/C G A G C G.

36. Overview Cytochrome P450 (CYP) 2C9
Vitamin K Epoxide Reductase, Complex 1 (VKORC1)
Derivation of pharmacogenetics-based warfarin dosing
Validation of pharmacogenetics-based warfarin dosing

37. Genes important for Warfarin Pharmacogenetics
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

38. Common VKORC1 non-coding SNPs
Individual Variability in Warfarin Dose
SENSITIVITY
CYP2C9 coding
SNPs
RESISTANCE
VKORC1 coding
SNPs
Frequency
Common VKORC1 non-coding SNPs
Amy
(*3/*3)
0.5 5 15
Warfarin maintenance dose (mg/day)
Adapted from Rettie and Tai, Molecular Interventions 2006

39. QC for Molecular Diagnostics
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.

40. Molecular diagnostics VS usual methods

41. QC for Molecular Diagnostics
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.

42. 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.