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BY LT.COL ZUJAJA HINA HAROON Molecular diagnostics is the ~$34 billion world-wide market 6-8% annual growth fastest growing segment of the diagnostics industry 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 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 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) 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 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 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 D3 VWA D8 Amelo D19 D16 D21 THO Size Standards D2 D18 FGA 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 * 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. Cytochrome P450 (CYP) 2C9 Vitamin K Epoxide Reductase, Complex 1 (VKORC1) Derivation of pharmacogenetics-based warfarin dosing Validation of pharmacogenetics-based warfarin dosing 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 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. 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.