“What I Do”

1. “What I Do” Bryan Betz PhD Clinical Assistant Professor Technical Director Molecular Diagnostics Laboratory

2. Molecular Diagnostics / Molecular Pathology Application of molecular biology techniques and knowledge to clinical laboratory diagnosis • Infectious disease • Inherited disease • Hematopathology • Solid Tumors • Identity Testing • Forensics • Pharmacogenomics Is and will continue to revolutionize the way in which diseases are diagnosed and therapeutics are employed

3. Molecular Pathology A Universal Discipline of Laboratory Medicine INFECTIOUS DISEASE FORENSICS HEMATO PATHOLOGY Molecular Pathology IDENTITY TESTING SOLID TUMORS GENETIC DISEASE

4. 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

5. UM Molecular Diagnostics Test Volume and TATJan 2002 – Dec 2009

6. Applications of Molecular Diagnostics in Clinical Laboratory Medicine • Infectious Disease • Qualitative and quantitative detection • Microbial identification • Drug resistance • Identity Testing • Determining familial relationships • Bone marrow engraftment analysis • GVHD monitoring • Laboratory specimen identification • Forensics • Genetics (inherited disease) • Diagnosis of: • Single gene disorders • Complex polygenic disorders • Chromosomal disorders • Oncology – Solid Tumor and Hematologic • Diagnosis • Prognosis • Predict response to therapy • Monitor residual disease • Pharmacogenomics • Drug metabolism • Determine drug dosage

7. UM Molecular Diagnostics Test Menu 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 Genetics/Pharacogenomics 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 Warfarin Sensitivity Analysis Identity Testing Bone Marrow Transplant Engraftment Analysis

8. My Day to Day Activities • Clinical • • Direct the development, validation, and implementation of new molecular tests • • Technical oversight and trouble shooting of existing tests • • Maintain quality control and quality assurance standards • • Daily review of test data • Teaching • • MGP fellows and block E residents • Research/Academic • • Novel molecular diagnostic tests and technologies • • Collaborations with AP and HP faculty

9. New Tests Since July 2007 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 Tumor 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 Genetics/Pharacogenomics 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 Warfarin Sensitivity Analysis Identity Testing Bone Marrow Transplant Engraftment Analysis

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

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

12. 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. Prognosis - Normal Karyotype AML CEBPAmut NPM1mutFLT3-ITDneg Other genotypes Schlenk RF et al. NEJM 358(18):1909, 2008

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

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

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

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

19. Monitoring Residual Disease – UroVysion FISH Malignant Urothelial Cell Normal Urothelial Cell CEP 3 LSI 9p21 CEP 7 CEP 17

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

21. Thanks!