1. INTERPRETING GENETIC MUTATIONAL DATA FOR CLINICAL ONCOLOGY Ben Ho Park, M.D., Ph.D. Associate Professor of Oncology Johns Hopkins University May 2014

2. DNA, Genes and Cancer  DNA is a cellular “blueprint” using four different bases: Adenine, Cytosine, Guanine, Thymine (A,C,G,T)  DNA  RNA  Protein  Two copies (generally) of DNA in each cell

3. Why look for mutated genes?  Because most human cancers arise from somatic mutations, this makes a physical change in the cancer cell that is different from normal cells, i.e. good target for therapy  Also because mutations leading to cancer are somatic, it can in theory be a marker of cancer and used for detection and possible prognosis  Sequencing involves amplifying via polymerase chain reaction (PCR) a “coding” region of DNA, and then determining the base pairs that are present, e.g. A, C, G, T using a sequencing machine

4. EXONs Primers How is sequencing done?

5. Sequencing “trace”

6. Fig. 1. The genomic landscapes of colorectal and breast cancers. The genomes of human cancers contain mutations in many different genes at low frequency (hills) and only a few genes that are mutated at high frequency (mountains). From Wood et al. Science 2007. The problem

7. Next Generation Sequencing  Next generation sequencing involves cutting DNA into small fragments and sequencing these in parallel. With enough “coverage” the entire genome can be sequenced quickly

8. Next generation sequencing

9. Workflow (future)  Biopsy of lesion  NGS of tumor to find “actionable” mutations  Mutations with FDA approved drugs  Mutations with FDA approved drugs for other cancer types  Mutations that might match to a clinical trial  Convene tumor board to discuss results  Enter report with list of recommendations/suggestions prioritized by level of evidence  All within 28 days 9

10. Challenges to overcome  Need for fresh biopsy or not  May be able to use blood as liquid biopsy  Ability to sequence with acceptable time and costs  Commercial vs. in house  Levels of evidence for mutations, drugs, clinical trials…need for expert curation  Tumor board with broad expertise: oncology, genetics, molecular pathology, NGS, ethics, legal, patient advocate  Genetic Alterations In Tumors With Actionable Yields (GAITWAY) tumor board

11. Current Challenge with Metastatic Disease: Intratumoral Mutational Heterogeneity Gerlinger et al, NEJM 366:883, 2012

12. Exon Sequencing of ptDNA to test Heterogeneity of Metastatic Breast Cancer 12  Whole exome sequencing of ptDNA in metastatic disease is possible (Nature 2013)  Can sequencing of ptDNA yield additional mutations than sequencing of a single metastatic site? In collaboration with Foundation Medicine, we will obtain matched metastatic biopsies and plasma samples and compare mutational spectra of one metastasis to ptDNA. Not randomized

13. Exon Sequencing of ptDNA to test Heterogeneity of Metastatic Breast Cancer 13  J12129 protocol for 40 patients, IMAGE Study: Individualized Molecular Analyses Guide Efforts in Breast Cancer  Triple negative metastatic breast cancer patients eligible.  Biopsy of one lesion performed and sent for targeted gene sequencing (FoundationOne).  Blood collected and ptDNA sequenced for same Foundation One panel; results compared

14. Conclusions  Molecular genetics and genomic approaches have increased our understanding of breast cancer and our ability to make clinical decisions regarding risk, therapies and prognosis  The use of both germline and tumor DNA in particular has revealed new insights into the complexities of breast cancer that will pave the way for future studies towards individualizing treatment plans for each patient