Leumeta TM : (Plasma-based testing): New Paradigm for Clinical Testing in Leukemia & Lymphoma

1. LeumetaTM: (Plasma-based testing): New Paradigm for Clinical Testing in Leukemia & Lymphoma Maher Albitar, MD Quest Diagnostics, Nichols Institute San Juan Capistrano, CA 92690 (949)728-4784 Maher.x.Albitar@Questdiagnostics.com

2. Cancer is Not a Physiologic Process • High uncontrolled proliferation rate • High relative turnover of cells • Heterogeneity within the tumor cells

3. Circulating Cellular Debris Contains Fingerprints of Tumor Cells Bone Marrow Plasma

4. Hypothesis • Plasma (or serum) contains the “fingerprints” of cancer cells • Plasma (or serum) is enriched by tumor-specific DNA, RNA and cellular proteins. • Every cell-based test can also be performed using plasma (LeumetaTM).

5. Why Hematologic Diseases? • Neoplastic cells “swim” in plasma/serum. • Hematologic diseases can be patchy • Reticuloendothlial system is frequently damaged in hematologic diseases

6. Example of Heterogeneity in Bone Marrow in a Patients with CML

7. Marrow Fibrosis and Poor Aspiration

8. Plasma-Based (LeumetaTM ) DNA Tests • Mutations • Chromosomal translocations • Chromosomal aberrations (deletions, amplification) • Minimal Residual disease

9. Plasma is More Sensitive in Detecting JAK2 Mutation and Homozygous/Hemizygous Patients Patient #1 Patient #2 Forward Cells Reverse Forward Plasma Reverse

10. Patients with Homozygous/Hemizygous JAK2 as Detected Using Plasma Have More Aggressive Disease

11. Plasma is More Sensitive than Cells in Detecting TP53 Mutations

12. Plasma is More Sensitive in Detecting Ras Oncogene Mutations

13. Detection of 5q Deletion in the Plasma of a Patients with AML Using LOH

14. Plasma is more Sensitive than Peripheral Blood Cells in Detecting NPM1 Mutations in AML

15. Plasma-Based (LeumetaTM ) RNA Tests • Mutations • Chromosomal translocations • Expression and quantification • Minimal Residual disease

16. Plasma is More Sensitive than Cells in Detecting ABL Kinase Mutations 931T>C (F311L) 758A>T (Y253F) 944C>T (T315I) F PB R F BM R F Plasma R

17. Plasma is More Sensitive in Detecting T315I Mutation Using ASO Assay

18. Plasma is More Sensitive in Monitoring BCR-ABL Transcript in Patients with CML Plasma

19. Plasma-Based (LeumetaTM ) Protein Tests • Protein levels • Phosphorylation and modification • Enzymatic activity • Tumor markers • Diagnosis and classification

20. Membrane Attachment of CD20 and CD52 Carbohydrate CD52 GPI Anchor CD20 Cell Membrane N Cell Membrane C

21. High levels of cCD20 in NHL but not in Hodgkin’s Disease

22. High Levels of cCD52 in NHL but not in Hodgkin’s Disease

23. Shorter Survival in Patients with Lymphoma and High Levels of cCD20

24. Levels of cCD52 (23n Cut-off) and b2M (3.5 cut-off) Are Useful in Stratifying Patients with CLL

25. High Levels of cCD20 in CLL Correlate with Short Survival

26. Cell-Free Circulating Stem Cell Marker (CD34) in Patients with Leukemia

27. Cell-Free Circulating CD4 as a Biomarker in Patients with Myelodysplastic Syndrome

28. Plasma Proteasome Activity as a Biomarker in Patients with Acute Myeloid Leukemia

29. Plasma BCR-ABL Protein in Patients with CML (Pre-Rx)

30. Plasma Levels of Phospho-BCR-ABL Protein in Patients with CML (Pre-Rx)

31. Currently Available Leumeta Tests PlasmaCells 16031X/ 16029X abl Kinase Domain Mutation in CML 17679X/ 14991X t(11;14) bcl-1/JH(MCL) Real Time PCR 17690X/ 15007X t(14;18) bcl-2/JH Real Time PCR 17702X/ 15480X IgVH Mutation Analysis 16101X/ 16102X JAK2 Mutation (V617F) 16104X/ 16106X c-kit Mutation Analysis 16119X/ 14868X B-cell gene rearrangement, Qual., PCR 16118X/ 16005X B-cell gene rearrangement, Quant., PCR 16127X/ 16128X ras Mutation Analysis, Plasma-based, 17862X/ 15930X T-Cell Receptor (TCR) Gene Rearrangement, Qualitative PCR 17861X/ 16025X T-Cell Receptor (TCR) Gene Rearrangement, Quantitative PCR 17853X/ 15052X bcr/abl Gene Rearrangement, Quantitative PCR, Plasma-based 19783X/ 19782X ABL T3151 Mutation in CML 16159X/ 16158X NPM(Exon 12) Mutation Analysis

32. Leumeta™ - Summary New Paradigm in Testing • Plasma contains the fingerprint of tumor cell (Leukemia/lymphoma) • Plasma is frequently more sensitive than cell samples (less dilution by normal cells). • Plasma-based testing reduce the need for biopsies • Most of Plasma-based assays are quantitative, easier to standardize and more objective. • There is a need for the development of more plasma-based tests to more significantly reduce the need for biopsies.

33. References 1.Manshouri T, Do KA, Wang X et al. Circulating CD20 is detectable in the plasma of patients with chronic lymphocytic leukemia and is of prognostic significance. Blood. 101(7), 2507-2513 (2003). 2.Giles FJ, Vose JM, Do KA et al. Circulating CD20 and CD52 in patients with non-Hodgkin's lymphoma or Hodgkin's disease. Br J Haematol. 123(5), 850-857 (2003). 3.Albitar M, Do KA, Johnson MM et al. Free circulating soluble CD52 as a tumor marker in chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies. Cancer. 101(5), 999-1008 (2004). 4.Jilani I, Kantarjian H, Faraji H et al. Measurement of free circulating Bcr-Abl fusion protein and its phosphorylation in patients with chronic myeloid leukemia. Blood. abstract 2006. 5.Rogers A, Joe Y, Dey A et al. Relative increase in leukemia-specific DNA in peripheral blood plasma from patients with acute myeloid leukemia and myelodysplasia. Blood. 103(7), 2799-2801 (2004). 6.Ahmed M, Giles F, Joe Y et al. Use of plasma DNA in detection of loss of heterozygosity in patients with multiple myeloma. Eur J Haematol. 71(3), 174-178 (2003). 7.Ma W, Kantarjian H, Verstovsek S et al. Hemizygous/homozygous and heterozygous JAK2 mutation detected in plasma of patients with myeloproliferative diseases: correlation with clinical behaviour. Br J Haematol. 134(3), 341-343 (2006). 8.Ma W, Tseng R, Gorre M et al. Plasma RNA as an alternative to cells for monitoring molecular response in patients with chronic myeloid leukemia. Haematologica. 92(2), 170-175 (2007). 9.Ma W, Kantarjian H, Jilani I et al. Heterogeneity in detecting Abl kinase mutations and better sensitivity using circulating plasma RNA. Leukemia. 20(11), 1989-1991 (2006). 10.Ma W, Jilani I, Gorre M et al. Plasma as a source of mRNA for determining IgVH mutation status in patients with chronic lymphocytic leukaemia. Br J Haematol. 133(6), 690-692 (2006).