1. Multi-dimensional Genomic Profiling of Acute Leukemias Characterized by MLL gene rearrangements Eunice S. Wang MD (Medicine) and Norma J. Nowak PhD (Cancer Genetics) Roswell Park Cancer Institute

2. 2008 Estimated US Cancer Cases* *Excludes basal and squamous cell skin cancers and in situ carcinomas except urinary bladder. Source: American Cancer Society, 2008. Men 745,180 Women 692,000 26%Breast 14%Lung & bronchus 10%Colon & rectum 6%Uterine corpus 4%Non-Hodgkin lymphoma 4% Thyroid 4%Melanoma of skin 3%Ovary 3%Kidney & renal pelvis 3%Leukemia 23%All Other Sites Prostate25% Lung & bronchus15% Colon & rectum10% Urinary bladder7% Non-Hodgkin5% lymphoma Melanoma of skin5% Kidney & renal pelvis4% Oral cavity3% Leukemia 3% Pancreas3% All Other Sites20%

3. Jordan C et al. N Engl J Med 2006;355:1253-1261 Blood cancers are normal blood cells gone “bad”

4. Acute Lymphocytic Leukemia (ALL) Clonal expansion of immature lymphoblasts

5. Acute Lymphocytic Leukemia (ALL) Blood cancer characterized by uncontrolled growth of immature abnormal white blood cells. The most common pediatric cancer with 75% of cases occurring in children < 6 yrs old Comprise up to 20% of acute leukemia in adults Cure rates following standard chemotherapy and bone marrow transplantation depend on the particular genetics of the ALL cells.

6. Diagnosis of blood cancers Cancer cell 1. Appearance (morphology) Protein 2. Abnormal protein expression (flow cytometry) (histochemistry) Protein 3.Chromosome abnormalities (cytogenetics) X X 4. Specific gene mutations

7. Chromosomes, genes, and DNA 22 chromosomes, X and Y

8. Mixed Lineage Leukemia (MLL) Gene Rearrangement 4 and 11 11 and 19 Transfer of genes from chromosome 11 to another one

9. Acute leukemias associated with MLL gene rearrangements do poorly with therapy No MLL MLL positive

10. MLL fusion proteins function to modify DNA transcription and induce leukemia

11. Need for new therapies for ALL

12. Multi-dimensional genomic profiling of acute leukemia characterized by MLL gene rearrangements The goal of this project is to identify and characterize the key genes and pathways underlying MLL-associated leukemia for future translation into novel clinical therapies.

13. Multi-dimensional genomic profiling Using cutting-edge multi-dimensional genomic profiling, we will perform in depth genetic analyses of MLL leukemia patient cells from individuals treated at RPCI: - Change in expression of all known human genes - Submicroscopic DNA gains/losses (aCGH) - Alterations in non-coding RNAs regulating genes (microRNA or miRs) - Single nucleotide DNA sequence variations (SNPs) - Attachment of DNA methyl groups (methylation)

14. Studies of Copy Number Variation (CNV) Differences in the number of copies of large segments of DNA have been found between individuals at many sites of the human genome. Studies of CNV have revealed that: –CNV can drive genetic disease; –parts of the genome are polymorphic for copy number. Cataloging of these variants in disease and in reference populations is underway, using both sequencing and microarray hybridization.

15. Traditional CGH Array CGH Control Test Control Test Picture courtesy of Brynn Levy, Ph.D.

16. Metaphase CGH to aCGH Oligonucleotide arrays (25 – 85mer) BAC arrays (175Kb) G-Banding Diagnostics Discovery Resolution Coverage >10Mb 100% >175Kb 96% >40Kb <1%

17. Single Nucleotide Polymorphisms

18. Illumina HumanCytoSNP-12 BeadChip All Markers299,671 Markers within 10kb of a Gene148,666 nsSNPs3,480 MHC / ADME / Indel SNPs761 / 2,382 / 0 Spacing kb (Mean/Median)9.6 / 6.2 SNPs in Mitochondrial Genome0 X-chr / Y-chr / PAR Loci15,056 / 2,679 / 10 DNA Input Requirement200 ng Number of samples per BeadChip12 ContentNumber

19. A Wide Spectrum of Aberrations Can Be Detected A C B D

20. MicroRNAs (Mirs) Single-stranded RNAs capable of regulating gene expression in cells

21. Illumina MicroRNA Profiling Human –1146 human mature miRNA sequences are targeted Content –Sanger miRBase v12 (September 2008 Release) and recent literature –Targeted Sample –200ng total RNA input –R2 >0.99 even for FFPE samples

22. 24 MLL Leukemia Patient Samples Frozen Samples RNA & DNA extraction -aCGH -microRNA -SNPs -methylation

23. Pts 24 MLL Patient CNV/SNP Data

24. 24 MLL Patient MicroArray Data

25. Statistical Analysis Results of these arrays will be -Compared with normal cells (when available) from the same patients -Subjected to statistical analysis using specialized software in order to determine which corresponding target genes and pathways represent potential therapeutic targets.

26. Jacquie Hirsch for ALL Foundation Research