1. Challenges and Considerations in Linking Adult and Pediatric Leukemias David G. Poplack M.D. Texas Children’s Cancer Center Baylor College of Medicine

2. Explore the relationship between pediatric and adult leukemias Explore the relationship between pediatric and adult leukemias Compelling biological evidence of similarities or differences useful in guiding the drug development process Compelling biological evidence of similarities or differences useful in guiding the drug development process

3. Are there defined subsets of adult and pediatric leukemias that share biologically relevant features that would mandate they be commonly studied? Are there defined subsets of adult and pediatric leukemias that share biologically relevant features that would mandate they be commonly studied?

4. Brief overview Brief overview Discuss rationale and promise Discuss rationale and promise Highlight Challenges Highlight Challenges Linking Adult and Pediatric Leukemias

5. Adult and Pediatric Leukemias Adult and Pediatric Leukemias ADULTS CHILDREN ADULTS CHILDREN Acute Lymphocytic11% 75-80% Acute Myeloid32%20% Chronic Myeloid15%<5% Chronic Lymphocytic26%N/A

6. Adults have a worse prognosis!

7. Survival in Acute Leukemias Survival in Acute Leukemias AdultsChildren Acute Lymphoid50-60%80-85% Acute Myeloid10-20%40-50%

8. ACUTE LEUKEMIAS A heterogeneous group of disorders!

9. Survival of Children with Acute Lymphoblastic Leukemia 80 60 40 20100 Years of Number of 1972-75 1972-75 02 4 8610 Years from Diagnosis % %SurvSurvIIVaVall%SurvSurvIIVaVallIl 1989-93 1983-89 1978-83 1970-72 1968-70 3,080 3,712 2,979 1,313 936 499 402 DIagnosisChildren 12,921 Total Number of Patients Treated: C C G Bleyer

10. Evidence for Heterogeneity in ALL Evidence for Heterogeneity in ALL Clinical Behavior Clinical Behavior Morphology Morphology Cytochemistry Cytochemistry Immunophenotype Immunophenotype Cytogenetics Cytogenetics Molecular Phenotyping Molecular Phenotyping

11. Impact of Heterogeneity on ALL Therapy “Staging” of patients according to risk; therapy “tailored” accordingly “Staging” of patients according to risk; therapy “tailored” accordingly Low risk patients -effective, less toxic therapies Low risk patients -effective, less toxic therapies High risk patients - more intensive treatment High risk patients - more intensive treatment Successful strategy Successful strategy Many prognostic criteria provided little biological insights Many prognostic criteria provided little biological insights

12. Technologic Advances Improvements in molecular cytogenetics and genomics Improvements in molecular cytogenetics and genomics A new paradigm A new paradigm More biologically relevant basis for classification More biologically relevant basis for classification Identifying molecular targets for therapy Identifying molecular targets for therapy

13. Cytogenetics Cytogenetics Giemsa banding Giemsa banding FISH FISH Molecular cytogenetics (PCR/RTPCR) Molecular cytogenetics (PCR/RTPCR) Spectral karyotyping (SKY) Spectral karyotyping (SKY) Comparative genomic hybridization (CGH) Comparative genomic hybridization (CGH)

14. Case 169 Pseudo-tetraploid with two 12-15 translocations

15. Molecular Geno- and Phenotyping Molecular Geno- and Phenotyping Southern, Northern, and Western Blots Southern, Northern, and Western Blots PCR technology (single, multiplex, real-time) PCR technology (single, multiplex, real-time) Differential RNA expression technologies (cDNA microarray, SSH) Differential RNA expression technologies (cDNA microarray, SSH) CGH and SKY on arrays CGH and SKY on arrays Single Nucleotide Polymorphism (SNPs) chips Single Nucleotide Polymorphism (SNPs) chips Proteomics Proteomics

16. Genome-wide analysis of gene expression using cDNA microarray technology

17. CDNA Array in ALL CD34 (7.4) CD34 (7.4) FL Cytokine receptor (5.6) FL Cytokine receptor (5.6) CD72 (4.6) CD72 (4.6) NADH ubiquinone oxidoreduct.15kDA protein(4.2) NADH ubiquinone oxidoreduct.15kDA protein(4.2) C-Fos (3.5) C-Fos (3.5) Multiple ESTs (3-4) Multiple ESTs (3-4) Insulin-like growth factor (2.3) Insulin-like growth factor (2.3) Elevated in t(4:11)

18. cDNA Array in ALL Membrane metallo-endopeptidase (enkephalinase/CALLA CD10) (18.6) Membrane metallo-endopeptidase (enkephalinase/CALLA CD10) (18.6) Arachidonate 5-lipoxygenase (8.5) Arachidonate 5-lipoxygenase (8.5) CGD gene (8.0) CGD gene (8.0) CD20 (7.0) CD20 (7.0) Hemoglobin beta (5.7) Hemoglobin beta (5.7) CD1c antigen (5.7) CD1c antigen (5.7) Human cerebellar degeneration associated protein (4.1) Human cerebellar degeneration associated protein (4.1) Elevated in pre-B ALL

19. What subsets of adult and pediatric leukemias might be appropriate for common therapeutic studies?

20. Acute Lymphoblastic Leukemia t (9;22)/Ph+ - BCR-ABL t (9;22)/Ph+ - BCR-ABL t (4;11) - MLL-AF4 - and other 11q23 abnormalities t (4;11) - MLL-AF4 - and other 11q23 abnormalities t (8;14) - B-cell t (8;14) - B-cell Hypodiploidy (<45 chromosomes) Hypodiploidy (<45 chromosomes) Hyperdiploidy (>50 chromosomes) Hyperdiploidy (>50 chromosomes) t (12;21) - TEL-AML1 t (12;21) - TEL-AML1

21. Acute Myeloblastic Leukemia Acute Myeloblastic Leukemia True De novo AML True De novo AML - t (15;17) and other APL variants - t (8;21), inversion 16, trisomy 8 Therapy related AML Therapy related AML - Monosomy 7, 5q-, and 11q23 MDS MDS

22. The Era of Molecular Targeting BCR-ABL t(9;22) - target for treatment of Ph 1 positive disease - e.g. STI 571 BCR-ABL t(9;22) - target for treatment of Ph 1 positive disease - e.g. STI 571 t(15;17) - APL treatment with ATRA t(15;17) - APL treatment with ATRA Provide compelling arguments: Provide compelling arguments: - confirm validity of targeting relevant molecular lesions - testing targeted agents in all of the relevant populations

23. Can we develop a general principle that might guide the identification of biological subsets that would be suitable for study both in adults and children?

24. Should be based on characteristics believed to be associated with the establishment, maintenance or progression of the malignant phenotype or linked to its development of resistance to specific treatments.

25. Examples of Biological Features that may be Shared by Adult and Pediatric leukemias Cytogenetic abnormalities Fusion proteins - downstream events Gene expression patterns Receptors - e.g. growth, anti-apoptosis Drug resistance genes Tumor suppressor genes

26. Challenges to the Design of Common Adult and Pediatric Leukemia Trials Biologic basis for selecting grouping may turn out to be incorrect Biologic basis for selecting grouping may turn out to be incorrect Toxicity Issues Toxicity Issues Small Patient Numbers (geographical accrual problems) Small Patient Numbers (geographical accrual problems) Impact of Transplantation Impact of Transplantation Requires extraordinary cooperation Requires extraordinary cooperation

27. Heterogeniety in location of actual fusions leads to heterogeneity in resulting proteins: Heterogeniety in location of actual fusions leads to heterogeneity in resulting proteins: t (9;22) resulting in p190 and p210 kinases Heterogeneity in mRNA expression: Heterogeneity in mRNA expression: t (1;19) +/- RNA expression t (1;19) +/- RNA expression Impact of other genetic changes: Concomitant aneuploidy Impact of other genetic changes: Concomitant aneuploidy Beyond the Breakpoint

28. Benefits of Designing and Implementing Common Adult and Pediatric Leukemia Trials New and improved therapies for our patients New and improved therapies for our patients Better understanding of the underlying biology of these diseases Better understanding of the underlying biology of these diseases

29. Development of common,comprehensive prospective biological studies should be encouraged Development of common,comprehensive prospective biological studies should be encouraged It is important to study both poor and good prognostic groups. It is important to study both poor and good prognostic groups.

30. Summary Summary Using current classification techniques there are adult and pediatric leukemia subsets in which joint biology and treatment protocols are justified Using current classification techniques there are adult and pediatric leukemia subsets in which joint biology and treatment protocols are justified Significant caveats to this strategy exist Significant caveats to this strategy exist Development of coordinated, prospective biological and classification studies of adult and pediatric leukemias, using the latest genomic technologies, is of critical importance. Development of coordinated, prospective biological and classification studies of adult and pediatric leukemias, using the latest genomic technologies, is of critical importance.

31. Cytogenetics in Adult and Pediatric ALL B Cell t(8:14) B Cell t(8:14) B Cell precursor B Cell precursor Hyperdiploidy (>50) TEL-AML1 t(12;21) E2A-PBX1 t(1;19) BCR-ABL t(9;22) MLL-AF4 t(4;11) Hypodiploidy (<45) T Cell T Cell 30-50@ 3 yr80-90@ 5 yr Unknown85-90@ 5 yr 20-40@ 3 yr70-80@ 5 yr <10@ 3 yr20-40@ 5 yr 10-20@ 3 yr10-35@ 5 yr 10@ 3 yr25-40@ 3 yr 50@ 3yr65-75@ 5yr ADULTSCHILDREN 50-55@ 4 yr75-85@ 5 yr

32. Cytogenetics in Adult & Pediatric AML Cytogenetics in Adult & Pediatric AML t(8;21) t(8;21) t(15;17) t(15;17) 11q23 11q23 Inv (16) Inv (16) -7 or del(7q) -7 or del(7q) -5 or del(5q) -5 or del(5q) AML1-ETO AML1-ETO PML-RARalpha,etc. PML-RARalpha,etc. MLL fusions MLL fusions CBFbetaMYH11 CBFbetaMYH11 Abnormality Molecular Lesion