1. CLL Research Consortium FISH studies, Core C June, 2005 NCI Submission

2. Specific Aims Goal 1: Standardize CRC FISH for clinical trials. Goal 1: Standardize CRC FISH for clinical trials. Goal 3: Use FISH testing for CRC clinical trials Goal 3: Use FISH testing for CRC clinical trials Goal 2: Utilize FISH to study the genetic origin and progression of CLL. Goal 2: Utilize FISH to study the genetic origin and progression of CLL.

3. Goal 1: Standardize interphase FISH for CLL among CRC institutions Rationale/hypothesis: 1. This is a new initiative for the CRC. 2. FISH methods for CLL vary among CRC laboratories. 3. National standards for CLL FISH studies not available. 4. Seven CRC institutions will work together to standardize and validate FISH studies for CLL. 5. Without standardized FISH results will be difficult to interpret in various clinical trials and biological studies.

4. CRC team to standardize FISH testing 1.Gordon Dewald PhD, Mayo Clinic, Project leader 2.Marie Dell'Aquila PhD, University of California at San Diego 3.Paola Dal Cin PhD, Brigham and Women's Hospital 4.Lynne Abruzzo MD, MD Anderson 5.Constance Griffin MD, Johns Hopkins University 6.Nyla Heerema PhD, Ohio State University 7.Prasad Kodura PhD, Long Island Jewish Medical Center Laura Rassenti PhD, (CRC Tissue Core Director) Andrew Greaves (CRC Bioinformatics Director) Donna Neuberg (CRC Biostatistician) Carlo Croce, MD (CRC Project 1, PI)

5. Process to standardize FISH for the CRC In Part 1, CRC cytogeneticists will complete a questionnaire to explore variations in equipment, methods, and experience with FISH for CLL. In Part 2, CRC cytogeneticists will study a set of blood and/or bone marrows representing known normal and abnormal FISH patterns. In Part 3, CRC cytogeneticists will evaluate a series of patients to study various FISH anomalies and different percentages of abnormal nuclei. In Part 4, each CRC approved laboratory will test two unknown specimens once per year to help ensure they maintain proficiency. Results of each Part will be analyzed by the CRC statistical center and summarized in writing by one of the cytogeneticists. This information will be shared with other participants. Discrepancies will be resolved via teleconference, email and/or other forms of communication.

6. Rationale/hypothesis: Goal 2: Utilization of FISH to study the genetic origin and progression of CLL Detection of chromosome anomalies by interphase FISH is an important new technology to study CLL. Genetic basis for the origin of CLL is unknown and chromosome anomalies described so far are associated with chromosome evolution and disease progression. The largest panel FISH assays for interphase analysis in clinical practice today look at only 12 chromosomal loci. There is considerable need to continue the search for the genetic basis for the origin and progression of CLL.

7. Outline of work 1. Utilize various approaches to detect significant genes that have not yet been associated with CLL. a. Periodically analyze CRC conventional cytogenetic database for clues to critical chromosomal loci. b. Work closely with Dr. Carlo Croce (Project 1 PI) to study candidate genes that he discovers. c. Utilize novel mitogens to stimulate CLL cells and perform conventional chromosome analyses. d. Use various DNA chip techniques to look for critical chromosomal loci in patients with CLL. e. Based on results of all these approaches, Mayo will build suitable FISH probes for chromosomal anomalies of potential significance in CLL.

8. Outline of work con’t 2. Begun work with Dr. Croce a. Dr. Croce provided DNA and sequence information for miR15 and miR16. a. Dr. Dewald will try to build FISH probes for miR15 and miR16 b. This will be challenging as miR15 and miR16 contain < 1 kb.. c. We may need to expand the probes by adding adjacent DNA or use multiplex ligation dependant probe amplification methods to study miR15 and miR16. d. We hope to define the precise critical deletion region of chromosome 13 associated with CLL b. Dr. Croce is studying abnormalities of chromosome 6 in CLL. a. DNA probes for 6q will be used in interphase FISH studies. b. These results will be compared to other FISH probes for chromosome 6 to look for the critical deletion region.

9. Outline of work con’t 3. Study 10 patients with CLL using various forms of DNA chip analyses. a. SNP chip to detect gains and losses of genes linked within 100 Kb of any SNP. b. CGH DNA array analysis for gains and/or losses of tumor suppressor genes and oncogenes. c. CGH array analysis to detect gains and losses of DNA segments at 1000 Kb intervals throughout the genome. d. This work will all be done at Mayo Clinic. They have the equipment and capability to analyze the data.

10. Outline of work con’t 4. Applications of novel FISH probes a. Any novel FISH probes with value for routine clinical applications will be validated by the tissue core and included with FISH studies for patients enrolled in clinical trials. b. New FISH probes will be used to perform retrospective studies of the biology of CLL by using stored CRC blood and/or bone marrow. c. We expect that novel FISH probes would increase the proportion of patients with CLL in whom chromosome anomalies are detected. These novel probes may also provide clues to new targets for CLL therapy.