CLL Research CLL Research Consortium Consortium

FISH studies, Core CFISH studies, Core C

June, 2005June, 2005

NCI SubmissionNCI Submission

Specific AimsSpecific Aims Goal 1: Standardize CRC FISH Goal 1: Standardize CRC FISH

for clinical trials. for clinical trials. Goal 3: Use FISH testing for Goal 3: Use FISH testing for

CRC clinical trialsCRC clinical trials Goal 2: Utilize FISH to study Goal 2: Utilize FISH to study

the genetic origin and the genetic origin and progression of CLL.progression of CLL.

Goal 1: Standardize interphase FISH Goal 1: Standardize interphase FISH for CLL among CRC institutionsfor 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.

CRC team to CRC team to standardize FISH testingstandardize FISH testing

1. Gordon Dewald PhD, Mayo Clinic, Project leader2. Marie Dell'Aquila PhD, University of California at San Diego3. Paola Dal Cin PhD, Brigham and Women's Hospital4. Lynne Abruzzo MD, MD Anderson5. Constance Griffin MD, Johns Hopkins University6. Nyla Heerema PhD, Ohio State University7. 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)

Process to standardize Process to standardize FISH for the CRCFISH 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.

Rationale/hypothesis:

Goal 2: Utilization of FISH to Goal 2: Utilization of FISH to study the genetic origin and progression of CLLstudy 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.

Outline of workOutline of work1.1. Utilize various approaches to detect significant genes Utilize various approaches to detect significant genes

that have not yet been associated with CLL. that have not yet been associated with CLL.

a.a. Periodically analyze CRC conventional cytogenetic Periodically analyze CRC conventional cytogenetic database for clues to critical chromosomal loci. database for clues to critical chromosomal loci.

b.b. Work closely with Dr. Carlo Croce (Project 1 PI) to study Work closely with Dr. Carlo Croce (Project 1 PI) to study candidate genes that he discovers. candidate genes that he discovers.

c.c. Utilize novel mitogens to stimulate CLL cells and perform Utilize novel mitogens to stimulate CLL cells and perform conventional chromosome analyses. conventional chromosome analyses.

d.d. Use various DNA chip techniques to look for critical Use various DNA chip techniques to look for critical chromosomal loci in patients with CLL. chromosomal loci in patients with CLL.

e.e. Based on results of all these approaches, Mayo will build Based on results of all these approaches, Mayo will build suitable FISH probes for chromosomal anomalies of suitable FISH probes for chromosomal anomalies of potential significance in CLL. potential significance in CLL.

Outline of work con’tOutline of work con’t2.2. Begun work with Dr. Croce Begun work with Dr. Croce

a.a. Dr. Croce provided DNA and sequence information for miR15 and Dr. Croce provided DNA and sequence information for miR15 and miR16.miR16.

a.a. Dr. Dewald will try to build FISH probes for miR15 and miR16Dr. Dewald will try to build FISH probes for miR15 and miR16

b.b. This will be challenging as miR15 and miR16 contain < 1 kb. . This will be challenging as miR15 and miR16 contain < 1 kb. .

c.c. We may need to expand the probes by adding adjacent DNA or use We may need to expand the probes by adding adjacent DNA or use multiplex ligation dependant probe amplification methods to study multiplex ligation dependant probe amplification methods to study miR15 and miR16. miR15 and miR16.

d.d. We hope to define the precise critical deletion region of We hope to define the precise critical deletion region of chromosome 13 associated with CLLchromosome 13 associated with CLL

b.b. Dr. Croce is studying abnormalities of chromosome 6 in CLL. Dr. Croce is studying abnormalities of chromosome 6 in CLL.

a.a. DNA probes for 6q will be used in interphase FISH studies. DNA probes for 6q will be used in interphase FISH studies.

b.b. These results will be compared to other FISH probes for These results will be compared to other FISH probes for chromosome 6 to look for the critical deletion region.chromosome 6 to look for the critical deletion region.

Outline of work con’tOutline of work con’t3.3. Study 10 patients with CLL using various Study 10 patients with CLL using various

forms of DNA chip analyses. forms of DNA chip analyses.

a.a. SNP chip to detect gains and losses of genes SNP chip to detect gains and losses of genes linked within 100 Kb of any SNP. linked within 100 Kb of any SNP.

b.b. CGH DNA array analysis for gains and/or losses CGH DNA array analysis for gains and/or losses of tumor suppressor genes and oncogenes.of tumor suppressor genes and oncogenes.

c.c. CGH array analysis to detect gains and losses CGH array analysis to detect gains and losses of DNA segments at 1000 Kb intervals of DNA segments at 1000 Kb intervals throughout the genome.throughout the genome.

d.d. This work will all be done at Mayo Clinic. They This work will all be done at Mayo Clinic. They have the equipment and capability to analyze have the equipment and capability to analyze the data. the data.

Outline of work con’tOutline of work con’t4. 4. Applications of novel FISH probesApplications of novel FISH probes

a.a. Any novel FISH probes with value for routine Any novel FISH probes with value for routine clinical applications will be validated by the tissue clinical applications will be validated by the tissue core and included with FISH studies for patients core and included with FISH studies for patients enrolled in clinical trials. enrolled in clinical trials.

b.b. New FISH probes will be used to perform New FISH probes will be used to perform retrospective studies of the biology of CLL by retrospective studies of the biology of CLL by using stored CRC blood and/or bone marrow. using stored CRC blood and/or bone marrow.

c.c. We expect that novel FISH probes would increase We expect that novel FISH probes would increase the proportion of patients with CLL in whom the proportion of patients with CLL in whom chromosome anomalies are detected. These chromosome anomalies are detected. These novel probes may also provide clues to new novel probes may also provide clues to new targets for CLL therapy.targets for CLL therapy.