1. CRC Core C FISH STUDIES Progress report (Old & New Aims) Dec 3, 2009 CRC Meeting ASH09

2. Specific Aims, 2005   Aim 1: Standardize CRC FISH for clinical trials   Aim 2: Validate CRC cytogenetic data for use in clinical studies   Aim 3: Use FISH to study 1000 stored specimens (clinical & prognostic study)   Aim 4: Study the genetics of CLL

3. Aim 1: Standardize CRC FISH Smoley et al., Validation of CLL FISH Panel Scoring by Members of the Chronic Lymphocytic Leukemia Research Consortium. ASH 2008 and Manuscript in preparation (Dan Van Dyke), will be done by 1/1/2010 The CRC cytogenetics labs used varying probe sets, number of cells scored, normal cut-offs, & scoring criteria After working together for several years including a workshop: 1) CRC labs now use similar probe sets including CCND1/IGH (to rule out MCL) 2) CRC labs are now much more comparable in FISH scoring criteria based on very good agreement in the last proficiency test

4. Aim 2: Validate FISH data   Entire FISH database has been QC’d !!   All CRC labs use same format to submit cytogenetic data   New FISH data are now actively validated in real time by standardized process

5. Aim 3: Clinical & Prognostic Studies 3870/5300 CRC patients have FISH data 5-fold increase in 4 years Targeted studies were conducted instead of “1000 stored” CRC specimens requested and used for cytogenetic studies: CpG & FISH interlab comparisons miRNA FISH development 15% 73%

6.   Rassenti et al., Correlations of genomic aberrations and prognostic markers in chronic lymphocytic leukemia in relation to the intensity of the clonal abnormality. ASH 2007   Rassenti et al., TCL1 expression in chronic lymphocytic leukemia correlates with the intensity of 11q deletions and ZAP-70. ASH 2007   Tam et al., De novo deletion 17p13.1 chronic lymphocytic leukemia shows significant clinical heterogeneity: the M. D. Anderson and Mayo Clinic experience. ASH 2009 & Blood 114:957-64, 2009   Van Dyke et al., A comprehensive evaluation of the prognostic significance of 13q deletions in patients with B-chronic lymphocytic leukaemia. ASH 2008 & Br J Haematol, in press Aim 3: Clinical & Prognostic Studies (related to CRC FISH data)

7. Aim 4a-c: Genetics of CLL   Novel mitogens: CpG oligonucleotide   DNA Chip analysis   10 patients at 1Mb intervals oligo-array   Candidate loci: CpG, Chips, & Croce (Proj 1)   Evaluate new FISH targets   Develop FISH for microRNA sites of interest

8. Aim 4a: Novel mitogens: CpG oligonucleotide Heerema et al., Karyotype Results From CpG Oligodeoxynucleotide Stimulated Chronic Lymphocytic Leukemia (CLL) Cultures Are Consistent Among Laboratories: a CLL Research Consortium (CRC) Study. ASH 2009 & Manuscript in preparation (Nyla Heerema) CpG mitogen identifies abnormalities much more often than traditional cytogenetics Approximately 80% of CLL patients abnormal Interlaboratory comparison demonstrated similar results on same patient samples cultured and analyzed in each CRC cytogenetics laboratory

9. Aim 4b: DNA Chip (array CGH) analysis   Plan:   10 patients at 1000 kb interval backbone by array CGH   Actual:   >50 pts at 5 kb interval backbone by array CGH   Leis et al., Impact of IGVH Gene Mutational Status and VH Family Usage on Chromosomal Aberrations Detected by High-Resolution Array-CGH in B-CLL. Internat Workshop On CLL, Barcelona, 2009.   Eckel-Passow et al., 1M oligoarray study of 52 newly diagnosed CLL patients (half are CRC patients). Manuscript in preparation (Dan Van Dyke)

10. Chromosome region sites where recurrent abnormalities were seen in 3 or more CLL patients using a 1M array CGH assay

11. Array CGH complexity # abnormalitiesN Median months from diagnosis to CGH assay 1 <73114.6 7 to 14721.2 >14545.1 1 Array CGH was done at time of entry into therapy This implies that genetic abnormalities accumulate with time in CLL patients Preliminary data: Response to chemo-immunotherapy declined as complexity went up (p=0.017)

12. Aim 4c: Genetics of Candidate loci: CpG, Chips, & Croce Project 1   Evaluate new FISH targets   Array CGH will replace this approach   see New Aims 3 & 4 – limited number of targets with FISH; unlimited number & much smaller size targets with aCGH; cost ~ or <   FISH probes for microRNA (Project 1)   Many attempts – miR15-16a   <1 kb target too small to use FISH probes   CGH array will be better than FISH

13. NEW Specific Aims, 2010   Aim 1: Interrogate FISH database   Clinical studies are planned or to be planned   Aim 2: CpG oligonucleotide stimulation   Do karyotype & complexity predict prognosis independent of other parameters?   Aim 3: Validate array CGH for CLL   1M oligo array on 100 newly diagnosed patients   Develop a ~100 kb CLL-specific array   Croce Tcl-1 mouse will be compared to human CLL   Aim 4: CGH to study CLL evolution & stable vs aggressive CLL

14. Aim 1: Interrogate FISH database FISH data (& CRC FISH labs) are ready for clinical trials – now standardized & QC’d   Does % of FISH-abnormal cells influence clinical course or prognosis?   Are there modifier effects between genetic abnormalities in relation to clinical outcome?   Dohner et al tested prognosis one FISH probe at a time   Validate moderating effect of 13q- on 11q- and 17p-   Test for other FISH-detected interactions   Can cytogenetic progression predict disease progression?

15. Aim 2: CpG oligonucleotide stimulation   What CpG parameters are independent of other lab parameters & correlate with disease state?   Complexity?   Unbalanced rearrangements?   Nonclonal cells?   Does CpG karyotype evolve and correlate with disease progression?

16. Aims 3 & 4 array CGH studies   Aim 3: Validate array CGH for CLL   Aim 4: array CGH to study genetic evolution & stable vs aggressive CLL

17. Cytogenetics: Past, Present and Future Chromosome analysis Whole genome coverage Low resolution (~5 Mb) FISH analysis Site-specific High resolution (~100 Kb) Array CGH analysis Whole genome coverage High resolution (5 Kb-500 Kb) Molecular karyotype

18. Array CGH Technology (Oligos or SNPs or both) Arrayed features Control or patient’s normal genomic DNA is fluorescently labeled CLL genomic DNA is fluorescently labeled Digital Image Processing and Automated Analysis

19. Pericentromeric regions cenqterpter Array Design Known clinically relevant regions (recurrent gain/loss, oncogene, microRNA) del/dup Baldwin et al., Genet Med. 10:415-429, 2008

20. Pericentromeric regions cenqterpter Array Design Known clinically relevant regions del/dup ~50 kb interval backbone for 100k array ~5 kb (functional) interval backbone for 1M array Baldwin et al., Genet Med. 10:415-429, 2008

21. Aim 3: Validate array CGH for CLL   Test 100 newly diagnosed patients   Use Agilent 1M oligo array   Examine and follow those with uncommon recurrent gain or loss   Use to design CLL-specific ~100k array   Target probes to known & new CLL-associated sites   Target probes to Croce-selected miRNA sites   Validate CLL-specific array on 50 selected cases   Does B-cell sorting increase sensitivity? (n=10)   Interspecies comparison with Croce (Tcl-1 mouse model) for genetic basis of CLL

22. B-cell sorting to increase sensitivity High purity tumor sample Low purity or subclonal population (FISH 13q- = 21.5%)

23. Aim 4: array CGH to study evolution & Stable Versus Aggressive CLL   Cytogenetic evolution   20+ of the original 100 newly diagnosed patients at diagnosis, every 2-4 yr, & at progression   FISH, CGH, & CpG at each time point   Does watch&wait lead to greater complexity & less OR?   Array CGH vs FISH vs CpG   Which best predicts progression & survival?   Which is most cost-effective?   Stable disease vs de novo aggressive   CGH at diagnosis & progression, n=20 each   What genetic changes influence aggressiveness?

24. CRC Core C FISH studies Cytogenetics Team CRC Cytogenetics Lab Directors: Daniel Van Dyke Mayo Clinic Paola Dal Cin Brigham and Women's Marie Dell'Aquila UC San Diego Nyla Heerema Ohio State University Ayala Aviram Long Island Jewish CRC Coordinators/Interactions: Laura Rassenti Tissue Core Director (UCSD) Andrew GreavesBioinformatics Director(UCSD) Donna NeubergBiostats Director (DF) Carlo CrocePI of Project 1(OSU) All CRC sites for Clinical data correlations & FISH data mining