FISH and CHIPS for CLL Patients Angela Douglas Consultant Cytogeneticist and Directorate Lead Cheshire and Merseyside Genetics Service CLL - Heterogeneous Disease Most common Leukaemia in western world 25% of all Leukaemias Harbours various Genetic Aberrations

Derived from malignant transformation of either B or T Cells Disorder of morphologically mature but immunologically less mature lymphocytes Manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues Shows a highly variable clinical course Early Intermediate Advanced stage Also variability within each stage Need for reliable prognostic factors to; identify high risk patients; initiate therapy; and/or guide treatment choice

Identifying Genetic Markers Conventional Cytogenetics FISH Molecular Genetics Multiplex Ligation-dependant Probe Amplification (MLPA) Microarrays (CGH / Oligos) Conventional Cytogenetics

Difficult to culture - metaphase cells Additives used to aid stimulation of CLL cells CD40 ligand Combination of CpG-oligodeoxynucleotides (DSP30) and IL-2 Pokeweed or TPA (12-O-TetradecanoylPhorbol-13Acetate) Mitogens Long term cultures >4 days The most common Cytogenetic abnormalities include: Deletion 13q14 (51%) Del/t involving 11q22-q23 (20%) Trisomy 12 (15%) Deletion 17p13 (7%) Del/t involving 14q32 (7%) Deletion 6q21-q23 (<5%)

32% CLL cases abnormal by Conventional Cytogenetics Cytogenetics in CLL More recently +19, 2p16 loss, 2p24 gains, loss of 2q and 3q (Schwaenen et al 2004, Veelken et al 2007) Clonal Evolution thought to predict disease progression (Oscier et al 2007)

Complex Karyotypes shown to confer poor prognosis (LRF CLL4 Clinical Trial) Translocations have been shown to confer poor prognosis Thought that CLL characterised mainly by Genomic alterations (del/amp) but reports have shown up to 30% of patients with a Translocation. Most unique, possibly familial ?predisposition to CLL (Michaux et al 2005, Mayr C et al 2006). FISH

Interphase cells Quick, easy (scan 100s cells), directed to specific areas (targeted), costly. CLL Probe Panel comprising of; p53/ spectrum orange (17p13) and ATM (11q22.3)/spectrum green probe set. Locus Specific (LSI) D13S319 (13q14.3, DLEU1 & 2))/ spectrum orange, LSI LAMP1 (13q34)/spectrum Aqua with CEP 12 (+12)/ spectrum green probe set. In addition the following Fusion/ Break apart probes used for: The IgH/CCND1 dual fusion probe [t(11;14)] (MCL)

The Immunoglobulin Heavy chain gene (IgH) [14q32] The MYB gene [6q23] 80% Cases (selected) abnormal by FISH (cryptic) Guidance at moment is for all cases to be monitored for 17p deletion affects treatment choice, patients resistant to purine analogues, poor prognosis if deletion present in >20% of cells (Pettitt AR, 2007). Molecular Genetics

PCR (QF, QRT, RT), multiplex, (MLPA) Southern Blot Electrophoresis Gel Sequence Genetic Analyser Used to detect copy number variation (del/amp), mutations and translocations Can detect: Cryptic deletions/mutations on, 11q, 13q, 14q, 17p mono-allelic and bi-allelic Minimal critical region identified Genes with Oncogenic / Tumour Suppressor function Role in development and progression of CLL Molecular Genetics

(P53 and ATM mutations), which cause dysfunction of P53 Pathway DNA damage response pathway, correlate to increase in DNA dependant protein kinase levels and impairment of apoptosis, DNA repair and Cell cycle arrest (Poor Prognosis): Apparently Normal 17p13/11q22.3 Cytogenetically IgVH Mutational status (14q), Unmutated poor - ? associated with unique mutations in BCL6 lead to more aggressive clones and progressive disease. Mutated good. (Complex, time consuming and costly) ZAP 70 Surrogate marker for IgVH, easier to look for, positivity

associated with poor response to therapy and shorter PFS. May reflect defective pathways of B-CLL cells rather than being a marker of malignancy per se. ?TCL1, ?BCL2,?REL, ?BCL11A, Lipoprotein Lipase (LPL), PEG 10, Septin 10 and Dystrophin, AID.. Could be used to detect MRD Although can predict relapse in Autografted patients, in allografted patients does not necessarily predict clinical relapse Molecular Level mi-RNAs

Class of small endogenous RNAs Play important regulatory roles by targeting mRNAs for cleavage or translational repression Act in diverse biological processes including development, cell growth, apoptosis and haematopoiesis. 50% of known miRNAs are located at known cancer associated genomic regions Association with Cancer MiRNAs expression profiles shown to distinguish normal B-cells from

abnormal B-cells miR15a, miR16-1, miR331, miR29a, miR195, miR34a, miR34c highly expressed in CLL miR15a and miR-16-1 (on critical region of 13q14) abnormally expressed Target BCL2 gene (inverse correlation in expression) CLL patients with unmutated IgVH and high levels of expression of ZAP-70 have high levels of TCL1 due to low levels of expression of miR29 and miR181 miR-29 and miR-181 deregulate/regulate TCL1 gene, ?causal event in

the pathogenesis of aggressive form of B-CLL. Expression levels inversely correlated to TCL1 expression Possible candidates for Therapeutic agents? Multiplex Ligation-dependent Probe Amplification (MLPA) Easy to perform, sensitive, specific, robust and cost effective, it offers fast high throughput testing (24-48hrs) CLL kit can be used to determine the loss or gain of: TP53 (17p13; 8 probes),

The RB1 / DLEU / MIR15-16 region on 13q14 (12 probes), The ATM gene on 11q23 (7 probes), The PTEN gene on 10q23 (2 probes), The MYCN gene 2p24 (3 probes), The 8q24 MYC region (3 probes), The 6q23-26 region (3 probes), The CDKN2A-2B genes on 9p21 (2 probes) Presence of trisomy 12 (10 probes) Trisomy 19 (5 probes) Genomic Arrays Genomic arrays More sensitive than CC Less targeted than FISH/MLPA More specific information on Breakpoints

Detection of new non-random changes High density CHIPS will detect cryptic losses and gains Genomic imbalances detected by arrays have lead to the discovery of the location of dosage sensitive genes in CLL Efficient aCGH Array CGH allows the rapid detection of copy number changes across the whole genome

At present, array CGH is still a relatively expensive technique to perform Automated DNA extraction Role Redesign/Skill mix DNA quality and purity measured by spectrophotometer degradation identified by running on agarose gel: only perform test on Cases pass criteria - Improves success rate Hybridisation Automated Consistency in process

Analysed using Automated Scanner/Computer Software Standardisation of Interpretation / Results More Science less Art Platforms Available BAC Arrays 1Mb 0.5Mb Whole Tiling Path Arrays (100Kb) Oligo Arrays Higher resolution 15K, 44K, 105K, 244K aCGH-Liverpool

Implemented aCGH for Constitutional analysis 0.5Mb array Cytochip v.2 Tailored for Constitutional 4400 BAC clones spaced 565kb x4 400 disease specific clones for 90 conditions spaced 100kb x6 Sub-telomere clones spaced 250kb x4 Two hybridisation areas (sub-arrays)-Dye-Swap Can detect abnormal clones present as low as 10%

Not an Oncology Specific Array Used as initial screen of CLLs, as the protocol working well, to see if we were missing abnormalities CLL Case Case Male: 59y @ Early Stage CLL By FISH (CLL Panel ): +12 Cep 12 (Centromeric); del13q14 D13S319; del13q34 LAMP1; del17p13 p53; 11q22.3 ATM Detected: del17(p13) (97%), mono-allelic del13q14 (38%) and Bi-allelic del13q14 (10%) By aCGH: Detected: amp 3p; del 4p; amp 5q; amp 8q; -9; del 17p; amp 17p; del 18q; Missed Mono-allelic and Bi-allelic del13q14 CLL Patient 1

Whole Genome View 3q amp Del 4p14 - 4p16 Amp 5q 8q24 Amplification Including amplification of c-myc Monosomy 9 Del17p13/amp 17p11 Deletion Detected by FISH

del18q Why didnt aCGH Pick Up the del13q14? D13S319 on 13q14.3 - 135Kb probe situated between RB-1 Gene and D13S25 Marker Wheres the del13q14? D13S319 Solution is more clones less gaps Oligonucleotide Arrays

Higher Resolution than CGH Arrays Able to pick up smaller abnormalities present in smaller clones (7-10%) Gives better definition of abnormality; specific breakpoints and gene content Gives information on potential genes for therapeutic agents, CLL ideally suited for therapeutic vaccines, blood readily available Identifies pathways important in Pathogenesis of disease As well as highlighting prognostic markers Same methodology/equipment/cost cheaper Whats Important? Patient Management Paramount

Reliability of testing, standardisation Clinical Context Clinical Usefulness Therapeutic Context Understand the Disease Identifying new prognostic biomarkers Better classification of disease Understand pathogenetic pathways Identify information for therapeutic treatment

Determine likelihood of treatment success Monitor disease Predict clinical progression We need to identify the promising biomarkers to deliver the above and inform Clinical trials Recommendations

Del 17p13 (p53)/11q22.3 (ATM) cause dysfunctional p53 pathway Poor Prognosis Del13q14.3 - Good prognosis; bi-allelic del13q - Less Favourable Acquired Translocations - Poor (Constitutional Predisposition; 8-9% of CLL cases familial) Complex Karyotypes Poor Clonal Evolution - Predict disease progression +12 alone Good +12 /CK/CE (indicator of disease progression) - Poor Newer non-random changes del 2p16, amp 2q24, amp 3q, +19 - Prognosis unknown (Change Prognostic Hierarchy) CCND1 and CCND2 help in classification of B-Cell LPD Definitely is a need for additional testing and new technologies that are timely and cost effective that will enable more sensitive and specific chromosome analysis in CLL

patients Acknowledgements Genetics Laboratories Peter Howard Una Maye Microarrays Mark Atherton Oncology Section Roger Mountford David Gokhale

Lindsey Bradley Julie Sibbring RLUBH Prof. Andy Pettitt Ke Lin MC(Cl)CN Haematologists

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