FISH and CHIPS for CLL Patients

Angela DouglasConsultant 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-13-

Acetate) 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 100’s 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 Levelmi-RNA’s

• Class of small endogenous RNA’s• Play important regulatory roles by targeting mRNA’s for cleavage or

translational repression• Act in diverse biological processes including development, cell

growth, apoptosis and haematopoiesis.• 50% of known miRNA’s are located at known cancer associated

genomic regions • Association with Cancer• MiRNA’s 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 CLL’s, 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 1Whole Genome View

3q amp

Del 4p14 - 4p16

Amp 5q

8q24 Amplification

Including amplification of c-myc

Monosomy 9

Del17p13/amp 17p11Deletion Detected by FISH

del18q

Why didn’t aCGH Pick Up the del13q14?

D13S319 on 13q14.3 - 135Kb probe situated between RB-1 Gene and D13S25 Marker

Where’s 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

What’s 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