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 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 Level
mi-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 @ D, 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 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