Improved detection of
chromosomal abnormalities in
CLL by G-band analysis
David Bohanna
West Midlands Regional Genetics Laboratory
CLL
Most frequent leukaemia in the Western
world
Characterised by accumulation of long lived
B-lymphocytes in the blood
Most (~80%) patients diagnosed early
without symptoms
– Detected as a result of a routine blood test
– Require markers that can predict the prognosis in these patients
West Midlands Regional Genetics Laboratory
Prognostic Markers
IGH mutational status
– Mutated have superior survival
– High expense and expertise
CD38 and ZAP70 expression
– Increased expression correlates with unmutated IGH (although
not perfectly)
Cytogenetics
– G-band analysis (hampered by low MI)
– Prognostic information obtained from interphase FISH studies
West Midlands Regional Genetics Laboratory
Interphase FISH prognostic markers
Byrd et al, 2004
Poor: 17p (TP53) deletion, 11q
(ATM) deletion
Intermediate: trisomy 12
Good: 13q mono allelic
deletion*
13q14.3
13q34
* Prognostic significance of bi allelic deletions of 13q uncertain
West Midlands Regional Genetics Laboratory
Techniques at the WMRGL
Standard
– 1. Interphase FISH studies
Mainly TP53 and ATM
Not a whole genome screen like G-band analysis
– 2. Set up B-cell mitogen (TPA) stimulated
cultures
~40-50% abnormality rate
Experimental
– Use of alternative B-cell mitogens
West Midlands Regional Genetics Laboratory
Alternative B-cell mitogen
DNA fractions from Mycobacterium
bovis shown to have mitogenic effect
for B-cells in mice (Tokunaga et al,
1984)
Mycobacterium bovis
Synthetic CpG oligonucleotides (ODN)
later shown to mimic these effects
ODN that contain cytosine nucleotide
next to guanine nucleotide in the linear
DNA sequence
CpG ODN
West Midlands Regional Genetics Laboratory
DSP30 and CLL
DSP30 (CpG ODN)
– Used to stimulate B-cells in CLL by Claudia Haferlach’s group
– Interleukin-2 (IL-2) required
– 72hr ONC (overnight colcemid) culture
Haferlach et al (2007)
– 415/500 (83%) showed abnormalities visible by G-band analysis.
– 392/500 (78%) were abnormal by interphase FISH (with an 8
probe panel).
– 38% showed abnormalities by G-banding in addition to those
covered by the FISH panel (including complex karyotypes).
West Midlands Regional Genetics Laboratory
Aims
Improve the G-band abnormality rate
achieved by WMRGL
Compare the G-band abnormality rate of
DSP30/IL-2 cultures with TPA cultures
Compare G-band abnormality rate of
DSP30/IL-2 cultures with interphase FISH
West Midlands Regional Genetics Laboratory
Methodology
35 patients with confirmed diagnosis of CLL
72hr DSP30/IL-2 (ONC) culture
6 day TPA (1hr colcemid) culture
Interphase FISH on unstimulated culture
–
–
–
–
13q14.3
ATM (11q22.3)
TP53 (17p13.1)
CEP 12
West Midlands Regional Genetics Laboratory
Methodology
G-band analysis of 20 cells from each of
the stimulated cultures
100 cells (approx.) scored by interphase
FISH
West Midlands Regional Genetics Laboratory
Results Summary
DSP30/IL-2
ONC Cultures
TPA Cultures
Interphase FISH
(ATM,13q,p53,+12)
Success Rate
94% (33/35)
66% (23/35)
100% (35/35)
Abnormality
Rate
67% (22/33)
57% (13/23)
74% (26/35)
Abnormality
Rate
(Including
fails)
63% (22/35)
37% (13/35)
74% (26/35)
West Midlands Regional Genetics Laboratory
Comparison of G-band analysis of
DSP30/IL-2 cultures and TPA cultures
22 cases failed/normal in 6DTPA cultures
10/22 abnormal in the DSP30/IL-2 culture
13 cases failed/normal in DSP30/IL-2
cultures
1/13 abnormal in the 6DTPA culture
West Midlands Regional Genetics Laboratory
Comparison of G-band analysis of
DSP30/IL-2 cultures with
interphase FISH
13 cases failed/normal in DSP30/IL-2 cultures
7/13 abnormal by interphase FISH
9 cases normal by interphase FISH
4/9 abnormal in DSP30/IL-2 cultures
11/26 cases abnormal by interphase FISH show
additional abnormalities in DSP30/IL-2 cultures
West Midlands Regional Genetics Laboratory
Abnormalities detected by FISH and
G-band analysis
18
No. of cases
16
14
12
FISH
10
8
DSP30/IL-2
TPA
6
4
2
0
del (13q)
trisomy 12
del (11q)
del (17p)
Abnormality
• 8/16 13q deletions detected in
DSP30/IL-2 cultures
• 8/9 +12 clones detected in DSP30/IL-2
cultures
• 4/4 11q (ATM) and 17p (TP53) deletions
detected in DSP30/IL-2 cultures
West Midlands Regional Genetics Laboratory
Deletions of 13q
10 mono allelic deletions detected by FISH
del(13)(q12q14)
4/10 were visible on G-banding in DSP30/IL-2 cultures
– 3/4 had one additional abnormality (undetected by interphase
FISH)
? Different prognosis
6/10 had an apparently normal karyotype
– are the deletions sub-microscopic or under-represented in
metaphases? *
0/10 cases were associated with loss of 11q (ATM) or 17p (TP53)
* Metaphase FISH of DSP30/IL-2 cultures showed abnormal
population to be significantly under represented or not present in the
metaphases.
West Midlands Regional Genetics Laboratory
Deletions of 13q
6 bi allelic deletions detected by FISH
del(13)(q12q14)
4/6 visible as mono allelic deletions in DSP30/IL-2 cultures
– 3/4 had additional abnormalities (2 ‘complex’)
2/6 had an apparently normal karyotype
– are the deletions sub-microscopic or under-represented in
metaphases?*
3/6 were associated with either loss of 11q (ATM) or 17p (TP53)
*Metaphase FISH of DSP30/IL-2 cultures showed abnormal population to
be significantly under represented or not present in the metaphases.
Trisomy 12
8/9 trisomy 12 clones were detected by Gband analysis of DSP30/IL-2 cultures
4/9 trisomy 12 clones had
a similar del/add(14q)
Del/add (14q) not
detected in any other
abnormal karyotypes
Potential sub group of +12
patients with different
prognosis?
Case 1
Case 4
Case 10
Case 29
West Midlands Regional Genetics Laboratory
11q and 17p deletions
2/2 cases had loss of 11q (ATM) by G-band analysis
of DSP30/IL-2 cultures.
del(11)(q14q23)
2/2 cases had loss of 17p (TP53) by G-band analysis
of DSP30/IL-2 cultures.
add (17)(p1)
In 4/4 cases with a poor cytogenetic marker, the
abnormality was visible on G-band analysis of
DSP30/IL-2 cultures.
West Midlands Regional Genetics Laboratory
Is a ‘complex’ karyotype in CLL
associated with a poor prognosis?
Haferlach et al defined a ‘complex’ karyotype as
≥3 chromosome abnormalities
– Correlation with unmutated IGH
Poor prognosis
3/35 cases in our study had a ‘complex’
karyotype of ≥3 abnormalities
– 2 cases had an 11q (ATM) or 17p (TP53) deletion
Poor prognosis using interphase FISH data
– 1 case had +12
Intermediate prognosis using interphase FISH data; now
poor prognosis?
West Midlands Regional Genetics Laboratory
Conclusions
For the 35 cases studied, the overall abnormality
rate in DSP30/IL-2 stimulated cultures (63%) is
higher than in TPA stimulated cultures (37%).
Standard practice at WMRGL has been changed
to include a DSP30/IL-2 stimulated culture for all
CLL referrals.
The abnormality rate for interphase FISH studies
was 74%, which was higher than that obtained
using DSP30/IL-2.
West Midlands Regional Genetics Laboratory
Conclusions
Additional abnormalities were seen using Gbanding compared to interphase FISH. These
included:
– del/add(14q) within trisomy 12 clones
– additional abnormalities with del(13q)
– ‘complex’ karyotypes
“Prospective clinical trials should evaluate
the prognostic impact of newly available data
from G-band analysis of CLL cases”.
Haferlach et al, 2007
West Midlands Regional Genetics Laboratory
Acknowledgements
Susan Rose Project Supervisor
Mike Griffiths Head of WMRGL Oncology Section
Mary Strachan WMRGL Oncology Section
WMRGL Oncology section
WMRGL FISH section
Professor Paul Moss University Hospital Birmingham
West Midlands Regional Genetics Laboratory