1. West Midlands Regional Genetics Laboratory
Improved detection of chromosomal abnormalities in CLL by G-band analysis David Bohanna
West Midlands Regional Genetics Laboratory

2. 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

3. 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
IGH mutational status considered most reliable prognostic marker
West Midlands Regional Genetics Laboratory

4. 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
FISH studies carried out using an 8 probe panel
* Prognostic significance of bi allelic deletions of 13q uncertain
West Midlands Regional Genetics Laboratory

5. 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
TPA is a carcinogen
West Midlands Regional Genetics Laboratory

6. Alternative B-cell mitogen
DNA fractions from Mycobacterium bovis shown to have mitogenic effect for B-cells in mice (Tokunaga et al, 1984)
Synthetic CpG oligonucleotides (ODN) later shown to mimic these effects
ODN that contain cytosine nucleotide next to guanine nucleotide in the linear DNA sequence
Mycobacterium bovis .
CpG ODN
West Midlands Regional Genetics Laboratory

7. 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

8. 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

9. 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

10. 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
Abnormalities were recorded according to international standards
West Midlands Regional Genetics Laboratory

11. Interphase FISH (ATM,13q,p53,+12) Abnormality Rate (Including fails)
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)
Reason for high failure rate of TPA, TPA could be getting old
Colc on day as opposed to ONC, less mets obtained
West Midlands Regional Genetics Laboratory

12. 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

13. 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

14. West Midlands Regional Genetics Laboratory
Note. Deletions of 6q, deletions of 14q and translocations involving 14q not tested for by FISH.
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

15. West Midlands Regional Genetics Laboratory
Deletions of 13q
10 mono allelic deletions detected by FISH
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)
del(13)(q12q14)
Metaphase FISH showed one of the 13q deletions with ANK to be sub microscopic. Other under represented in the metaphases.
Although associated with poor markers and complex karyotypes, the significance of bi allelic 13q deletions when found alone is unclear.
In the case where it was in the metaphases, only found in poor morphology metaphases.
* 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

16. Deletions of 13q 6 bi allelic deletions detected by FISH
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.
del(13)(q12q14)

17. West Midlands Regional Genetics Laboratory
Trisomy 12
8/9 trisomy 12 clones were detected by G-band 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?
This has been reported and we are going to do IGH studies.
Case 1
Case 4
Case 10
Case 29
West Midlands Regional Genetics Laboratory

18. 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.
2/2 cases had loss of 17p (TP53) by G-band analysis of DSP30/IL-2 cultures.
In 4/4 cases with a poor cytogenetic marker, the abnormality was visible on G-band analysis of DSP30/IL-2 cultures.
del(11)(q14q23)
add (17)(p1)
West Midlands Regional Genetics Laboratory

19. 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?
Small data set but complex karyotypes may well confer a poor prognosis
West Midlands Regional Genetics Laboratory

20. 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.
13q deletions not picked up very well by DSP30/IL-2 cultures.
West Midlands Regional Genetics Laboratory

21. West Midlands Regional Genetics Laboratory
Conclusions
Additional abnormalities were seen using G-banding 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”.
As G-band is whole genome screen
Haferlach et al, 2007
West Midlands Regional Genetics Laboratory

22. 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