1. Complex karyotype in CLL
Arnon Kater, Panagiotis Baliakas, Alexander Leeksma MD
On behalf of the ERIC ”complex karyotype project”
Good afternoon everyone, today I will give a short update of the complex karyotype project in CLL on behalf of the ERIC complex karyotype working group.

2. Introduction Accepted diagnostic workup CLL prior to treatment
FISH 13q, tris 12, 11q and 17p; TP53 mutation according to ERIC guidelines
Since 2006 data individual groups of predicitive value of complex karyotype (CK), never integrated in guidelines
New interest in CK in era of novel agents (Thompson, Cancer 2015)

3. Questions How to define CK? Predictive value of CK?
Differences in methodology?
Prospective analysis?
ERIC wide registry
Group of dedicated researches
First meeting at MLL Munich fall 2016
Grant Janssen

4. Overview
Short summary results complex karyotype (CK) classical cytogenetics study (on behalf of Panagiotis Baliakas/Kostas Stamatopoulos)
Preliminary results CK CGH array study
Because Panagiotis is not attending this meeting I will give a short summary of the data of the ERIC classical cytogenetics study. After the first part I will show you some preliminary results of the CGH array study

5. Overview of the cohort 3656 3580 ≥10 metaphases N=3580 N, % Male
Female
2252, 63%
1328, 37%
Median age diagnosis
<55
>70
65.6 years
698/3560, 20%
1184/3560, 33%
MBL
Binet A
Binet B
Binet C
258/2863, 9%
2098/2893, 73%
357/2893, 12%
258/2893, 9%
M-CLL
1222/2051, 60%
TP53abs
299/3308, 9%
del(11q)
377/3256, 12%
Trisomy 12
507/3260, 16%
del(13q)
1769/3271, 54%
Treated (median FU: 4.6 years)
1413/3393, 42%
3656
3580
≥10 metaphases
This is an overview of the cohort consisting of more than 3000 CLL patients. As you can see it is a reflexion of a normal cohort.

6. Method and timing of testing
CPG/IL2
n=379, 11%
TPA,
n=1846, 52%
TPA, CPG+IL2
n=1355, 37%
3580
Tested within 6 months from diagnosis
N=2684/3549, 76%
Tested within 1 year from diagnosis
N=2814/3549, 79%
Tested before treatment (treated cases)
N=1238/1406, n=88%

7. Overview of chromosomal aberrations
3580
3179 (89%)
Non-CK
741 (23%)
1 aberration
[non-del(13q)]
406 (13%)
2 aberrations
401 (11%) CK
2032 (64%)
Normal/del(13q)
No difference between cell stimulation protocols

8. CK (≥3 aberrations) vs non CK
P-value
Male
275/401, 69%
1977/3179, 63%
0.012
<55 years
81/397, 20%
617/3163, 20%
0.67
>70 years
144/397, 36%
1040/3163, 33%
0.17
MBL
Binet A
Binet B
Binet C
13/295, 4%
186/295, 63%
66/295, 22%
30/295, 10%
245/2568, 10%
1912/2568, 74%
291/2568, 11%
120/2568, 5%
0.003
<0.0001
M-CLL
98/254, 38%
1124/1797, 63%
TP53abs
117/381, 31%
182/2927, 6%
del(11q)
106/3691, 29%
271/2887, 9%
Trisomy 12
98/3682, 27%
409/2892, 14%
del(13q)
237/3913, 60%
1532/2900, 53%
1: 14/105, 13% TP53abs, 2: 17/97 TP53abs, 39/97: +12,+19,
3: 71/235 (30%) negative for TP53abs, del(11q), +12

9. CK and OS Selected cases karyotyped before treatment
We first looked at overall survival in complex vs non-CK patients. The definition for a complex CLL is 3 or more chrom aberrations 13q excluded. As expected complex karyotype is associated with worse survival
Selected cases karyotyped before treatment

10. CK and TP53mut Multivariate analysis CK Age Mutation TP53
If we go further into this and look at te effect of a p53 mutation you can see that this is still the case with a p53 mut.
Multivariate analysis CK
Age
Mutation
TP53

11. 3abs vs 4abs vs ≥5abs
Complex karyotypes with more abs are doing worse than the ones with less abs.

12. Are all CK equal?
Interestingly a complex karyotype with trisomy 12 and 19 can be seen as a prognostically favorable subgroup. The question is of course if we should make another risk stratification based on this result.

13. Preliminary conclusions Classical cytogenetics (retrospective analysis)
Different mitogens homogeneous results
CK independent risk factor but:
different subgroups can be found (e.g. +12,+19)
exact definition of CK to be determined
Submitted to IWCLL on behalf of ERIC

14. Overview
short summary results complex karyotype (CK) classical cytogenetics study (on behalf of Panagiotis Baliakas/Kostas Stamatopoulos)
preliminary results CK CGH array study
Because Panagiotis is not attending this meeting I will give a short summary of the data of the ERIC classical cytogenetics study. After the first part I will show you some preliminary results of the CGH array study

15. Preliminary results CK CGH array study (first analysis)
ERIC survey: 25 centers will provide data (different sizes)
Very preliminary results of first 9 centers
Methods:
For del13q, del11q, del17p: no cut-off
All other abberations: cut-off of >5MB (Schoumans diagnostic guidelines)
1. effect complex karyotype (CK) on survival
2. effect different aberrations on survival
As promised I will show you some preliminary results of the CK CGH array study. Thus far we only have the results of 9 out of the 25 centers involved in this large study. The first analysis we performed is about the CGH array data only.

16. Overview of the CGH array cohort
N=1322
N, %
Male
Female
892, 67,5%
427, 32,3%
Median age diagnosis
<55
>70
63 years
146/677, 22%
168/677, 25%
MBL
Binet A
Binet B
Binet C
13/594, 2%
432/594, 73%
102/594, 17%
47/594, 8%
M-CLL
311/554, 56%
TP53abs
55/618, 9%
del(11q)
182/1294, 14%
Trisomy 12
174/1294, 13%
del(13q)
584/1293, 45 %
1322
This is an overview of the CGH array cohort. The cohort is lower in number but still a good reflexion of the general population of CLL patients.

17. Overview of chromosomal aberrations
1322
1148 (87%)
Non-CK
552 (42%)
1 aberration
191 (14%)
2 aberrations
174 (13%) CK
405 (31%)
Normal
This is a schematic overview of the subdivision of the CK and non-CK based on CGH arrays. The CK percentages are a little bit higher compared to the CK status defined by classical cytogenetics in the other cohort.

18. Overview effect recurrent aberrations
The cohort is in line with earlier studies which show an effect of del11q and 17p on overall survival. For del13q and trisomy 12we could not find any differences in overall survival although there is a trend towards a lower overall survival if you look at trisomy 12.

19. Overview effect other aberrations
Besides the recurrent aberrations we looked with CGH arrays at other less recurrent aberrations not regularly tested/included/analyzed by the standard FISH panels. Trisomy 18 and 19 alone do not have an effect on survival as well as duplication of 8q. A duplication of 2p and especially a deletion of 6q seem associated with an inferior prognosis.

20. Dissecting CK
If we dissect the CK in this cohort we get this pie chart.

21. Dissecting CK
For the array analysis we see the same picture if we compare non-CK with complex karyotypes if we look at overall survival.

22. Dissecting CK
If we subdivide te complex cases we see that especially 5 or more aberrations are associated with a worse survival while 3 or 4abs are somewhere in between.

23. Effect TP53mutation
And if we compare non complex karyotype with complex karyotype in the presence of a p53 mutation you can see that the CK patients do worse.

24. Preliminary conclusions Array-based (work in progress)
Complex karyotype is a prognostic factor in CLL
The presence of ≥5abs rather than >3abs is associated with the worst clinical outcome
Genomic complexity is associated with worse clinical outcome even amongst cases harboring TP53mut

25. Acknowledgements ERIC CK working group Sabine Franke, Liege, Belgium
Panagiotis Baliakas, Uppsala , Sweden
Richard Rosenquist, Uppsala, Sweden
Sabine Franke, Liege, Belgium
Anh Nhi Tran, Stockholm, Sweden
Florence Nguyen-khac, Paris, France
Claudia Haferlach, München, Germany
Loïc Ysebaert, Toulouse, France
Karl-Anton Kreuzer, Cologne Germany
David Oscier, Bournemouth, Great Britain
Douka Vasiliki, Thessaloniki, Greece
Constantine Tam, Melbourne, Australia
Kostas Stamatopoulous, Thessaloniki, Greece Maria Laura Blanco, Barcelona, Spain
Hidde Posthuma, Amsterdam, the Netherlands
Anna Puiggros, Barcelona, Spain
Clemens Mellink, Amsterdam, the Netherlands
Blanca Espinet, Barcelona, Spain
Arnon Kater, Amsterdam, the Netherlands
Jacqueline Schoumans, Lausanne, Switzerland
Ana Eugenia Rodriguez, Salamanca, Spain
Sarka Pospisilova, Brno, Czech Republic
Karla Pevlova, Brno, Czech Republic
Kaja Malinka, Brno, Czech Republic
Sabine Jeromin, München, Germany
Marian Stevens-Kroef, Nijmegen, the Netherlands
I would like to thank all the members of the complex karyotype working group. It is really a pleasure to work with you all. Thank you very much for your attention. I am open to answer questions.
(If you are not on board and want to be part of this large initiative to address the impact of genomic complexity in CLL please send an to

26. CK and IGHV status (classical cytogenetics

27. CK and IGHV status (classical cytogenetics)

28. CK and IGHV status (CGH array)

29. Dissecting CK (CGH array)
If we compare IGHV UM and look at the effect of a complex karyotype we do not see any difference if we compare UM cases non complex with a complex karyotype. This means that mutation status in CLL is a better prognostic marker than an complex karyotype.

30. CK and IGHV status (classical cytogenetics)

31. Are all TP53mut equal?
noTP53abs, n=2974

32. Are all TP53mut equal?
noTP53abs, n=2974

35. Dissecting CK
This picture does not change if we change the criteria for a complex karyotype when we do not include 13q deletions as aberrant.

36. Effect of IGHV
The complex karyotypes are seen much more frequently in UM CLL and in UM CLL the CKs are associated with a worse survival compared to the M-CLL patients.

37. Effect TP53 mutation
As expected for p53 mutations we see that in the absence of a functional p53 survival is worse compared to a normal p53.

38. Effect TP53 mutation
But if we look at complex karyotypes and look at the effect of a p53 mutation we see that within the complex karyotypes p53 mutations still do worse.

39. CGH array vs FISH recurrent aberrations
Male
Female
177, 69%
79, 31%
Median age diagnosis
<55
>70
65 years
43/248, 17%
86/248, 35%
MBL
Binet A
Binet B
Binet C
13/209, 6%
146/209, 70%
32/209, 15%
18/209, 9%
M-CLL
47/119, 39%
aberration
CGH array
FISH
13qmono
110
122
13qbi 29 21
trisomy12 37 38
del11q 52 56
del17p 16 22
Total
244
259
This a sneak preview of the next part of the CGH array project.
The idea is to compare CGH array with classical cytogenetics and FISH. Some labs nowadays only use CGH instead of CK in combination with FISH. The question is or FISH is still useful or that we can do without. In the first analyzed patients we see some differences. With FISH we detect a little bit more aberrations if we compare this with CGH array without a size cut-off. We need more data to say something about these differences.