1. Nyla A. Heerema The Ohio Sate University Email: nyla.heerema@osumc.edu
CpG Stimulated Karyotypic Analysis of Chronic Lymphocytic Leukemia (CLL) Is a Significant Prognostic Factor and Should Be Performed on all Patients
Nyla A. Heerema
The Ohio Sate University

3. Objectives Know the significance of CpG stimulated karyotypes in CLL
Know the significance of the presence of a translocation within 1 year of diagnosis of CLL
Know the significance of a complex karyotype detected within 1 year of diagnosis of CLL
Understand how a CpG stimulated karyotype can show significant additional aberrations as CLL treatments change.

4. CLL
Clonal proliferation and accumulation of B- lymphocytes in blood, bone marrow, lymph nodes and spleen
Inhibition of apoptosis
Cells accumulate
BCL2 consistently overexpressed
>90% circulating lymphocytes in G0
Cytokines involved in regulation of growth and differentiation expressed
Median age at diagnosis: 72 years
Cells CD5+ and CD19+

5. Prognosis Median survival: 9 years – prior to imatinib Primary factors
Great variability: few months - >20 years
Primary factors
Clinical (Rai/Binet) stage
Bone marrow histopathology
Peripheral white blood cell counts
Lymphocyte doubling time
Age
Gender
IGHV mutational status
Genetics
Diffuse BM pattern> worse outcome
Rozman et al. N Engl J Med. 1995;333: Cheson et al. Blood. 1996;87:

6. High Risk Cytogenetics
Traditional
del(17p13.1): interphase FISH for TP53
del(11q22.3): interphase FISH for ATM
Newer: Complex karyotype/translocations: metaphase analysis using newer stimulants, e.g. CpG oligodeoxynucleotides
DELETE?

7. Metaphase Cytogenetics - Requires Culture of CLL Cells
Apoptose in culture
B-cell stimulation usually required
Typical B-cell mitogen response weak
Oligodeoxynucleotide (CpG) stimulation results in much higher detection of abnormalities
Mitogens currently used (OSU)
CpG oligodeoxynucleotide + pokeweed mitogen + phorbal myristal acetate
CpG alone
Oligonucleotides: single stranded DNA ~20+ bp long

8. What are Oligodeoxynucleotides?
Short single strands of DNA or RNA, approximately base pairs
One with 20 base pairs would be called a 20-mer
Can be longer, bp
Natural or synthesized
Activate cells of immune system in a sequence- dependent manner
Used to enhance some cancer therapies
Cause proliferation, cytokine production, regulation of surface molecules
Used in oligonucleotide arrays

9. CpG Used at OSU (CpG ODN-685)
A 21-mer fully phosphorothioated unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide that targets TLR9
TLR9: toll-like receptor 9, involved in pathogen recognition and activation of innate immunity
Five CG sequences, all unmethylated
5’-TCG TCG ACG TCG TTC GTT CTC 3’
21 bp
5 CG sequences – NOT methylated
Patent pending as adjuvant in Hepatitis B virus and rabies vaccine.
Liang, et al., Blood 2010; 115(24):
Detailed procedure available upon request

10. 44,X,-Y, t(1;6)(p32;p23),add(2)(q21),add(7)(p21),-8,-9,
der(9)t(8;9)(q23;p21)dup(8)(q23q24),+12,der(17)add(17)(p13)add(17)(q21),-22,+r

11. Complexity (>3 independent aberrations) within 1 Year of Diagnosis and Time to Treatment

12. Del(17p) & Complexity within One Year: Prognosis
Subset: Not del(17p)
Subset: del(17p)

13. Patient Characteristics (n=329) N % HR 95% CI p-value
Age at diagnosis, years Mean(sd)
  60 (11)
Median(Range)
60 (34-88)
1.00
0.99
1.02 
 0.64
Sex
Male
211
64.1
Female
118
35.9
0.95
0.67
1.34
0.77
Complexity
1 (0-17) 
< 3 Abnormalities
276
83.9
>3 Abnormalities 53
16.1
2.92
1.98
4.31
<0.001
Translocation
Neither
244
74.2
Balanced 30
9.1
2.64
1.87
3.71
Unbalanced 55
16.7
Rai Stage at Diagnosis
0-2
296
90.2
3-4 32
9.8
3.73
2.32
5.99
IGHV unmutated No
144
49.3
Yes
148
50.7
3.48
2.38
5.08
trisomy 3
312
94.8 17
5.2
1.05
0.49
2.24
0.91
trisomy 8
313
95.4 15
4.6
2.53
1.40
4.59
0.002
trisomy 12
251
76.3 78
23.7
1.23
0.84
1.81
0.29
del13q
155
47.1
174
52.9
0.78
0.56
1.09
0.15
del17p
298
90.6 31
9.4
2.10
1.31
3.37
del6q
315
95.7 14
4.3
1.69
0.82
3.45
del11q
285
86.6 44
13.4
Need chart with P-values!

14. Multivariable Analysis
Characteristics HR
95% CI
p-value
IGHV mutated: Translocation present vs. absent
3.59
1.80
7.19
 0.002
IGHV unmutated: Translocation present vs. absent
1.03
0.63
1.67
Complexity, >3 abnormalities: present vs. absent
1.70
2.80
0.037
del(11q): present vs. absent
1.57
1.04
2.36
0.030
Rai Stage: 3-4 vs. 0-2
1.94
1.18
3.17
0.009

15. Translocation Negates Good Prognosis of Mutated IGHV

16. Both Complexity and IGHV Are Prognostically Significant
IGVH mutated, no complexity
IGVH mutated with complexity
IGVH unmutated, no complexity
IGVH unmutated with complexity

17. Progression-free Survival with Ibrutinib Treatment
Use only all patient chart?
All patients
By del(17p) or del(11q)
Byrd, et al, Blood, 2015

18. Variables Contributing to Ibrutinib Discontinuation

20. Near-tetraploidy Is Associated with Richter’s Transformation

21. Richter’s transformation
Near-tetraploidy Is Associated with an Increased Incidence of Richter’s Transformation
Cumulative incidence
Years
Near-tetraploid (n=9)
Not near-tetraploid (n=291)
Gray’s Test, p<0.0001
Richter’s transformation
Total transformations
Tetraploid negative
Tetraploid positive 28
 22 6

22. Conclusions
Stimulation of CLL cells with CpGs greatly enhances detection of prognostic aberrations.
Translocations in IGHV-mutated patients and complex karyotypes detected within one year of diagnosis of CLL are associated with a poor prognosis.
Pre-treatment near-tetraploidy is highly associated with ibrutinib discontinuation via Richter’s transformation.

23. Thanks to: Qiuhong Zhao Amy S. Ruppert Heather Breidenbach
Leslie A. Andritsos
Michael R. Grever
Jeffrey A. Jones
Kami J. Maddocks
Jennifer Woyach
Farrukh Awan
Meixiao Long
Lynne Abruzzo
Amber Gordon
Caitlin Coombes
John C. Byrd
Natarajan Muthusamy
Cecelia Miller

25. Overall TFT 329 patients within 1 year of diagnosis (includes former CPX patients)
Median follow-up for censored patients was 30 months (range months). Median TFT for the entire cohort was 47 months (95% confidence interval (CI) months).

26. Near-tetraploidy N ≈ 92 chromosomes in a cell
Reported in various lymphomas
Incidence has not been described in CLL
Promotes chromosome instability

28. CpGs Stimulate CLL Cells
In mice, CpGs are taken in by endosomes, within minutes B-cells enter the cell cycle, secrete immunoglobulin and are rescued from apoptosis
Similar response in human B and B-CLL cells (Decker et al, Blood 95:999,2000)
CpGs enter the cell and cause immune stimulations, which include expression of IL2
Mechanism not clear, specific sequence is important
CpG is very simple to use in a clinical setting
CpGs enter the cell and cause immune stimulations, which includes expression of IL2

29. New Treatment - Ibrutinib
The BCR pathway v imp. BTK an imp member of pathways and is proximal in the pathway. It’s imp in survival & proliferation of malignant B-cells. Ibrutinib is a co-valent inhibitor of BTK.
Front line FCR: Fludarabine, cyclophosphamide, and rituximab

30. Ibrutinib Relapse/Richter’s Transformation
Progression with CLL is associated with mutations in BTK and/or PLCG2
87% had detectable mutation at relapse
Mutations can be detected prior to clinical overt relapse
Mutations are rare in patients who have Richter's transformation
Can we identify a biomarker specifically associated with the development of Richter’s transformation?
Woyach et al., JCO, 2017

31. Richter’s transformation
Near-tetraploidy Is an Independent Risk Factor for Richter’s Transformation
Cumulative incidence
Years
Not near-tetraploid/Not complex (n=119)
Not near-tetraploid/Complex (n=163)
Near-tetraploid/Complex (n=9)
Richter’s transformation
p<0.0001
(hazard ratio=8.66, 95% CI , p<0.0001) and complex karyotype (hazard ratio=4.78, 95% CI , p=0.01)
Near-tetraploidy HR=8.66, 95% CI
Complex karyotype HR=4.78, 95% CI