Cp G Stimulated Karyotypic Analysis of Chronic Lymphocytic

Cp. G 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 Email: nyla. heerema@osumc. edu

Objectives § Know the significance of Cp. G 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 Cp. G stimulated karyotype can show significant additional aberrations as CLL treatments change.

CLL § Clonal proliferation and accumulation of Blymphocytes in blood, bone marrow, lymph nodes and spleen § Inhibition of apoptosis - Cells accumulate - BCL 2 consistently overexpressed § >90% circulating lymphocytes in G 0 § Cytokines involved in regulation of growth and differentiation expressed § Median age at diagnosis: 72 years

Prognosis § Median survival: 9 years – prior to imatinib - 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 Rozman et al. N Engl J Med. 1995; 333: 1052 -1057. Cheson et al. Blood. 1996; 87: 4990 -4997.

High Risk Cytogenetics § Traditional § del(17 p 13. 1): interphase FISH for TP 53 § del(11 q 22. 3): interphase FISH for ATM § Newer: Complex karyotype/translocations: metaphase analysis using newer stimulants, e. g. Cp. G oligodeoxynucleotides

Metaphase Cytogenetics - Requires Culture of CLL Cells § § Apoptose in culture B-cell stimulation usually required Typical B-cell mitogen response weak Oligodeoxynucleotide (Cp. G) stimulation results in much higher detection of abnormalities § Mitogens currently used (OSU) - Cp. G oligodeoxynucleotide + pokeweed mitogen + phorbal myristal acetate - Cp. G alone

What are Oligodeoxynucleotides? § Short single strands of DNA or RNA, approximately 19 -25 base pairs § One with 20 base pairs would be called a 20 -mer § Can be longer, 160 -200 bp § Natural or synthesized § Activate cells of immune system in a sequencedependent manner § Used to enhance some cancer therapies § Cause proliferation, cytokine production, regulation of surface molecules § Used in oligonucleotide arrays

Cp. G Used at OSU (Cp. G ODN-685) § A 21 -mer fully phosphorothioated unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide that targets TLR 9 § TLR 9: toll-like receptor 9, involved in pathogen recognition and activation of innate immunity § Five CG sequences, all unmethylated 5’-TCG ACG TTC GTT CTC 3’ Liang, et al. , Blood 2010; 115(24): 5041 -5052 Detailed procedure available upon request

44, X, -Y, t(1; 6)(p 32; p 23), add(2)(q 21), add(7)(p 21), -8, -9, der(9)t(8; 9)(q 23; p 21)dup(8)(q 23 q 24), +12, der(17)add(17)(p 13)add(17)(q 21), -22, +r

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

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

Patient Characteristics (n=329) N Age at diagnosis, years Mean(sd) Sex Complexity Translocation Rai Stage at Diagnosis IGHV unmutated trisomy 3 trisomy 8 trisomy 12 del 13 q del 17 p del 6 q del 11 q Median(Range) Male Female Median(Range) < 3 Abnormalities >3 Abnormalities Neither Balanced Unbalanced 0 -2 3 -4 No Yes No Yes No % 60 (11) 60 (34 -88) 211 64. 1 118 35. 9 1 (0 -17) 276 83. 9 53 16. 1 244 74. 2 30 9. 1 55 16. 7 296 90. 2 32 9. 8 144 49. 3 148 50. 7 312 94. 8 17 5. 2 313 95. 4 15 4. 6 251 76. 3 78 23. 7 155 47. 1 174 52. 9 298 90. 6 31 9. 4 315 95. 7 14 4. 3 285 86. 6 HR 95% CI p-value 1. 00 0. 95 2. 92 0. 99 0. 67 1. 98 1. 02 1. 34 4. 31 2. 64 1. 87 3. 71 <0. 001 3. 73 2. 32 5. 99 <0. 001 3. 48 1. 05 2. 53 1. 23 0. 78 2. 10 1. 69 2. 38 0. 49 1. 40 0. 84 0. 56 1. 31 0. 82 5. 08 2. 24 4. 59 1. 81 1. 09 3. 37 3. 45 <0. 001 0. 91 0. 002 0. 29 0. 15 0. 002 0. 15 0. 64 0. 77 <0. 001

Multivariable Analysis Characteristics IGHV mutated: Translocation present vs. absent IGHV unmutated: Translocation present vs. absent Complexity, >3 abnormalities: present vs. absent del(11 q): present vs. absent Rai Stage: 3 -4 vs. 0 -2 HR 95% CI 3. 59 1. 80 7. 19 1. 03 0. 63 1. 67 1. 70 1. 57 1. 94 1. 03 2. 80 1. 04 2. 36 1. 18 3. 17 p-value 0. 002 0. 037 0. 030 0. 009

Translocation Negates Good Prognosis of Mutated IGHV

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

Progression-free Survival with Ibrutinib Treatment All patients By del(17 p) or del(11 q) Byrd, et al, Blood, 2015 18

Variables Contributing to Ibrutinib Discontinuation 19

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Near-tetraploidy Is Associated with Richter’s Transformation 21

Near-tetraploidy Is Associated with an Increased Incidence of Richter’s Transformation 0. 8 Gray’s Test, p<0. 0001 0. 4 0. 6 Near-tetraploid (n=9) 0. 2 0. 0 Cumulative incidence 1. 0 Richter’s transformation Not near-tetraploid (n=291) 0 1 2 3 Years 22 Total transformations Tetraploid negative Tetraploid positive 28 22 6 4 5 6

Conclusions § Stimulation of CLL cells with Cp. Gs 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

24 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

Overall TFT 329 patients within 1 year of diagnosis (includes former CPX patients)

Near-tetraploidy • N ≈ 92 chromosomes in a cell • Reported in various lymphomas • Incidence has not been described in CLL • Promotes chromosome instability 27

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Cp. Gs Stimulate CLL Cells § In mice, Cp. Gs 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) § Cp. Gs enter the cell and cause immune stimulations, which include expression of IL 2 § Mechanism not clear, specific sequence is important § Cp. G is very simple to use in a clinical setting

New Treatment - Ibrutinib

Ibrutinib Relapse/Richter’s Transformation § Progression with CLL is associated with mutations in BTK and/or PLCG 2 § 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

Near-tetraploidy Is an Independent Risk Factor for Richter’s Transformation 0. 2 0. 4 0. 6 0. 8 Not near-tetraploid/Not complex (n=119) Not near-tetraploid/Complex (n=163) p<0. 0001 Near-tetraploid/Complex (n=9) 0. 0 Cumulative incidence 1. 0 Richter’s transformation 0 1 2 3 Years 4 5 Near-tetraploidy HR=8. 66, 95% CI 3. 83 -19. 59 Complex karyotype HR=4. 78, 95% CI 1. 42 -15. 94 32 6