1. Clonal Evolution in CLL: Impact on timing of therapy Nicholas Chiorazzi The Feinstein Institute for Medical Research North Shore – LIJ Health System Manhasset, NY

2. At all points in time, CLL clones are heterogeneous based on a number of criteria CLL worsens as subclones exhibiting new or different DNA mutations and hence biology emerge over time – “clonal evolution” The occurrence of new structural abnormalities in the DNA of leukemic subclones requires that new strands of DNA be made, i.e., for cells to divide and proliferate Three principles relevant for a discussion of clonal evolution

3. Intra-clonal heterogeneity All CLL clones are heterogeneous based on:  Surface membrane and intracellular phenotypes  CD38 – Damle et al. Blood 1999; Ghia et al. Blood 2003  ZAP-70 – Rassenti et al. N Engl J Med 2005  CD49d – Gattei et al. Blood 2008  Telomere length and telomerase activity  Bechter et al. Cancer Res 1998  Damle et al. Blood 2004  Roos et al. Blood 2008  Survival and growth requirements  Pepper et al. Leukemia 2006  Ongoing IGHV mutations  Gurrieri et al. J Exp Med 2002  Volkheimer et al. Blood 2007  Sutton et al. Blood 2009  Chromosomal and specific gene differences  Shanafelt et al. J Clin Oncol 2006  Landau et al. Leukemia 2013  Time since birth/last replication – “Age”  Calissano et al. Mol Med 2011

4. Gruber and Wu. Semin Hematol 51:177-187, 2014 Evolution and growth in our understanding of CLL heterogeneity over time

5. Evidence for clonal evolution occurring in CLL A. Sequential analyses of:  Karyotype and FISH abnormalities  Shanafelt et al. J Clin Oncol 2006  Global DNA abnormalities by comparative genomic hybridization and SNP profiling  Grubor et al. Blood 113: 1294-1303, 2009  Braggio, Kay et al. Leukemia 2102 B. Analyses of DNA abnormalities by next generation sequencing of CLL genomes/exomes

6. Clonal evolution A. Sequential analyses of FISH abnormalities, microRNA abnormalities, and global DNA abnormalities ~25% of patients develop a new genetic abnormality over time in coding or non-coding genes  Occurs more frequently in:  U-CLL clones and in M-CLL clones of patients that eventually require therapy  CD38 + clones  ZAP-70 + clones  CD49d + clones  Most common new lesions:  del(13q)  del(17p) – harbinger of accelerated disease  Greater the number of clonal aberrations the shorter the time to treatment and survival

7. Karyotype evolution and survival Shanafelt et al. J Cin Oncol;26:e5-e6, 2008

8. The greater the genomic complexity, the shorter progression-free survival (CGH) Kay et al. Cancer Genet Cytogenet 203:161-8, 2010

9. Evidence for clonal evolution occurring in CLL A. Sequential analyses of:  FISH abnormalities  Shanafelt et al. J Clin Oncol 2006  Global DNA abnormalities by comparative genomic hybridization and SNP profiling  Braggio, Kay et al. Leukemia 2102 B. Analyses of DNA abnormalities by next generation sequencing of CLL exomes and genomes

10. Clonal evolution in CLL B.Analyses of DNA abnormalities by next generation sequencing of CLL exomes and genomes Initial studies in 2011:  Puente et al. Nature 475: 101-105, 2011  Fabbri et al. J Exp Med 208: 1389-1401, 2011  Wang et al. N Engl J Med 365: 2497-2506, 2011 Since then a number of additional and more intricate studies:  Quesada et al. Nat Genet 44: 47–52, 2012  Schuh et al. Blood 120, 4191–4196, 2012  Landau et al. Cell 152: 714–726, 2013

11. Summary of consistent findings B.Analyses of DNA abnormalities by next generation sequencing of CLL exomes and genomes  Genomic complexity exists in CLL of a degree less than that of solid tumors and DLCBL; similar to AML  Over 20 recurrent mutations were identified. Most common abnormality is in NOTCH1  Specific mutations associate with at least 7 biological pathways  Mutations appear to fall into two categories: initiating clonal driver mutations and secondary, subclonal mutations  Subclonal mutations often emerge after therapy but many/most exist prior to therapy

12. Significantly mutated genes and associated pathways Landau et al. Cell 152, 714–726, 2013

13. Associations between specific gene mutations and other characteristics Wang et al. N Engl J Med 365: 2497-2506, 2011

14. Genetic Evolution and Clonal Heterogeneity Result in Altered Clinical Outcome Genetic evolution and clonal heterogeneity result in altered clinical outcome Landau et al. Cell 152, 714–726, 2013

15. Frequency of genetic alterations in CLL, early and later in disease progression Gruber and Wu. Semin Hematol 51:177-187, 2014

16. A model for the stepwise evolution of CLL Landau et al. Cell 152, 714– 726, 2013

17. Clonal evolution in CLL C.Acquisition of therapy resistance as a consequence of clonal evolution Example: resistance to ibrutinib treatment  Woyach et al. N Engl J Med 370: 2286-2294, 2014  Furman et al. N Engl J Med 370: 2352-2354, 2014

18. Effect of C481S mutation of BTK on ibrutinib binding and the ability of ibrutinib to inhibit BTK phosphorylation Furman et al. N Engl J Med 370: 2352-2354, 2014

19. Functional characterization of PLCγ2 with the R665W and L845F mutations Woyach et al. N Engl J Med 370: 2286-2294, 2014

20. A model for the stepwise evolution of CLL Landau et al. Cell 152, 714– 726, 2013

21. Background All CLL clones are heterogeneous based on:  Surface membrane and intracellular phenotypes  Telomere length and telomerase activity  Survival and growth requirements  Ongoing IGHV mutations  Chromosomal and specific gene abnormalities  Time since last replication – “Age”  Calissano et al. Mol Med 2011 This type of heterogeneity is not “fixed” and “mutant” but is “dynamic” and “physiologic ”

22. Deuterated (“heavy”) water - 2 H 2 O

23. HydrogenDeuterium 2H2O2H2O Gas chromatography/ Mass spectrometry CLL cells DNA In vivo “pulse-chase” study

24. What can these studies tell us?  Birth and death/elimination rates of CLL clones  Messmer et al. J Clin Invest 115: 755, 2005  van Gent et al. Cancer Res 68: 10137, 2008  deFoiche et al. Br J Haematol: 143: 240, 2008  Means to indirectly identify cells that have most recently been born/divided in patients  Calissano et al. Blood 114: 4832-4842, 2009  Calissano et al. Mol Med 17: 1374-82, 2011 Cell fractions with more cells with 2 H-labeled DNA contain the most recently replicated/born cells

25. CXCR4 CD5 19 + 3 - CXCR4 bright CD5 dim CXCR4 int CD5 int CXCR4 dim CD5 bright Gas chromatography/ Mass spectrometry Deuterium content of fractions sorted based on reciprocal densities of CXCR4 and CD5

26. The CXCR4 dim CD5 brite fraction is significantly enriched in cells with 2 H-labelled DNA 02142 0 10 20 30 Days 2 H enrichments in DNA PROL INT/BULK REST CXCR4 int CD5 int : “INT” (tumor bulk) CXCR4 dim CD5 bright : “DIM” (proliferative) CXCR4 bright CD5 dim : “ BR” (resting)

27. G0G0G0G0 G1G1G1G1S G2G2G2G2M Ki67 P<0.01 The CXCR4 dim CD5 brite fraction is significantly enriched in cells expressing Ki-67 N=13 CXCR4brCD5dimCXCR4intCD5intCXCR4dimCD5br0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 CXCR4 br CD5 dim CXCR4 int CD5 int CXCR4 dim CD5 br % Ki-67 positive cells

28. Stromal cell Nurse like cell SDF-1(CXCL12) Solid Tissue Blood Exit Blood BCR signaling TLR signaling CD5 CLL CXCR4 CD38 BCR CLL CXCR4 CD5 CD38 BCR CLL CXCR4 CD5 CD38 BCR CLL CXCR4 CD5 CD38 BCR CLL CXCR4 CD5 CD38 BCR CLL CXCR4 CD38 BCR CLL CXCR4 CD38 BCR Release DeathLife Proliferativecompartment Intraclonal heterogeneity – time since birth/replication Resting, re-entry compartment CD5 Bulk Re-initiate or survive/rest

29. Why should we care about the “proliferative fraction” if it represents only ~1% of a CLL clone? CLL patients progress to more severe disease when members of the clone develop new DNA mutations over time – “clonal evolution”  Permanent new DNA mutations can only occur when new strands of DNA are made, as cells divide and proliferate  Hence, the “proliferative fraction” contains potentially very dangerous CLL cells since they just replicated their DNA

30. What is the “mutational process” that can induce somatic point mutations and DNA deletions as causes of ongoing genomic lesions in CLL? Is the “mutational process” more active in the proliferative fraction of CLL cells?

31. Activation-induced cytidine deaminase (AID) 1.Essential and sufficient to initiate DNA point mutations that lead to repair with different nucleotides during a germinal center reaction. 2. Essential and sufficient to initiate DNA deletions that are an intimate component of IGH class switch recombination, although other elements are required to repair the break points and seal off the deletion 3. Shown to have 3. Shown to have “off target activity” (i.e., mutate or delete outside the IGV locus) in a wide range of both hematological and non-hematological cancers -AID can act as an oncogenic enzyme

32. Sort Strategy:ProliferativefractionRestingfraction CD5 CXCR4 Proliferative Fraction Resting Fraction Intermediate Fractions AID Beta Actin PCR FOR AID CD23CXCR4 CD5 AID mRNA is enriched in the peripheral blood proliferative fraction Patten et al. Blood 120:4802, 2012

33. Clear: CD5 + CD19 + Cells Tinted: CD19 - Cells AID protein expression is inducible in peripheral blood CLL co-cultures t= 0 hrs AID + cells<1% t= 168 hrs AID + cells 65% CD23 RPA AID Composite Stimulation strategy: CLL PBMCS cultured with murine fibroblasts (L cells) plus anti-CD40 and IL-4 x630 t= 72 hrs AID + cells 27% Patten et al. Blood 120:4802, 2012

34. Multiple Divisions PBMCs with L-cells plus anti-CD40 and IL-4 0.1% PBMCs with L cells No Division IMC: Red 0.1% 75%0.1% AID: Blue AID protein is expressed primarily in dividing cells CD5 + CD19 + cells D7 culture UNSTIMULATED STIMULATED CD5 + CD19 + cells D7 culture

35. Is this inducible AID functional? Confocal assay for the presence of double strand DNA breaks within cell nuclei – Anti-phospho-histone H2A.X staining (pH2A.X) Evidence of immunoglobulin class switching Appearance of new mutations in IGHV/D/J transcripts by single cell PCR

36. Increased double strand DNA breaks are seen in divided CLL cells: anti-pH2A.X staining Stimulated Cells: D14 of culture CD23:Red Unstimulated Cells: D14 of culture CD23 pH2A.X (DSBs)CFSE x630 All x630 pH2A.X (DSBs)

37. % Stimulated cells expressing pH2A.X greater than unstimulated cells % of cells CD5 + CD19 + Cells CFSE AID IMC AID expression by stimulated cells CFSE (Log Scale)n=3 Increased double strand breaks are seen in divided CLL cells: anti-pH2A.X staining

38. Confocal assay for the presence of double strand breaks within cell nuclei – Anti-phospho-histone H2A.X staining (pH2AX) Evidence of immunoglobulin class switching Appearance of new mutations in IGHV/D/J transcripts by single cell PCR Is this inducible AID functional?

39. Immunoglobulin class switching STIMULATION: for up to 14 days RT PCR with clone specific VH and CH primers UNSWITCHED:  transcripts SWITCHED:  transcripts (  transcripts) Sequencing: analysis only on clone specific V-D-J Sort 2 populations:UNDIVIDED or or MULTIPLY DIVIDED 20 cell aliquots CFSE FSC-A CD5 + CD19 + Cells Patten et al. Blood 120:4802, 2012

40. p=0.0002 Divided cells contain Ig class switched transcripts Wells without switched transcripts Wells with switched transcripts Divided11322 Undivided891 Unstimulated450 (n=3) % Positive wells for switched transcripts Percentage

41. % IgG expression vs division Surface IgG expression increases with cell division Undivided <0.1% CD19 IgG Multiply Divided IgG CD19 1% CFSE FSC-A CD5 + CD19 + Cells CLL1299

42. Confocal assay for the presence of double strand breaks within cell nuclei – Anti-phospho-histone H2A.X staining (pH2AX) Evidence of immunoglobulin class switching Appearance of new mutations in IGHV/D/J transcripts by single cell PCR Is this inducible AID functional?

43. Sorted single cells: same strategy as for class switch analysis High fidelity reverse transcriptase and polymerase The experimental ERROR RATE following both steps: – less than 6 x 10 -6 per base pair Mutations in IGHV/D/J genes

44. Mutations in IGHVDJ rearrangements No of mutations per 10 4 base pairs UNMUTATED IGHV CLL1278: UNMUTATED IGHV MUTATED IGHV CLL1082: MUTATED IGHV

45. Lymph node CLL cells can express AID protein x630 Red-CD23;Blue-Ki67;Green-AID AID + cells are Ki-67 + ; many Ki-67 + cells are AID - x100DAB staining anti-AID 10 Cases: 5 demonstrate scattered AID + cells Patten et al. Blood 120:4802, 2012

46. Lymph node: flow cytometry CD19 CD5 93% AID 0.02%1.2% AID + Blocking PeptideAID Alone MFI AID + Cells MFI All CD5 + CD19 + Cells of surface markers for the proliferative and resting CLL phenotypes % Greater in all CD5 + CD19 + Cells % Greater in AID + Cells

47. Does AID expression correlate with clinical course in patients?

49. AID + CLL patients correlate with increased numbers of cytogenetic aberrations and worse clinical outcomes P = 0.02P = 0.001 P = 0.02P = 0.001 P = NS Patten et al. Blood 120:4802, 2012

50. Summary All CLL clones are heterogeneous at all points in time This heterogeneity can be genetic/fixed or physiologic/dynamic Those clonal submembers that divide are more likely to upregulate AID and therefore develop new genetic changes The degree of intraclonal genetic heterogeneity correlates with CLL disease progression and shorter time-to-treatment and length of survival Over time, and especially with therapy, these intraclonal genetic variants can outcompete the initial major clonal submembers – Clonal Evolution