Clonal Evolution in CLL:
Impact on timing of therapy
Nicholas Chiorazzi
The Feinstein Institute for Medical Research
North Shore – LIJ Health System
Manhasset, NY
Three principles relevant for a discussion
of clonal evolution
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
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
Evolution and growth in our understanding of
CLL heterogeneity over time
Gruber and Wu. Semin Hematol 51:177-187, 2014
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
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:





Most common new lesions:



U-CLL clones and in M-CLL clones of patients that eventually
require therapy
CD38+ clones
ZAP-70+ clones
CD49d+ clones
del(13q)
del(17p) – harbinger of accelerated disease
Greater the number of clonal aberrations the shorter the time to
treatment and survival
Karyotype evolution and survival
Shanafelt et al. J Cin Oncol;26:e5-e6, 2008
The greater the genomic complexity,
the shorter progression-free survival (CGH)
Kay et al. Cancer Genet
Cytogenet 203:161-8, 2010
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
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
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
Significantly mutated genes and associated pathways
Landau et al. Cell 152, 714–726, 2013
Associations between specific gene mutations
and other characteristics
Wang et al. N Engl J Med 365: 2497-2506, 2011
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
Frequency of genetic alterations in CLL,
early and later in disease progression
Gruber and Wu. Semin Hematol 51:177-187, 2014
A model for the stepwise evolution of CLL
Landau et al.
Cell 152, 714–
726, 2013
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
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
Functional characterization of PLCγ2
with the R665W and L845F mutations
Woyach et al. N Engl J Med 370: 2286-2294, 2014
A model for the stepwise evolution of CLL
Landau et al.
Cell 152, 714–
726, 2013
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 ”
Deuterated (“heavy”) water - 2H2O
2H O
2
DNA
Hydrogen
Deuterium
In vivo “pulse-chase” study
CLL cells
DNA
Gas chromatography/
Mass spectrometry
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 2H-labeled DNA contain
the most recently replicated/born cells
Deuterium content of fractions sorted
based on reciprocal densities of CXCR4 and CD5
CXCR4brightCD5dim
CXCR4
CXCR4intCD5int
CXCR4dimCD5bright
CD5
Gas chromatography/
Mass spectrometry
The CXCR4dimCD5brite fraction
is significantly enriched in cells with 2H-labelled DNA
CXCR4dim
CD5bright:
“DIM”
(proliferative)
30
PROL
INT/BULK
REST
CXCR4bright
CD5dim:
“BR”
(resting)
2H
CXCR4int
CD5int :
“INT”
(tumor bulk)
enrichments in DNA
20
10
0
0
21
Days
42
% Ki-67 positive cells
The CXCR4dimCD5brite fraction
is significantly enriched in cells expressing Ki-67
P<0.01
4.5
4.0
3.5
3.0
2.5
2.0
CXCR4 br CD5 dim
CXCR4 int CD5 int
CXCR4 dim CD5 br
N=13
1.5
1.0
0.5
0.0
CXCR4 br CD5
dim
CXCR4 int CD5
int
CXCR4 dim CD5
Ki67
G0
G1
S
G2
M
br
Intraclonal heterogeneity – time since birth/replication
Resting, re-entry
compartment
Blood
CD38
Life
Death
Bulk
CD38
BCR
CLL
CXCR4
CD5
CLL
CXCR4
CD5
BCR
CD38
Solid Tissue
Re-initiate or survive/rest
CD38
CD38
CLL
CD5
SDF-1
CXCR4
Release
BCR
(CXCL12)
CXCR4
BCR
CD38
CLL
Stromal cell
Nurse like cell
CD5
BCR
CD38
CXCR4
BCR signaling
TLR signaling
CLL
BCR
CD5
CD5
Blood
CLL
CXCR4
CLL
Exit
CXCR4
BCR
Proliferative
compartment
CD5
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
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?
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 “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
AID mRNA is enriched in the peripheral
blood proliferative fraction
Sort Strategy:
CXCR4
Proliferative
fraction
CD5
CXCR4
CD23
Resting
fraction
CD5
AID
PCR FOR AID
Beta
Actin
Patten et al. Blood 120:4802, 2012
AID protein expression is inducible in
peripheral blood CLL co-cultures
AID+
t= 0 hrs
t= 72 hrs
cells<1%
+ Cells
Clear: CD5+AID
CD19
+ cells
27%
Tinted: CD19 Cells
t= 168 hrs
AID+ cells 65%
Stimulation strategy:
CLL PBMCS cultured with murine fibroblasts (L cells) plus
anti-CD40 and IL-4
CD23
Composite
RPA
AID
x630
Patten et al. Blood 120:4802, 2012
AID protein is expressed
primarily in dividing cells
UNSTIMULATED
STIMULATED
PBMCs with L cells
PBMCs with L-cells
plus anti-CD40 and IL-4
0.1%
0.1%
75%
0.1%
AID: Blue
IMC: Red
CD5+CD19+ cells D7 culture
No Division
CD5+CD19+ cells D7 culture
Multiple
Divisions
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
Increased double strand DNA breaks are seen in
divided CLL cells: anti-pH2A.X staining
Stimulated Cells: D14 of culture
x630
CD23
pH2A.X
CD23:Red
(DSBs)
CFSE
All
CFSE
All
Unstimulated Cells: D14 of culture
x630
CD23
pH2A.X (DSBs)
CD23:Red
Increased double strand breaks are seen
in divided CLL cells: anti-pH2A.X staining
% Stimulated cells expressing pH2A.X
greater than unstimulated cells
AID expression by
stimulated cells
CFSE
% of cells
AID
IMC
CD5+CD19+ Cells
CFSE (Log Scale)
n=3
Is this inducible AID functional?
• 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
Immunoglobulin class switching
CD5+CD19+ Cells
Sort 2 populations:
UNDIVIDED
or
MULTIPLY DIVIDED
FSC-A
STIMULATION:
for up to 14 days
20 cell aliquots
CFSE
UNSWITCHED:
m transcripts
SWITCHED:
 transcripts
(a transcripts)
Sequencing: analysis only on clone specific V-D-J
RT PCR with clone
specific VH and
CH primers
Patten et al. Blood 120:4802, 2012
Divided cells contain Ig class switched transcripts
Percentage
% Positive wells for
switched transcripts
Wells without
switched
transcripts
Wells with
switched
transcripts
Divided
113
22
Undivided
89
1
Unstimulated
45
0
p=0.0002
(n=3)
Surface IgG expression increases with cell division
CD5+CD19+ Cells
CLL1299
FSC-A
% IgG expression vs division
CFSE
CD19
1%
IgG
Undivided
<0.1%
CD19
Multiply Divided
IgG
Is this inducible AID functional?
• 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
Mutations in IGHV/D/J genes
• 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
No of mutations per 104 base pairs
Mutations in IGHVDJ rearrangements
CLL1278: UNMUTATED IGHV
CLL1082: MUTATED IGHV
Lymph node CLL cells can express AID protein
Red-CD23;Blue-Ki67;Green-AID
10 Cases:
5 demonstrate
scattered AID+cells
x630
AID+ cells are Ki-67+;
many Ki-67+ cells are AIDDAB staining anti-AID
x100
Patten et al. Blood 120:4802, 2012
Lymph node: flow cytometry
AID + Blocking Peptide
CD5
93%
AID
0.02%
AID Alone
1.2%
CD19
MFI AID+ Cells
MFI All CD5+CD19+ Cells
% Greater in all
CD5+CD19+ Cells
% Greater in
AID+ Cells
of surface markers
for the
proliferative and resting
CLL phenotypes
Does AID expression correlate with clinical
course in patients?
AID+ CLL patients correlate with increased numbers
of cytogenetic aberrations
and worse clinical outcomes
P = 0.02
P = 0.001
P = 0.001
P = 0.02
P = NS
P = NS
P = 0.001
P = 0.001
Patten et al. Blood 120:4802, 2012
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