clonal evolution in cll: impact on timing of therapy nicholas chiorazzi the feinstein institute for...
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Clonal Evolution in CLL: Impact on timing of therapy
Nicholas Chiorazzi
The Feinstein Institute for Medical ResearchNorth Shore – LIJ Health System
Manhasset, NY
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 discussionof clonal evolution
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
Gruber and Wu. Semin Hematol 51:177-187, 2014
Evolution and growth in our understanding of CLL heterogeneity over time
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:
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
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 inAltered 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
Hydrogen Deuterium
2H2O
Gas chromatography/Mass spectrometry
CLL cells
DNA
DNA
In vivo “pulse-chase” study
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
C
XCR4
CD519+ 3-
CXCR4brightCD5dim
CXCR4intCD5int
CXCR4dimCD5bright
Gas chromatography/Mass spectrometry
Deuterium content of fractions sorted based on reciprocal densities of CXCR4 and CD5
The CXCR4dimCD5brite fraction is significantly enriched in cells with 2H-labelled DNA
0 21 420
10
20
30
Days
2 H e
nric
hmen
ts in
DN
A
PROL
INT/BULK
REST
CXCR4int
CD5int :“INT”(tumor bulk)
CXCR4dim
CD5bright:“DIM”(proliferative)
CXCR4bright
CD5dim:“BR” (resting)
G0 G1 S G2 M
Ki67
P<0.01
The CXCR4dimCD5brite fraction is significantly enriched in cells expressing Ki-67
N=13
CXCR4br CD5 dim CXCR4int CD5 int CXCR4dim CD5 br
0.00.51.01.52.02.53.03.54.04.5
CXCR4br CD5dim
CXCR4int CD5 int
CXCR4dim CD5br
% K
i-67
posi
tive
cells
Stromal cellNurse like cell
SDF-1(CXCL12)
Solid Tissue
Blood
Exit
Blood
BCR signalingTLR signaling
CD5CLL
CXCR4
CD38BCR
CLL
CXCR4
CD5CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLLCXCR4
CD38
BCR
CLL
CXCR4
CD38
BCR
Release
Death
Life
Proliferativecompartment
Intraclonal heterogeneity – time since birth/replication
Resting, re-entrycompartment
CD5
Bulk
CD5Re-initiate or survive/rest
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
Sort Strategy:
Proliferative fraction
Resting fraction
CD5
CXCR4Pro
liferative
Fracti
on
Resting F
raction
Intermediate Fr
actions
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
Clear: CD5+CD19+ CellsTinted: CD19- Cells
AID protein expression is inducible in peripheral blood CLL co-culturest= 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
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
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
CD23:Red
CD23:Red
Unstimulated Cells: D14 of culture
CD23
CD23
pH2A.X (DSBs) CFSE
CFSE
x630 All
Allx630 pH2A.X (DSBs)
% Stimulated cells expressing pH2A.X greater than unstimulated cells
% o
f cel
ls
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
• 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?
Immunoglobulin class switching
STIMULATION:for up to 14 days
RT PCR with clone specific VH andCH primers
UNSWITCHED:m transcripts
SWITCHED:g transcripts(a transcripts)
Sequencing: analysis only on clone specific V-D-J
Sort 2 populations:
UNDIVIDED orMULTIPLY DIVIDED
20 cell aliquots
CFSE
FSC-
A
CD5+CD19+ Cells
Patten et al. Blood 120:4802, 2012
p=0.0002
Divided cells contain Ig class switched transcripts
Wells without switched transcripts
Wells with switched transcripts
Divided 113 22
Undivided 89 1
Unstimulated 45 0
(n=3)
% Positive wells for switched transcripts
Perc
enta
ge
% IgG expression vs division
Surface IgG expression increases with cell division
Undivided
<0.1%
CD19
IgG
Multiply Divided
IgG
CD19
1%
CFSE
FSC-
ACD5+CD19+ Cells
CLL1299
• 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?
• 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
Mutations in IGHVDJ rearrangements N
o of
mut
ation
s pe
r 104
base
pai
rs
CLL1278: UNMUTATED IGHV CLL1082: MUTATED IGHV
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
Lymph node: flow cytometry
CD19
CD5 93%AID
0.02% 1.2%
AID + Blocking Peptide AID Alone
MFI AID+ Cells
MFI All CD5+CD19+ Cells
of surface markersfor the proliferative and resting CLL phenotypes% Greater in all
CD5+CD19+ Cells% Greater in
AID+ Cells
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 = 0.02P = 0.001 P = 0.001
P = NS
P = NS
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