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