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Novel Insights into the Biology of CLL

Mark C. Lanasa1

1Division of Medical Oncology, Duke University Medical Center, Durham NC

Significant advancements in the care of patients with chronic lymphocytic leukemia (CLL) have occurred over the pastdecade. Nonetheless, CLL remains incurable outside of allogeneic transplantation. CLL is the most common leukemia

in the United States and Europe, and new treatments and therapeutic strategies are clearly needed. To address this

need, the pathogenesis of CLL has been an area of intense ongoing investigation. These international efforts illuminate

a complex biology that is reliant on the interplay of inherited, environmental, and host factors. This broad review will

discuss the recent advances in our understanding of CLL biology including the elucidation of inherited and acquired

genetic changes; the role of the B-cell receptor and B-cell receptor signaling; CLL cell kinetics; and the interactions in

the microenvironment between CLL cells, other immune cells, and stromal elements. This improved understanding of

disease pathogenesis is facilitating the development of novel therapeutic treatment strategies.

Chronic lymphocytic leukemia (CLL) remains an enigmatic dis-

ease. Although the first clinical description of chronic lymphoidleukemia was published over 150 years ago, the etiology of CLL is

unknown, the cell of origin of CLL is unknown, and CLL remains

incurable outside of allogeneic transplantation. CLL is the most

common leukemia in the United States, with approximately 15,500

new diagnoses per year. Because of the relatively long survival of

patients with CLL, it is by far the most prevalent leukemia, with an

estimated 95,000 Americans living with CLL.1 CLL lymphocytes

have a characteristic immunophenotype: CD5, CD19, CD20dim,

CD23, and surface immunoglobulindim. Although the majority of

patients are asymptomatic at diagnosis, the relentless accumulation

of CLL lymphocytes leads to symptomatic disease, need for

CLL-directed therapy, disease-related complications, and approxi-

mately 4500 CLL attributable deaths per year in the United States.

Several characteristics of CLL facilitate basic and translational

research: (i) the high population prevalence; (ii) the malignant cells

are easily obtained through venous phlebotomy; (iii) most patients

have an asymptomatic phase that allows for longitudinal evaluation;

and (iv) CLL is has a relatively long disease-specific survival.

Therefore, CLL has become a model system for the investigation of

B-cell lymphoproliferative disorders. In the 5 years since the

biology of CLL was last broadly reviewed in the American Society

of Hematology education session, tremendous progress has been

made in the understanding of CLL disease biology, and this review

will focus on these discoveries. Specifically, important advances

have been made in identifying inherited and acquired genetic

mutations, the role of B-cell receptor (BCR) signaling, and the

interplay between the malignant B cells and the tumor microenviron-ment. These advances reveal CLL to be a disease that is dependent

on on the interplay of inherited, environmental, and host factors.

Inherited Genetic FactorsThe most important risk factor for the development of CLL is a

family history of CLL. Among patients with newly diagnosed CLL,

8% to 10% have a family history of CLL. CLL has a heritability that

is twice that of common solid tumors with known low-prevalence,

high penetrance causative genes, such as breast and colon cancer. 2,3

Pedigree analysis using data from the Swedish Family Cancer

Database showed the relative risk of CLL among first-degree

relatives of persons with CLL to range between 7.0 and 8.5. This

equivalent risk across all first-degree relatives suggests an inherited,rather than environmental, basis for familial CLL. 4 Though families

with four or more cases of CLL have been reported, they are

extremely rare. Some CLL kindreds suggest a dominant inheritance,

therefore, genome-wide linkage studies in familial CLL have been

performed. The largest and most recent reported study added 101

new cases of familial CLL to a previously reported cohort of 105

families.5 This study was the first statistically significant genome-

wide linkage scan and identified chromosome 2q21.2 as associated

with inheritance of CLL. However, no causative genes have been

identified at this locus. Overall, linkage studies in CLL have been

limited by the low number of affected individuals per family, late

age of onset, and presumed genetic heterogeneity.

Complete sequencing of the human genome revealed large numbers

of common single nucleotide polymorphisms (SNPs). This discov-

ery led to the hypothesis that the inheritance of complex diseases

may be due to the coinheritance of these common variants. Di

Bernardo et al6 performed a multistage genome-wide SNP associa-

tion (GWA) study to evaluate the contribution of common variants

to the inheritance of CLL. This study identified six novel loci

associated with the development of CLL, thus providing the first

evidence for the contribution of common (population prevalence

5%) genetic variants to the development of CLL. Five of these SNPs

have been validated in an independent GWA.7 Subsequently, four

additional susceptibility loci have been identified through follow-up

analyses from the initial Di Bernardo study.8 Identification of these

genes informs our understanding of CLL biology. For example, theGWA linked interferon regulatory factor 4 (IRF4), a key regulator

of lymphocyte maturation and proliferation, with risk of developing

CLL. The 10 loci identified to date individually confer a small risk

of disease, with relative risks ranging from 1.2 to 1.6. As a group,

these risk alleles account for 10% of the total heritability of CLL.

Because GWA can detect only those alleles with a population

prevalence of 5%, it is possible that much of the inherited risk of

CLL is due to uncommon inherited variants (prevalence 5%).

Massively parallel sequencing technologies may enable the discov-

ery of these rare variants and illuminate the hidden heritability

in CLL.

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Acquired Genetic FactorsIn a landmark report by the German CLL Study Group, Dohner et

al9 showed that acquired chromosomal abnormalities involving

chromosomes 11, 12, 13, and 17 are common in CLL, and that these

abnormalities predict both time to first treatment and CLL-specific

survival. The genes principally responsible for the adverse progno-

sis associated with del 17p13 and del 11q22 were recognized to be

TP53 and ATM, respectively. The genes that contribute to CLL

pathogenesis in trisomy 12 remain unknown. There is ongoing

debate regarding whether trisomy 12 confers an increased risk of

disease progression; unlike the Dohner study, our institutional

experience is that patients with trisomy 12 do follow a somewhat

more aggressive disease course than those patients with normal

FISH (fluorescence in situ hybridization).10 Deletion of 13q14 is the

most common and most favorable cytogenetic abnormality in CLL.

The responsible genes at this locus were initially unclear. In 2002,

Calin et al11 showed that the microRNA 15/16 (miR 15/16) cluster

were the critical deleted genes in this region. MicroRNAs are small

(approximately 20 nucleotides) nonprotein coding RNAs that

modulate the level of specific proteins by binding sequence-

complimentary mRNAs. miR-15 and miR-16 were subsequently

shown to be negative regulators of BCL2.12 This discovery is

notable in that it was the first association of clinical disease with

microRNAs.

The Dohner study9 established interphase cytogenetics (FISH) as

the clinical standard of care for evaluation of chromosomal aberra-

tions in CLL because the low proliferative capacity of most CLL

limits the clinical utility of metaphase cytogenetics. However,

emerging data shows that the evaluation of four (or five) chromo-

somal loci by FISH obscures the significant heterogeneity and

complexity of acquired chromosomal defects in CLL. The applica-

tion of high-density SNP arrays illuminates some of this complex-

ity. For example, copy number neutral loss of heterozygosity is

observed in a significant number of CLL cases, and this finding

cannot be detected with either FISH or standard karyotyping. Using

SNP arrays, Ouilette et al13 showed significant heterogeneity among

13q14 deletions, with some deletions extending centromeric to

include the retinoblastoma gene. A follow-up study showed that

13q14 deletions that include retinoblastoma are associated with

genomic complexity,14 a finding associated with aggressive disease

in prior clinical reports. Although data currently available are

inadequate to support the widespread application of SNP arrays to

routine clinical care, this technology may ultimately be a useful

adjunct to routine clinical FISH.

At the chromosomal level, del 17p13 is more homogenous than

other acquired genomic defects. High-density SNP arrays showed

that the deletion breakpoint typically falls within chromosomal band

17p11.2 and extends telomeric to include most of the p arm.15

Deletions of 17p13 are almost always monoallelic, and it was

initially unclear why monoallelic loss of TP53 would cause such a

dramatic change in CLL proliferative capacity, chemosensitivity,

and prognosis. It is now understood that 80% of cases with del

17p13 have single nucleotide somatic mutations of the TP53 allele

on the other chromosome 17 (the allele in trans), thereby causing

near complete loss of TP53 function. Although uncommon, TP53

inactivating mutations can occur in the absence of del 17p13, and

these mutations apparently confer an equivalent adverse risk as del

17p13.16 TP53 is a multimeric protein with four identical subunits,

and these inactivating mutations may act in a dominant negative

manner, thus abrogating TP53 function even in the presence of a

wild-type allele in trans. Although only 5% of CLL cases show

deletion or mutation of 17p13 at diagnosis, somatic mutations occur

in approximately one-third of relapsed or chemotherapy refractory

patients. Whether this represents clonal selection or clonal evolution

or both is an area of active investigation.

Epigenetic changes are also relevant to the pathogenesis of CLL.

Epigenetic changes are noninherited chromosomal modifications

that affect gene transcription, such as methylation of gene promoters

and acetylation of histone-bound DNA. An example of epigenetic

change, global hypomethylation of DNA, has been described in

CLL. Between 2% and 8% of CpG islands (gene promoter

elements) are aberrantly methylated in CLL when compared with

normal B cells, a finding that suggests DNA methylation broadly

affects the transcriptional profile of CLL. A germline mutation in

death-associated protein kinase 1 (DAPK1) that segregated with

risk of CLL was identified by Raval et al18 in a family with multiple

CLL cases. The authors showed that DAPK1 expression is silenced

through promoter methylation in the majority of CLL cases,

suggesting a central role for both epigenetic modification and

DAPK1 in CLL leukemogenesis.18 Using high-density methylation

microarrays evaluating over 27,000 CpG sites, Kanduri et al19

identified differential patterns of methylation that were dependent

on the mutation status of the BCR. Poor-risk CLL showed

methylation profiles that facilitated signaling through proliferative

cellular pathways, including MAPK (mitogen-activated protein

kinase) and NF-B (nuclear factor- light-chain enhancer of

activated B cells), a finding that directly relates the clinical

phenotype of poor-risk CLL to methylation of BCR-mediated

signaling pathways.

The Role of BCR in CLLInvestigation of the immunoglobulin gene has led to findings that

are central to the current understanding of CLL (Table 1). During

B-cell development, variable-diversity-joining (VDJ) recombina-

tion generates an immunoglobulin heavy chain (IGH), and an

immunoglobulin light chain (IGL) is created through isotype-

specific VJ recombination. There is tremendous combinatorial

potential in this process, with perhaps 1010 potential IGH-IGL

sequence combinations. However, patients with CLL use a highly

restricted set of BCRs.20 Approximately 14% of all CLL cases

express the heavy chain VH169, and an additional 18% express

VH434. This finding of marked restriction of the IGH repertoire

led to the hypothesis that tonic stimulation by specific auto or

alloantigens drives expansion of the malignant clone.21,22 Intra-

clonal diversification of some CLL further supports the antigen-

drive hypothesis.23

During the germinal center reaction, somatic mutations are gener-

ated in the BCR to increase affinity for target antigen. Approxi-

mately half of CLL cases show IGH mutations, and the other half

Table 1. The mutation status of the immunoglobulin heavy chain (IGVH)

as a central determinant of CLL biology

IGVH Unmutated IGVH Mutated

Cell of origin Mature (antigen experienced) B

cell

Mature (antigen experienced) B

cell

Postulated mechanism of

maturation

Germinal center, T-help

dependent

T-help independent, germinal

center independent (?)

Chromosomal abnormalities del 11q22, del 17p13 del 13q14 (sole)

Frequency of BCR stereotypy 40% 10%

Antigen recognition Polyreactive oligoreactive

Response to BCR cross-linking Activation, proliferation Anergy or no response

Hematology 2010 71


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show an unmutated IGH, typically defined as 2% deviation

from germline sequence. Patients with mutated IGH typically

follow a more indolent disease course than those with unmutated

IGH24. This initially led to the hypothesis that CLL may be two

distinct entities, one deriving from naive (IGH unmutated) B cells

and the other from postgerminal center memory (IGH mutated) B

cells. However, gene expression profiling studies in CLL show both

IGH mutated and unmutated CLL to be derived from memory B

cells.25,26 The mechanism by which some CLL escape IGH mutation

is unclear. One hypothesis proposes that certain antigens favor ahelper T-cell independent or germinal center independent matura-

tion that does not involve IGH mutation. Alternatively, the IGH

unmutated CLL cells may be autoreactive and targeted for apopto-

sis, but avoid cell death through either tonic BCR stimulation or

transforming genetic events.

Recent experiments using phage display libraries have attempted to

better define the types of antigens and epitopes recognized by IGH

mutated and unmutated CLL. Monoclonal antibodies (MAbs)

generated from the BCR of IGH-mutated CLL bound epitopes with

structurally related amino acid motifs, whereas unmutated CLL

recognized multiple different epitopes.27 This suggests that in vivo

multiple, potentially structurally divergent antigens can bind and

stimulate CLL cells through the BCR. This conclusion is provoca-

tive because six of eight of the CLL MAbs investigated had

stereotyped BCRs. Stereotypy is the observation of near complete

sequence homology of the complimentarity-determining region 3

(CDR3) of the IGH28; the CDR3 largely defines the antigen

specificity of the BCR. For example, approximately 1% of all CLL

express VH1 69 that are virtually identical within the CDR3.29 The

presence of a stereotyped BCR is associated with adverse risk,

independent of IGH mutation status, although stereotypy is more

commonly observed among unmutated (40%) than mutated CLL

(10%). The observation of stereotypy has been forwarded as

evidence of selective antigenic pressure in CLL; however, MAbs

derived from these BCRs may be oligo- or polyreactive in vivo.

MAbs derived from stereotyped BCRs have also been shown to

react with conserved epitopes exposed on apoptotic cells.30 The

selection of stereotyped BCRs may be related to a combination of

antigenic pressure and CDR3 sequence facilitated BCR signaling.

Given the clear importance of the BCR in the clinical behavior of

CLL, the role of BCR-mediated signaling is another area of

significant interest. The surface density of the BCR is lower in CLL

than in most B-cell subsets, suggesting CLL may have attenuated

signaling responses to antigen binding. Experimental evidence

shows that, in vitro, approximately half of all CLL retain BCR-

mediated signaling, and that the IGH mutation status and other

molecular characteristics used for clinical prognostication (eg,

CD38 and ZAP70 [zeta-associated protein 70]) are important

determinants of BCR responsiveness. After cross-linking the BCRs

with isotype-specific antibodies, Lanham et al31 showed global

increases in tyrosine phosphorylation, a marker of induction of

intracellular signaling. The responsiveness to cross-linking was

strongly associated with IGH mutation status (P .0005), as well as

expression of CD38 (P .05).31 Using gene expression profiling,

Guarini et al32 showed that BCR cross-linking with anti-IgM led to

the transcription of gene pathways regulating cell-cycle regulation,

cytoskeletal organization, and proliferation; but, these changes were

only observed among IGH unmutated cases. BCR ligation leads to

enhanced intracellular signaling among ZAP70 expressing CLL,

and the introduction of ZAP70 into CLL not otherwise expressing

ZAP70 augments BCR-mediated signaling, suggesting a direct role

for ZAP70 in BCR-mediated signaling.33 Muzio et al34 showed that

CLL B cells that do not respond to BCR ligation (typically

IGH-mutated cases) show activation of cellular pathways that

suggest anergy. BCR signaling in vivo is likely even more

complicated: assuming that there are multiple antigens capable of

binding the BCR, the binding affinity for a specific antigen likely

determines whether the response is activating or anergic. Although

this complexity in vivo remains incompletely understood, in

general, BCR ligation in IGH unmutated CLL leads to predomi-

nantly activating and proliferative responses, whereas BCR signal-ing in IGH-mutated CLL favors anergic and antiapoptotic re-

sponses. Additionally, the adverse risk associated with ZAP70 and

CD38 expression may be directly related to their effects on cell

signaling and activation.

CLL Cell KineticsThe conventional view of CLL had been that it is primarily a disease

of failed apoptosis and passive accumulation. This view is sup-

ported by the observation that the great majority ( 98%) of

peripherally circulating CLL cells are arrested in G0 or the early G1

phase and have overexpression of antiapoptotic proteins, such as

BCL2. Other observations suggest that CLL may have significant

proliferative capacity. As discussed in the prior section, IGH

unmutated CLL has the capacity for proliferative responses to BCR

ligation. Many CLL express markers of cellular activation (eg,

CD38, CD49d, and CD69), providing a link between CLL cell

biology and prognostics. Clonal evolution and Richters transforma-

tion are observed in some cases. In a series of elegant experiments,

Messmer et al 35 evaluated the in vivo kinetics of CLL by having

patients consume fixed doses of deuterated heavy water (2H2O)

for 84 days. The deuterium was incorporated into newly synthesized

DNA during the S phase. Using mass spectrometry, the birth and

death rates of CLL cells were then determined. Although there

was significant variability in birth rates (0.11%1.76%/day), all

patients had a rate of new cell formation of at least 10 9 new CLL

cells per day. Patients with higher birth rates (exceeding 0.35%/day)

were more likely to have symptomatic disease. Significant variabil-

ity was also observed in the cellular death rate, and the balance

between birth and death likely determines both white blood cell

trend and risk of progression.35

The deuterated water experiments also provided novel insights into

CLL intraclonal heterogeneity and CLL cell trafficking. By flow

sorting the CD38 and CD38 fractions from CLL patients receiv-

ing deuterated water, Calissano et al36 showed that the CD38

fraction proliferated at a greater rate than the CD38 fraction. It has

previously been shown that, regardless of the proportion of CD38

cells, these cells show significantly increased expression of prolifera-

tion factors ZAP70, Ki67, and telomerase when compared with the

CD38 fraction.37 In a more recent report, Calissano et al provided

preliminary evidence that new CLL cells have a distinct immuno-

phenotype: CD5hiCXCR4low, whereas resting CLL cells are

CD5lowCXCR4hi. Gene expression array analysis of flow sorted

new and resting cell fractions that showed that new cells

differentially express genes related to proliferation, cellular activa-

tion, and cell signaling, whereas resting cells predominantly

express genes involved in apoptosis, cell death, and migration. 38

These studies have led to the hypothesis that CLL cells in the blood

compartment eventually enter a pathway of cellular senescence.

Upregulation of CXCR4 (a CXC chemokine receptor) promotes

reentry into the tumor microenvironment, where the cells receive

prosurvival signals. Rather than undergoing apoptosis, a subset of

resting CLL cells are activated, proliferate, downregulate CXCR4,



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and are then released back into the peripheral blood. These studies

shed light on the phenotypic continuum of CLL cells and better

define the life cycle of a CLL B cell, as well as the central role of the

tumor microenvironment in maintaining the malignant clone.

The Tumor Microenvironment in CLLOver the past decade, the essential role of the tumor microenviron-

ment in the survival and progression of CLL has become increas-

ingly clear. The tumor microenvironment describes an admixture of

malignant cells with host immune cells, stromal elements, and

vascular cells that create a niche wherein signals can be transmitted

through antigen presentation, cellcell interactions, and paracrine

signaling. The microenvironments differ in the bone marrow and

secondary lymphoid organs, where the former contains mesenchy-

mal stromal cells (MSC) within vascular niches and in the latter

nurse-like cells (NLCs), T cells and follicular dendritic cells are

present.

NLCs are large CD14 mononuclear cells that are abundant in the

secondary lymphoid organs of patients with CLL.39 NLCs and

MSCs constitutively express the chemokines CXCL12 (stromal-

derived factor-1, Sdf-1) and CXCL13. CLL cells express CXCR4

(CD184), the receptor for Sdf-1, and in vitro studies have shown

that engagement of Sdf-1 with CXCR4 promotes cell survival

through activation of the signal transducer and activator of transcrip-

tion 3 (STAT3) and MAPK pathways. Blockade of this interaction

with a small molecule inhibitor leads to decreased CLL viability,

suggesting this interaction is a potential therapeutic target.40 Addi-

tional studies have shown that CLL cell viability can be significantly

enhanced in vitro with Sdf-1, although viability is further enhanced

with NLCs over Sdf-1, suggesting that NLCs also deliver additional

prosurvival signals. It is now understood that, in addition to Sdf-1,

NLCs also secrete B-cell activating factor (BAFF) and a proliferation-

inducing ligand (APRIL). Engagement of these ligands with CLL

cells induces intracellular NF-B1 and MCL-1, whereas sdf-1

induces ERK1/2 and AKT. As such, NLCs can deliver multiple

prosurvival signals that utilize distinct signaling pathways.41 Engage-

ment of CD40, expressed on the surface of CLL cells, with CD154

(CD40L), expressed on CD4 T cells found in CLL pseudofollicles,

also induces the NF-B pathway. These diverse interactions have

important implications for CLL therapy, particularly as mechanisms

of chemotherapy resistance. A recent report showed that in vitro

coculture of CLL cells with CD154 expressing fibroblasts induced

1000-fold resistance to ABT-737, a small molecular inhibitor of

BCL2 and BCL-XL.42 Similarly, coculture of CLL cells with MSCs

induces resistance to fludarabine, cyclophosphamide, and dexameth-

asone through maintenance of MCL-1.43 Taken together, these in

vitro data strongly suggest that the tumor microenvironment en-

hances CLL cell resistance to both apoptosis and chemotherapy.

Investigation of novel mechanisms of interaction between the tumor

microenvironment and CLL cells is an active area of research. CLL

cells have the capacity to affect local angiogenesis within the

vascular niche. Microvessel density is increased in bone marrow of

CLL patients, with highest densities found at the periphery of

lymphoid aggregates. CLL cells have the capacity to secrete a

variety of angiogenic cytokines, including vascular endothelial

growth factor (VEGF), basic fibroblast growth factor (bFGF), and

thrombospondin-1 (TSP-1).44 More recently, Ghosh et al45 de-

scribed the contribution of CLL cell-derived microvesicles as a

mechanism by which CLL cells can module the local microenviron-

ment. Microvesicles are cell membrane-derived particles that can

deliver cell surface receptors, activated signaling proteins, or nucleic

acids to target cells. The authors found that CLL cell-derived mi-

crovesicles were increased in patients with advanced-stage CLL, and

that microvesicles could activate AKT/mTOR signaling in MSCs.45

Thus, microvesicles represent a novel mechanism of CLL signaling

within the microenvironment. Finally, toll-like receptors (TLRs) are

cell surface receptors that are part of the innate immune system that

bind structurally conserved microbial antigens and activate immune

responses. Recent data shows that TLRs are expressed and func-

tional in CLL; binding of TLRs with cognate antigen-activated

NF-B expression and induced surface expression of activationmarkers CD25 and CD80.46 Given the architectural complexity,

number of different cell types present, and clinical importance of

these interactions, investigation of the tumor microenvironment will

continue to be an active and fruitful area of research in CLL.

T-Cell Abnormalities in CLLMost malignancies are associated with decreased numbers of

circulating T cells, but in CLL they are elevated 2.5 to 4 times

normal, at least pretherapy.47 Although T cells are increased in

number, the T-cell repertoire is significantly contracted in CLL,

with oligoclonal and monoclonal subsets.48,49 CLL cells secrete

immunomodulatory cytokines, such as interleukin-6, interleukin-

10, and tumor growth factor- (TGF-), which shift the helper

T-cell response from a Th1 response to an anergic Th2 response. 50,51

Suppressive regulatory T cells (Treg) are also increased in patients

with CLL.52 A mechanism for this increase may be direct CLLT-

cell contact through CD27 and CD70; this interaction also induces

BCL2 within the Treg, making the T reg cell population in CLL

patients more resistant to apoptosis than in normal individuals.53

These qualitative and quantitative T-cell abnormalities likely allow

the CLL lymphocytes to avoid cell-mediated immune responses,

despite the fact that CLL cells express tumor-specific antigens that

can be presented by major histocompatibility complex molecules.

To better understand the mechanism underlying impaired T-cell

responses in CLL, Gorgun et al54 performed gene expression array

analyses of purified CD4 and CD8 T cells. Among CD4 T cells,

genes related to cell differentiation were differentially expressed

between T cells derived from previously untreated CLL patients and

healthy controls. Gene pathways controlling cytoskeleton forma-

tion, vesicle trafficking, and cytotoxicity were differentially ex-

pressed in CD8 T cells. When CLL cells were cocultured with

CD4 or CD8 T cells obtained from healthy controls, the abnormal

patterns of gene expression were induced, suggesting that these

T-cell defects are due to direct cellcell contact. 54 In light of the

observed dysregulation of cytoskeleton assembly genes, a subse-

quent study by Ramsay et al55 evaluated the capacity of T cells to

form a normal immunological synapse. This study showed that T

cells derived from CLL patients could not form normal cellcell

contact and that recruitment of T-cell signaling proteins to the

synapse was blocked. Similarly, coculture of CLL cells with donor

T cells blocked the formation of an immunologic synapse.55 The

inability of T cells derived from CLL patients to form a normal

immunologic synapse likely impairs both antigen recognition and

cellular cytotoxicity. Taken together, this global impairment in

T-cell function may be an important cause of tumor progression,

increased susceptibility to infection, and secondary malignancies in

patients with CLL.

Using Novel Biologic Insights to Develop an

Integrated Model of Disease BiologyDetailed laboratory-based and translational investigation has yielded

tremendous progress in our understanding of CLL. One of the

Hematology 2010 73


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significant challenges at this time is to assemble this body of

knowledge into a unified model of disease pathogenesis. Epidemio-

logic data implies the importance of ethnicity (likely a marker of

genetic risk) and age (a marker of immune senescence). A proposed

model follows: in a genetically susceptible host, tonic stimulation

by a stereotyped antigenlikely a low-affinity autoantigen

initiates expansion of a premalignant clone. Through some combina-

tion of tonic antigenic stimulation, acquisition of somatic genetic

mutations, and timely support from the tumor microenvironment,

the potentially autoreactive clone escapes immune surveillance andbegins to expand. In the tumor microenvironment, cells receive

proliferative signals from CD4 T cells and antiapoptotic signals

from NLCs and MSCs. Initially supported by the tumor microenvi-

ronment, the increasing clone may begin to further modulate the

microenvironment and immune response to favor survival and

progression of the clone. Among IGH unmutated clones, BCR-

transmitted signals provide predominantly proliferative signals,

whereas IGH-mutated clones express gene pathways related to

cellular anergy. The CLL cells continue to receive antigenic

stimulation, and as the clone continues to proliferate, additional

somatic mutations are acquired, and this may diminish the reliance

of the clone on both antigenic stimulation and microenvironment

support.

Some central questions to be investigated include the molecular

basis of the 2:1 male:female gender bias in CLL, the mechanism by

which some CLL acquire somatic mutations and others do not (ie,

the molecular determinants of IGH mutation status), the mechanism

by which preemergent CLL clones escape immune surveillance and

deletion, and whether clonal evolution can be avoided through

suppression of the proliferative compartment of CLL.

Finally, this scientific progress has created numerous targets of

novel therapeutic interventions. Lineage-restricted cell surface

molecules, such as ROR1 and CD37, are targets of novel MAbs.

Lenalidomide has been shown to reverse the abnormal immunologic

synapse formation in CLL, and also modulates the microenviron-

ment through monocyte and NK cell activation. Novel small

molecule inhibitors of BCR-mediated signaling are currently being

tested in clinical trials. CAL101 selectively blocks a phosphatidyl-

inositol 3-kinase isoform related to downstream signaling from

CXCR4. Additional trials studying small molecular inhibitors of

antiapoptotic proteins, such as BCL2, are underway. Elucidation of

the mechanisms of chemotherapy resistance related to loss of p53

function has led to strategies to circumvent p53-dependent path-

ways. The advances in our fundamental understanding of the

mechanisms of disease in CLL will undoubtedly lead to improved

therapies for our patients.

DisclosuresConflict-of-interest disclosure: The author has consulted and been

affiliated with the Speakers Bureau for GlaxoSmithKline and

Genentech, and has received research funding from Celgene and

Eleos.

Off-label drug use: None disclosed.

CorrespondenceMark C. Lanasa, MD, PhD, Assistant Professor of Medicine,

Division of Medical Oncology, Duke University Medical Center,

DUMC Box 3872, 1 Trent Dr., Morris Building Room 25153,

Durham, NC 27710; Phone: (919) 684-8964; Fax: (919) 684-5325;

e-mail: [email protected]

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