Dieta y Leucemia

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Nat Rev Dis Primers. Author manuscript; available in PMC 2017 March 04.
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Published in final edited form as:

Nat Rev Dis Primers. ; 3: 16096. doi:10.1038/nrdp.2016.96.
Chronic lymphocytic leukaemia

Thomas J. Kipps1, Freda K. Stevenson2, Catherine J. Wu3, Carlo M. Croce4, Graham
Packham2, William G. Wierda5, Susan O’Brien6, John Gribben7, and Kanti Rai8
1Division of Hematology-Oncology, Department of Medicine, Moores Cancer Centre, University of
California, San Diego, 3855 Health Sciences Drive M/C 0820, La Jolla, California 92093, USA.
2Southampton Cancer Research UK Centre, Cancer Sciences Academic Unit, Faculty of
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Medicine, University of Southampton, Southampton, UK.

3Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
4Departmentof Molecular Virology, Immunology and Medical Genetics, Ohio State University,
Columbus, Ohio, USA.
5Department of Hematology, MD Anderson Cancer Centre, Houston, Texas, USA.
6Division of Hematology, Department of Medicine, University of California, Irvine, California, USA.
7Department of Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London,
London, UK.
8CLL Research and Treatment Program, Feinstein Institute for Medical Research, Northwell
Health, New Hyde Park, New York, USA.
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Abstract
Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized by
the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid
tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell
receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions
between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the
lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who
Correspondence to T.J.K. Division of Hematology-Oncology, Department of Medicine, Moores Cancer Centre, University of
California, San Diego, 3855 Health Sciences Drive M/C 0820, La Jolla, California 92093, USA. tkipps@ucsd.edu.
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Author contributions
Introduction (T.J.K.); Epidemiology (T.J.K.); Mechanisms/ pathophysiology (T.J.K., C.M.C., C.J.W., G.P. and F.K.S.); Diagnosis,
screening and prevention (K.R., W.G.W. and T.J.K.); Management (W.G.W., S.O., J.G. and T.J.K.); Quality of life (J.G. and T.J.K.);
Outlook (T.J.K.); Overview of Primer (T.J.K.).
Competing interests
J.G. has received honoraria for advisory boards from Roche, Genentech, AbbVie, Janssen, Pharmacyclics, Acerta, Gilead, TG
Therapeutics and Unum. S.O. has acted as a consultant for Amgen and Celgene, is a scientific advisory board member for CLL Global
Research Foundation and has received research support from Acerta, TG Therapeutics, Regeneron, Gilead, Pharmacyclics and
ProNAi. K.R. is a member of the medical advisory board for Celgene, Roche/Genetech, Pharmacyclics and Gilead. T.J.K. has received
honoraria for Ad boards, meetings, presentations and/or consulting from Gilead, Pharmacyclics, Celgene, Roche and AbbVie, and has
received research funding from AbbVie and Celgene. C.J.W. is a co-founder and a member of the scientific advisory board of Neon
Therapeutics. W.G.W, G.P., C.M.C. and F.K.S. declare no competing interests.
SUPPLEMENTARY INFORMATION
See online article: S1 (table) | S2 (table)
Kipps et al. Page 2
require treatment soon after diagnosis to others who do not require therapy for many years, if at
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all. Several factors, including the immunoglobulin heavy-chain variable region gene (IGHV)
mutational status, genomic changes, patient age and the presence of comorbidities, should be
considered when defining the optimal management strategies, which include chemotherapy,
chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis,
such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify
patients who are at higher risk for disease progression and our capacity to treat patients with drugs
that selectively target distinctive phenotypic or physiological features of CLL. How these and
other advances have shaped our current understanding and treatment of patients with CLL is the
subject of this Primer.
Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized

by the accumulation of small, mature-appearing neoplastic lymphocytes in the blood,
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marrow and secondary lymphoid tissues, resulting in lymphocytosis, leukaemia cell

infiltration of the marrow, lymphadenopathy and splenomegaly. Genetic factors contribute to
the development of CLL; although CLL is the most common adult leukaemia in western
countries, it is less common in Asia and relatively rare in Japan and Korea, even among
Japanese people who immigrate to western counties.
CLL can be divided into two main subsets, which differ in their clinical behaviour. These
subsets are distinguished by whether CLL cells express an unmutated or mutated
immunoglobulin heavy-chain variable region gene (IGHV), reflecting the stage of normal B
cell differentiation from which they originate1,2. CLL cells that express an unmutated IGHV
originate from a B cell that has not undergone differentiation in germinal centres, which are
the sites in the lymph nodes where B cells experience somatic hypermutation in their
immunoglobulin variable region genes and selection during an immune response. Patients
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with CLL cells that express an unmutated IGHV typically have more-aggressive disease than
patients with CLL cells that express a mutated IGHV. CLL cells with mutated IGHV arise
from a post-germinal centre B cell that expresses immunoglobulin that has undergone
somatic hypermutation and, in some cases, also immunoglobulin isotype switching (FIG. 1),
similar to what occurs in normal B cells during an immune response to antigen. It should be
emphasized that the high level of somatic mutations that arise in IGHV in the germinal
centre are a natural part of affinity maturation of antibodies and, unlike mutations in other
genes, are not pathological. The tumours are simply reflecting the stage of maturation of the
parental B cell. In addition, some CLL cells have been described that are similar to
unmutated IGHV CLL, but originate from B cells with limited somatic mutation, such as
CLL with immunoglobulin heavy chains encoded by mutated IGHV3–21 and
immunoglobulin light chains encoded by unmutated IGLV3–21 (REFS 3,4).
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The repertoire of immunoglobin molecules produced by the CLL cells of all patients is
considerably more limited than the repertoire of immunoglobulin molecules that can be
made by the B cells of any one person5,6, reflecting the biased use in CLL of certain IGHV
genes that have restricted somatic mutation and limited junctional and heavy-light chain
combinatorial diversity. In as many as one-third of patients, the CLL cells express
immunoglobulin ‘stereotypes’, which are stretches of primary structure in the variable
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region that can also be identified in the immunoglobulins produced by the CLL cells of other
patients7. The restricted immunoglobulin repertoire in CLL is underscored by the finding
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that ~1 in 75 patients have CLL cells that express immunoglobulin molecules that are
virtually identical8. The limited immunoglobulin diversity provides compelling evidence that
CLL B cells are selected based on the binding activity of their expressed surface
immunoglobulin, suggesting that B cell receptor (BCR) signalling plays a crucial part in
CLL pathogenesis.
Several large genetic studies have revealed numerous genetic alterations in CLL, including
single- nucleotide polymorphisms (SNPs), chromosomal alterations and alterations in non-
coding RNA, such as microRNA (miRNA), some of which can be used to determine
prognosis and to guide management strategies. Interactions between CLL cells and their
microenvironment, including interactions with other cell types, such as T cells, nurse-like
cells and stromal cells, can induce B cell proliferation and contribute to disease.
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The distinctive cytogenesis of CLL contrasts with most other B cell malignancies, such as
follicular lymphoma, which is a germinal centre neoplasm, or myeloma (a post-germinal
centre neoplasm)9,10. However, diffuse large B cell lymphoma (DLBCL) resembles CLL in
consisting of two main subtypes: a germinal centre B-type DLBCL, which is derived from
germinal centre light zone B cells, and an activated B cell (or non-germinal centre) DLBCL,
which is derived from a later stage of germinal centre differentiation (before plasmablastic
differentiation)10. As in CLL, these two subtypes of DLBCL generally have distinctive
responses to therapy and clinical outcomes.
In this Primer, we describe the molecular pathogenesis of CLL and discuss the current
advances that are shaping our understanding and treatment of patients with this disease.
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Epidemiology
CLL is estimated to account for ~19,000 of all newly detected cancers in the United States
in 2016 (REF. 11). The average incidence of CLL varies between individuals in different
geographical regions and ranges from <0.01% of individuals in eastern Asia to ~0.06% of
individuals in Europe and the United States. The risk of developing CLL is about two-times
higher for men than for women and increases with age; the median age at diagnosis ranges
from 70 to 72 years11–14.
The US National Cancer Institute Surveillance, Epidemiology, and End Results programme
has estimated the number of new cases of CLL to be 6.3 per 100,000 men and 3.3 per
100,000 women. The incidence in white populations is estimated to be 6.8 per 100,000 men
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and 3.5 per 100,000 women, 4.9 per 100,000 men and 2.4 per 100,000 women in African
Americans, 2.7 per 100,000 men and 1.6 per 100,000 women in Hispanic Americans, 1.7 per
100,000 men and 1.3 per 100,000 women in Indigenous Americans, and 1.7 per 100,000
men and 0.3 per 100,000 women in people of Asian or Pacific Island descent in the United
States13.
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Hereditary factors
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Genetic factors contribute to disease susceptibility; among patients who are registered in the
CLL Research Consortium, 9% of patients have a relative with CLL. In addition, first-degree
relatives of patients with CLL have an 8.5-fold increased risk of developing this disease15,
and the concordance of CLL is higher among monozygotic twins than among dizygotic
twins16. Genome-wide association studies have identified SNPs in nearly 30 loci that are
associated with familial CLL, demonstrating that common genetic variation contributes to
heritable risk17–22 (see Supplementary information S1 (table)).
The altered expression of genes that are located in or near CLL-associated SNPs might
contribute to disease development. For example, a SNP in IRF4 is associated with low
expression of interferon regulatory factor 4; mice that are deficient in this protein can
develop CLL23, partly owing to hyperactivation of Notch signalling24. SNPs in LEF1 might
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be associated with high expression of lymphoid enhancer-binding factor 1, which is a

downstream effector of WNT signalling; normally, LEF1 is expressed at high levels in CLL
and, among other functions, can enhance resistance to cell death25. In addition, CLL-
associated SNPs have been found in BCL2, which encodes an anti-apoptotic protein that is
expressed at high levels in CLL, and in PMAIP1, which encodes a pro-apoptotic protein. A
SNP that is associated with reduced expression of mir-15a and mir-16-1 is associated with
familial CLL26,27. Because mir-15a and mir-16-1 repress the expression of BCL2 and
ZAP70 (REFS 26,27), reduced expression of these mi RNAs allows for the increased
expression of these genes, which encode proteins that respectively confer increased
resistance to cell death28 or enhanced BCR signalling29. Similarly, New Zealand black mice
have an allele at the mir-16-1 locus with shared synteny to human 13q14, which is
associated with low expression of mir-16-1; this allele is linked to the genetic propensity of
these mice to develop a B cell lymphoproliferative disease that resembles CLL30. Finally, a
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CLL-linked SNP in TERT is associated with a long leukocyte telomere length31,

conceivably contributing to the high rates of telomeric sister chromatid exchange observed
in CLL cells that could slow telomere erosion leading to cellular senscence32.
Environmental factors
The US Department of Veterans Affairs has accepted that exposure to Agent Orange is a risk
factor for CLL, which has enabled veterans with CLL to claim benefits if they were
previously exposed to Agent Orange while in military service33. In addition, evidence
suggests that exposure to insecticides might be a risk factor for CLL34. By contrast, little
evidence indicates that ionizing radiation can increase the risk of CLL35,36. Furthermore,
there is scant evidence that viral infections are risk factors, and epidemiological studies have
not found evidence that blood transfusions can transmit CLL37. No evidence suggests that
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dietary factors or lifestyle factors increase the risk of CLL.
Mechanisms/pathophysiology
Genetics
Genetic alterations in CLL can include chromosomal alterations, mutations, alterations in
the expression of mi RNAs and epigenetic modifications.
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Chromosomal alterations—Approximately 80% of patients with CLL carry at least one

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of four common chromosomal alterations: a deletion in chromosome 13q14.3 (del(13q)),

del(11q), del(17p) and trisomy 12 (REF. 38). Del(13q) is the most common chromosomal
alteration, evident in >50% of patients, and is associated with favourable prognosis. Within
this deleted region is the DLEU2-mir-15–16 cluster, which regulates the expression of
proteins that can inhibit apoptosis or that are involved in cell cycle progression39 (see
Supplementary information S2 (table)). Del(17p) is found in 7% of patients and is associated
with loss of the tumour suppressor gene TP53 (REF. 40), whereas del(11q) is found in 18%
of patients and is often associated with alterations in ATM (which encodes a protein
involved in DNA repair); each of these chromosomal alterations is associated with adverse
clinical outcome38, although this has improved in recent years41. Trisomy 12 is found in
16% of patients with CLL and is associated with an intermediate prognosis. Unlike the
neoplastic B cells in mantle cell lymphoma, CLL cells do not have the translocation t(11;14)
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(q13;q32) or other genetic alterations that enhance the expression of CCND1, which encodes
the cell cycle regulator cyclin D1.
Somatic mutations—The advent of massively parallel sequencing and the application of

whole-exome sequencing to CLL have transformed our understanding of the genetic
heterogeneity of CLL and have established that CLL harbours a high degree of genetic
variability42–45 (FIG. 2). From these studies, recurrent somatic mutations have been
consistently observed in genes that have a role in DNA damage (for example, TP53 and
ATM), mRNA processing (for example, SF3B1 and XPO1), chromatin modification (for
example, HIST1H1E, CHD2 and ZMYM3), WNT signalling, Notch signalling (for example,
NOTCH1) and inflammatory pathways (for example, MYD88). Other mutations, such as
those found in EGR2 or BRAF, can affect B cell-related signalling and transcription46 (FIG.
2).
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The functional role of several putative driver mutations has been confirmed; for example,
silencing mutated WNT pathway genes in primary CLL cells resulted in decreased cell
viability47. Mutations in POT1, which has a role in the protection of telomeres, prevented
the binding of protection of telomeres protein 1 to telomeric DNA, resulting in numerous
chromosomal abnormalities, in addition to the development of abnormal telomeres.
Mutations in SF3B1 have been found to be associated with aberrant RNA splicing45,48,49
and an altered DNA-damage response50. SAMHD1 encodes a protein involved in the
regulation of intracellular deoxy-nucleotide pools, which are recruited to the site of DNA
damage and are probably involved in the response to DNA double-strand breaks50.
The detection of somatic mutations and their relative frequencies is variable, which possibly
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reflects differences in the composition of the cohorts studied worldwide. Two seminal
studies have provided the largest sequenced collections to date51,52, in which >500 CLL
samples were characterized by whole-exome sequencing or whole- genome sequencing. The
clinical and/or biological features of the patients examined in each study were notably
distinct; one study analysed matched pretreatment samples from patients who required initial
treatment and noted mutations in SF3B1 (21% of patients), ATM (15% of patients), TP53
(7% of patients), NOTCH1 (6% of patients) or BIRC3 (4% of patients). The other study
assessed patients with earlystage CLL and patients with monoclonal B cell lymphocytosis
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and identified NOTCH1 (12.6% of patients), A T M (11% of patients), BIRC3 (8.8% of

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patients) and SF3B1 (8.6% of patients) as the most frequently mutated genes.
These large sample cohorts have provided the sensitivity to discover novel candidate cancer
genes that are altered in CLL. Both studies also identified somatic mutations in MGA and
PTPN11, which encode modulators of MYC, IKZF3, which encodes a key transcription
factor, and RPS15, which encodes 40S ribosomal protein S15 and is recurrently mutated in
~20% of patients who relapse after combination chemotherapy53. Other recurrent somatic
mutations include those in the 3′ untranslated region of NOTCH1 and a PAX5 enhancer,
which increases the expression of these B cell-associated transcription factors54,55. Patients
with mutations in the 3′ untranslated region of NOTCH1 have a shorter time from diagnosis
to treatment and poorer overall survival, similar to that of patients with non- synonymous
mutations, which alter the amino acid sequence of NOTCH1.
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Next-generation sequencing has revealed intra-tumoural heterogeneity in CLL. Some

somatic mutations, such as those in MYD88, or chromosomal abnormalities, such trisomy
12 or del(13q), are most often found in all the CLL cells of any one patient, indicating that
these genetic alterations occurred early in the evolution of the leukaemia. Other mutations,
such as those found in SF3B1 or NOTCH1, or chromosomal alterations, such as del(17p),
are typically found in only a fraction of the leukaemia cells and thus represent subclonal
events, which occur after the development of CLL. Across studies, subclonal driver
mutations are associated with more-aggressive disease, particularly when two or more are
found concurrent in a leukaemia cell population51,56,57. In addition, studies have
demonstrated that large clonal shifts can occur following chemotherapy, owing to increases
in the proportions of CLL cells that have a TP53 mutation or del(17p), indicating that such
genetic changes provide a strong fitness advantage in the setting of therapy51. By contrast,
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one study of CLL cells from patients treated with ibrutinib (an inhibitor of Bruton tyrosine
kinase (BTK)), revealed mutations associated with drug resistance that were distinct from
those observed in CLL cells of patients treated with standard chemotherapy58.
miRNA alterations—CLL was the first human disease that was found to be associated
with alterations in miRNA. Specifically, mir-15a and mir-16-1 (REF. 59) are deleted, altered
or downregulated in ~60% of patients with CLL and are dysfunctional in a few cases of
familial CLL26. mir-15a and mir-16-1 both target BCL2 and MCL1 (REF. 28), which
encode anti-apoptotic proteins of the BCL-2 family60; reduced expression or loss of these mi
RNAs can enhance the expression of these target genes. Attention has also focused on mi
RNAs that are dysregulated or that are differentially expressed in subgroups with distinctive
clinical and/or biological features61 (see Supplementary information S2 (table)). For
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example, miR-29a/b, miR-29c, miR-34b, miR-181b and miR-3676 target the 3’ untranslated
region of TCL1A62; loss or reduced expression of all or some of these miRNAs can lead to
enhanced expression of TCL1A, which, when constitutively expressed in mature B cells,
promotes the development of CLL in transgenic mice63. By contrast, increased expression of
mir-155 is associated with enhanced BCR signalling, B cell proliferation and
lymphomagenesis64,65.
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Epigenetic changes—The CLL epigenome shows global hypomethylation combined

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with local hypermethylation, as has been observed in other cancers66–68. Indeed,

comprehensive methylation profiling has demonstrated substantial intra-tumoural
methylation heterogeneity52,69–73. Increasing methylation heterogeneity has consistently
been associated with increased genetic complexity owing to the acquisition of subclonal
mutations, thus linking genomic and methylomic evolution in CLL52,70,71. Indeed, locally
disordered methylation in CLL might enhance the evolutionary adaptive capacity of CLL
cells by increasing the background ‘noise’ of the genome, thereby providing increased
opportunities for somatic mutations within the leukaemia clone. In support of this notion is
the observed association between methylation evolution and adverse clinical
outcome52,70,71.
Methylation signatures can classify distinct clinical CLL subgroups69,74. As these

methylation patterns are a heritable trait, they have been used to ‘trace’ back to the type of
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normal B cell from which the CLL cells were derived75. These studies revealed that the CLL
cells of different patients derive from a continuum of B cell maturation states, which are not
restricted to discrete maturation stages. Nevertheless, CLL cells that use unmutated IGHV
versus mutated IGHV generally have distinctive methylation patterns, which respectively
approximate to those of pre-germinal centre versus post-germinal centre memory B cells, as
depicted in FIG. 1. The diversity in the likely cells of origin of CLL cells highlights the
biological and phenotypic heterogeneity of this disease. These findings also suggest that
epigenetic programming that is dependent of transcription factors has a potentially important
role in the development of CLL.
BCR and B cell signalling

The BCR is composed of a ligand-binding trans-membrane immunoglobulin molecule
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(either IgA, IgD, IgE, IgG or IgM) and the signalling Igα (also known as CD79A)–Igβ
(also known as CD79B) heterodimer. CLL cells typically co-express IgD and IgM, although
at low levels compared with normal B cells; less than a few per cent of CLL cases express
class-switched isotypes, most commonly IgG. The CD79A and CD79B molecules contain
immunoreceptor tyrosine-based activation motifs, which can be phosphorylated following
the crosslinking of surface immunoglobulin, thereby triggering BCR signalling. A functional
BCR is required for the survival of mature B cells76 and is maintained in most mature B cell
malignancies, including CLL. In CLL, evidence suggests that the surface immunoglobulin of
CLL B cells is engaged by autoantigen, which leads to constitutive BCR signalling in
vivo77–79. The importance of this interaction is underscored by the clinical success of kinase
inhibitors that block BCR signalling (see Management), although effects on other receptors
might also have a role80.
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Like most cancers, CLL is heterogeneous and the outcome of BCR signalling ranges from
enhanced B cell activation to B cell anergy81,82. The main pathways that lead to cell survival
and proliferation downstream of the BCR are shown in FIG. 3, along with drugs targeted
against key signalling intermediates. BCR signalling that leads to anergy is less well defined,
but seems to involve biased activation of inhibitory molecules with only partial activation of
the pathways that are typically associated with B cell activation81. One important molecule
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that may be involved is the inositol lipid phosphatase SHIP1. SHIP1 is activated by the
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tyrosine-protein kinase LYN and may limit B cell activation by counteracting

phosphoinositide 3-kinase (PI3K) activity at both chronically engaged receptors and distant
non-ligated BCRs, rendering them insensitive to stimulation82,83.
Enhanced B cell activation is more commonly observed in CLL that expresses unmutated
IGHV, whereas anergy predominates in most cases of CLL that express mutated IGHV84.
Anergy is a state of cellular lethargy induced by chronic engagement of the surface antigen
receptors in the absence of adequate T cell help. Although capable of reversing their
phenotype, anergic cells are less likely to proliferate in response to BCR signalling than
more activated cells, which might, in part, account for the observation that patients with
CLL cells that express mutated IGHV generally have more indolent disease than patients
with CLL cells with unmutated IGHV85. The fate of the cell (activation versus anergy)
might be influenced by the CLL cell of origin (FIG. 1), as the cell types that can form CLL
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differ in their patterns of DNA methylation73, and are likely to respond differently to
autoantigens. An unresolved question is whether anergy can be reversed in vivo, mirroring
what occurs in vitro78.
The BCR also coordinates the activity of other cell surface receptors, including integrins,
such as α4β1 integrin. BCR stimulation can result in increased adhesion of CLL cells to
α4β1 integrin substrates, for example, fibronectin and vascular cell adhesion protein 1 (REF.
86). By contrast, CXC-chemokine receptor 4 (CXCR4) is downmodulated by BCR
engagement and both can trigger ‘inside-out’ signalling, resulting in the activation of α4β1
integrin87,88. Thus, recognized antigen encountered in lymphoid tissue is likely to affect
adhesion and migration of CLL cells. Modulation of these pathways, coupled with the role
of BTK and PI3K in chemokine receptor signalling89, contribute to the increased
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lymphocytosis observed in patients upon initiation of treatment with inhibitors of BTK or

PI3K (see Management).
Cancer microenvironment
CLL cells depend on survival signals that they receive in lymphoid tissues from
neighbouring non-neoplastic cells within the so-called cancer microenvironment. CLL cells
follow chemokine gradients into lymph nodes, where they form ‘proliferation centres’ (REF.
77), as opposed to normal germinal centres. In these proliferation centres, the CLL cells
contact non- malignant stromal cells, nurselike cells (also known as lymphoma-associated
macrophages), T cells and mesenchymal-derived stromal cells (FIG. 4). Engagement with
autoantigen may occur during this transit, thereby stimulating CLL cell activation and
proliferation if sufficient T cell help is available. Only a few per cent of the CLL cells
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undergo proliferation at any one time; the remainder of the cells are either unstimulated or
driven into anergy84. However, within such proliferation centres, all CLL cells are exposed
to chemokines, integrins, cytokines and survival factors (such as tumour necrosis factor
(TNF) ligand super-family member 13B (also known as BAFF) or TNF ligand superfamily
member 13 (also known as APRIL)), which activate canonical nuclear factor-κB (NF-κB)90,
before they exit to the blood. Activation of NF-κB can induce the expression of mir-155,
which enhances BCR signalling and activation by reducing the expression of INPP5D,
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which encodes SHIP1 (REF. 65). Cytokines that are secreted by T cells, such as IL-4, can
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upregulate surface IgM, which potentially facilitates the interaction of the CLL cell with
autoantigen91. In addition, the elaboration of various WNT proteins by cells in the
microenvironment can activate canonical and non-canonical WNT signalling pathways92,93.
Activation of the tyrosine-kinase-like transmembrane receptor ROR1 by WNT5A can induce
the activation of RAC1 and RHOA, and thereby enhance CLL cell proliferation and promote
migration in response to chemokines93; in part, for this reason, high-level CLL cell
expression of ROR1 is associated with accelerated disease progression94. Finally, Notch
signalling in response to Jagged, or Hedgehog signalling in response to Sonic Hedgehog or
Desert Hedgehog, can provide pro-survival stimulation for at least a subset of patients with
CLL, particularly those with trisomy 12 (REFS 95–97).
As CLL cells leave the tissue site, engagement with antigen will be transient and its effects
are likely to reverse in the blood, leading to variable increases in the expression of surface
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IgM and CXCR4 (REF. 98). CLL cell expression of CXCR4 is downmodulated upon
exposure to CXC-chemokine ligand 12 (CXCL12)99, which is produced by nurse-like cells
in the microenvironment100. Consequently, CLL cells in the blood that have just exited
lymphoid tissue express low levels of CXCR4 (known as CXCR4dim cells) and higher levels
of CD5 (known as CD5bright cells) relative to the CLL cells that are poised to re-enter
lymphoid compartments101. For unexplained reasons, a high level of expression of CXCR4
by circulating CLL cells is associated with poorer prognosis in patients with CLL that use
mutated IGHV102, possibly by influencing tissue re-entry. In terms of treatment effects,
kinase inhibitors, such as ibrutinib, inhibit BCR-associated pathways, which remain
important for cancer cells that are retained in lymphoid tissues, but can also directly inhibit
integrin-mediated and chemokine- mediated pathways, thereby contributing to the increased
lymphocytosis that occurs following the initiation of kinase inhibitor therapy103.
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Immune deficiency
One clinically important aspect of CLL is the development of hypogammaglobulinaemia
with consequent risk of infection. The mechanism involved is unclear, but IL-10, a known T
cell-derived immunosuppressive factor, might have a role104. For CLL, emerging evidence
suggests that the cancer cells themselves can produce IL-10 (REF. 105). Apparently, more
IL-10 is produced by CLL cells that express mutated IGHV than by CLL cells that express
unmutated IGHV. However, systemic levels of IL-10 and other suppressive factors can also
be influenced by the cumulative total-body numbers of cancer cells, which are often higher
in patients with CLL cells that express unmutated IGHV. This might account in part for the
finding of immune deficiency in patients with CLL cells that express either mutated IGHV
or unmutated IGHV. Furthermore, CLL cells express high levels of programmed cell death 1
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ligand 1 (PD-L1) and PD-L2, which suppress the effector responses of T cells that express
programmed cell death protein 1 (PD-1), leading to an ‘exhausted’ T cell phenotype and
impaired cellular immune function106.
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Diagnosis, screening and prevention

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Diagnostic work-up
Most often, patients with CLL are asymptomatic at the time of diagnosis and become aware
of the disease following the detection of lymphocytosis in a routine blood count. However,
CLL can have a range of clinical presentations; some patients feel well and are fully active,
but a minority have disease-related symptoms. The usual symptoms of CLL include fatigue,
involuntary weight loss, excessive night sweats, abdominal fullness with early satiety and
increased frequency of infections, which might be associated with
hypogammaglobulinaemia. Some patients can present with symptoms of an autoimmune
cytopenia (for example, autoimmune haemolytic anaemia or immune thrombocytopenic
purpura). Patients can also have or develop enlarged lymph nodes, hepatomegaly and
splenomegaly, which are palpable on physical examination. Enlarged lymph nodes can be
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easily palpable at three sites: the cervical, axillary and inguino-femoral regions.
Laboratory features—Laboratory assessment for CLL includes a full blood cell count
and flow cytometry. The most consistent laboratory abnormality observed is an increase in
the absolute number of blood lymphocytes above the normal adult upper limit of ~3,500
cells per µl, detected by a blood count. Most patients present with ≥10,000 cells per µl, but
some might have fewer numbers of blood lymphocytes upon relapse after therapy. The initial
diagnosis requires detection of ≥5,000 cells per µl of clonal CLL B cells107, which typically
express low levels of surface immunoglobulin with either κ-immunoglobulin or λ-
immunoglobulin light chains.
Flow cytometric or immunohistochemical analyses of the mononuclear cells in the blood,

marrow or lymph nodes can help to distinguish CLL from other types of lymphoma (BOX
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1). CLL B cells typically express CD5, CD19 and CD23 (also known as low-affinity
immunoglobulin-ε Fc receptor), and have low levels of CD20, but lack expression of CD10
and stain poorly, if at all, with the FMC7 monoclonal antibody, which recognizes an epitope
of CD20 (REF. 108). CLL cells also express CD200 (also known as OX-2 membrane
glycoprotein), which can help to distinguish CLL from mantle cell lymphoma109. In
addition, the CLL cells of >95% of patients express the onco-embryonic surface antigen
ROR1 (REFS 94,110).
Morphologically, CLL cells are small mature-appearing lymphocytes with dense chromatin,
a nucleus that virtually fills the cell with only a rim of visible cytoplasm and no (or
occasionally small) nucleoli (FIG. 5a). In CLL, the presence of smudged cells on the blood
smear is common and represents lymphocytes that were crushed in the process of making
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the slide (FIG. 5b). CLL cells also can appear as prolymphocytes, which are larger than
typical CLL cells, have less-condensed nuclei and a single prominent nucleolus (FIG. 5c).
However, if >55% of cells on the blood smear are prolymphocytes, a diagnosis of
prolymphocytic leukaemia should be considered111.
No abnormalities are considered specific for CLL in the blood chemistry panel. Quantitative
levels of serum immunoglobulins (for example, IgA, IgG and IgM) are usually normal at
diagnosis, but generally decline with disease progression. A direct Coombs test (which is
used to detect erythrocytes that are coated with anti-red blood cell autoantibodies) might be
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positive in the absence of overt autoimmune haemolytic anaemia in a large minority of

patients; however, patients with a positive direct Coombs test might be at increased risk of
developing this autoimmune disease.
Although not required for establishing a diagnosis of CLL, a marrow biopsy is often
performed; this usually shows hypercellularity owing to an increased percentage of mature-
appearing lymphocytes. Four patterns of lymphocytic infiltration in the marrow have been
described: nodular, interstitial, mixed (nodular and interstitial) or diffuse; the diffuse pattern
is typically associated with advanced disease112 (FIG. 6). In addition, the marrow usually
shows reduced numbers of myeloid and erythroid cells, which otherwise have normal
maturation.
A lymph node biopsy might be performed in a patient with an enlarged lymph node as part
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of a diagnostic evaluation for suspected lymphoma. Excised lymph nodes typically have a
diffuse infiltration of well-differentiated small lymphocytes, often obliterating the normal
nodal architecture, and scattered, vaguely nodular, pale haematoxylin and eosin-staining
areas, appearing as pseudo-follicles (FIG. 7a), which are enriched with prolymphocytes and
paraimmunoblasts (FIG. 7b); these areas comprise the proliferation centres113. The
pseudofollicles or proliferation centres are hallmark features in the lymph nodes of patients
with CLL or small lymphocytic lymphoma, as they are not observed in other types of
lymphomas.
Staging
Two clinical staging systems are widely used to divide patients with CLL into three broad
prognostic groups114,115. The Rai staging system (TABLE 1) is more commonly used in the
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United States, whereas the Binet classification (TABLE 2) is more commonly used in
Europe. The staging systems each recognize the importance of marrow function and define
late-stage, or high-risk, disease by the presence of pronounced anaemia or
thrombocytopenia.
Prognostic factors and nomograms

The clinical course of newly diagnosed CLL is extremely variable; some patients remain
free of symptoms and are fully active for decades, whereas others rapidly become
symptomatic or develop high-risk disease, which requires treatment soon after diagnosis and
might result in death due to therapy-related and/or disease-related complications. However,
most patients have a clinical course that is in between these two extremes.
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Prognostic factors that can help to identify patients who require therapy relatively soon after
diagnosis include certain clinical features and genetic, molecular and biochemical
characteristics of the CLL cell. Multivariable models, prognostic indexes116–118 and
nomograms119 have been developed to consolidate such prognostic factors so that they can
more robustly predict clinical outcome. Commonly used parameters that are associated with
poorer outcome are male sex, ≥65 years of age, poor performance status due to medical
comorbidities, certain CLL cell characteristics, such as the expression of unmutated
IGHV1,2, ZAP70 (REFS 120,121), CD49d (also known as integrin α4)122 or CD38 (REF.
2), the presence of del(17p)38 or del(11q)123, high serum levels of β2-microglobulin (>3.5
mg per l)124, complex karyotype (that is, the presence of three or more chromosomal
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aberrations observed on a karyotype test)125,126, or a high absolute lymphocyte count

(>50,000 cells per µl) and/or late-stage disease at initial presentation. Del(17p) is often
associated with inactivating mutations in TP53 and is a predictor of poor outcome to
treatment with regimens that involve conventional chemotherapy127.
Currently, the most reliable prognostic models are those developed for treatment-free
survival, as evolving treatments have yet to change the indications for therapy. Predictive
models to define overall survival with a given type of therapy are challenged by the
chronicity of CLL and the fact that patients often receive serial treatments, each of which
can affect outcome; moreover, death might be due to an indirect or unrelated cause.
Furthermore, treatment options are changing, with newly identified, highly effective agents
that are clearly prolonging survival and have activity among patients who would have been
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considered high risk when the only option was conventional chemotherapy.
Management
Generally, indications to initiate therapy include pronounced disease-related anaemia or
thrombocytopenia (patients with Rai stage III or stage IV disease, or Binet stage C disease),
symptomatic lymphadenopathy and/or symptoms that are associated with active disease,
such as night sweats, fatigue, unintentional weight loss or fever without evidence of
infection107. However, when basing a treatment decision on constitutional symptoms alone,
the physician should consider other medical conditions, such as hypothyroidism,
hyperthyroidism, hypoglycaemia, chronic inflammation, uncommon opportunistic infections
or sleep disorders, including sleep apnoea.
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No established absolute lymphocyte count or lymph node size alone should form the basis
for the initiation of therapy. Instead, patients who are asymptomatic with early-stage or
intermediate-stage disease (such as Rai stage I or stage II, or Binet stage A or stage B) are
not recommended for therapy unless they have symptomatic disease or evidence for disease
progression. Evidence for disease progression can include a lymphocyte doubling time of <1
year, progressive palpable lymphadenopathy and/or progressive palpable splenomegaly in
serial examinations. In the absence of indications for treatment, patients are examined for
palpable lymphadenopathy and splenomegaly and have complete blood counts at 3–12-
month intervals, the frequency of which depends on the presence of signs of disease
progression. Clinical or laboratory features of anaemia or thrombocytopenia should prompt
evaluation for autoimmune haemolytic anaemia or immune thrombocytopenic purpura,
respectively; such autoimmune cytopenias might require treatment that is independent of the
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consideration for therapy directed against the under lying CLL. Finally, patients should be
cautioned to seek prompt medical attention for signs or symptoms of infection; because of
the acquired immune deficiency associated with CLL, the threshold for considering the use
of antimicrobial therapy should be low. Nonetheless, development of frequent or serious
infections is not an indication for CLL-directed therapy.
For patients who need treatment, the presence of del(17p) or mutated TP53 are the most
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important features that are currently directing the choice of therapy (FIG. 8). Next, advanced
age of >65 years, the presence of medical comorbidities and the objectives of treatment have
substantial bearing on the choice of therapy. Increasingly, IGHV mutational status is
considered as a parameter when determining the type of therapy; for example,
chemotherapy-based regimens are reserved for patients with CLL and mutated IGHV.
Conversely, the specific Rai or Binet stage of the patient who requires treatment does not
necessarily influence the choice of therapy.
Systemic treatments
The treatment of patients with CLL can include chemotherapy, a combination of
chemotherapy and immunotherapy, or drugs that target the signalling pathways that promote
the growth and/or survival of CLL cells (for example, BCR signalling and BCL-2)128,129.
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Chemotherapy—Chemotherapy has been the mainstay of therapy for the past 50 years.
Purine analogues (most commonly fludarabine, but also pentostatin or cladribine) and
alkylating agents (including chlorambucil, cyclophosphamide or bendamustine) are used in
the treatment of CLL130–132. Chemotherapy-based regimens can cause myelosuppression, an
increased risk of infections and, in a small subset of patients, post-therapy myelodysplasia or
secondary cancers, such as acute myeloid leukaemia (see Secondary cancers).
Chemoimmunotherapy—Phase III clinical trials have validated the benefit of anti-CD20

monoclonal antibodies, such as rituximab, obinutuzumab or ofatumumab, in combination
with chemotherapy for the treatment of patients with CLL. In one trial (the CLL8 trial of the
German CLL Study Group), patients who received fludarabine and cyclophosphamide with
rituximab had higher response rates and a longer median progression-free survival (PFS)
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than patients who were treated with fludarabine and cyclophosphamide133. In a separate
study (the CLL11 trial), patients >65 years of age with medical comorbidities who were
treated with chlorambucil and either obinutuzumab or rituximab had improved response
rates and longer median PFS than patients who were treated with chlorambucil alone134.
However, the median PFS was significantly longer for patients who received obinutuzumab
(26.7 months) than in those who received rituximab (11.1 months). In a third phase III trial,
median PFS significantly improved from 13.1 months for patients treated with chlorambucil
to only 22.4 months for patients treated with chlorambucil and ofatumumab135. As a
consequence of these three trials, the US FDA approved the use of rituximab, obinutuzumab
or ofatumumab in combination with chemotherapy for the first-line treatment of patients
with CLL. The FDA also approved the use of ofatumumab as a single agent for the treatment
of patients with relapsed or refractory disease based on data from a phase II study136.
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Bendamustine is commonly used with rituximab and has good response rates in treatment-
naive patients without del(17p)137, although no randomized trials comparing bendamustine
and rituximab versus bendamustine alone have been conducted. Bendamustine has also been
used in combination with obinutuzumab, which showed highly encouraging results138 and is
being evaluated in larger clinical trials.
In a randomized trial, the rates of complete response and complete response without
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evidence for minimal residual disease (MRD) were higher in patients treated with
fludarabine, cyclophosphamide and rituximab than in those treated with bendamustine and
rituximab, and the median PFS was ~1 year longer139. However, patients in the
bendamustine and rituximab treatment subgroup were older and had a higher proportion of
patients who had CLL cells expressing unmutated IGHV, making this cohort at higher risk
for a poorer outcome than the cohort of patients treated with fludarabine, cyclophosphamide
and rituximab. It also should be noted that patients treated with fludarabine,
cyclophosphamide and rituximab had higher rates of neutropenia and infections than
patients treated with bendamustine and rituximab. Because of this, many physicians
currently provide patients with growth factors (for example, filgrastim or pegfilgrastim) and
prophylactic antimicrobial therapy when they are treated with the fludarabine,
cyclophosphamide and rituximab regimen, but such measures were not recommended for
patients treated in this trial139. In any case, there has not been significant difference observed
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in overall survival between the two treatment arms, but events are limited.
Some patients can experience a prolonged PFS following treatment with fludarabine,
cyclophosphamide and rituximab, particularly those with CLL with mutated IGHV that lack
del(17p) or del(11q), which are associated with chemotherapy resistance or relatively short
PFS, respectively. Long-term follow-up data on patient outcomes following therapy with this
regimen indicate that patients with mutated IGHV might achieve a long-term survival
benefit (and possible ‘cure’) with chemoimmunotherapy140–142.
Inhibitors of BCR signalling—Three main classes of drugs that each can inhibit BCR
signalling have been evaluated in patients with CLL: BTK inhibitors, PI3K inhibitors and
spleen tyrosine kinase (SYK) inhibitors86,143. CLL cells with unmutated IGHV seem to be
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more sensitive to inhibitors of BCR signalling than CLL cells with mutated IGHV144, but
whether inhibitors, such as ibrutinib, are more effective in patients with CLL and unmutated
IGHV, remains to be validated in clinical trials.
Ibrutinib has been approved in the United States and Europe for use as initial therapy, as
well as in patients with relapsed disease, which followed results from a randomized trial that
showed a significantly higher response rate to therapy with ibrutinib than with
ofatumumab145. In addition, with continuous therapy, patients treated with ibrutinib had a
significantly longer median PFS and overall survival than patients treated for 8 months with
ofatumumab. Approval of ibrutinib as initial therapy was based on the results of a
randomized trial that showed a significant improvement in median PFS and overall survival
in patients ≥65 years of age without del(17p) who were treated indefinitely with ibrutinib
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than in patients treated for up to 48 weeks with chlorambucil146.
Upon initiation of treatment with ibrutinib, lymphadenopathy is rapidly reduced, which is

associated with a concomitant increase in absolute lymphocyte count147. The rise in absolute
lymphocyte count is related to the inhibition of chemokine receptor signalling, which
inhibits the migration of CLL cells from the blood into the lymphoid tissues. This resulting
lymphocytosis should not be considered a sign of progression; over time, the lymphocytosis
subsides as the overall tumour burden decreases with continued therapy.
Adverse effects of ibrutinib include fatigue, diarrhoea, bleeding, ecchymoses, rash,

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arthralgia, myalgia, increased blood pressure and atrial fibrillation. Clinical trials are
currently evaluating second-generation BTK inhibitors (for example, acalabrutinib148, ONO/
GS-4059 (REF. 149) or BGB-3111) to determine whether any one of these drugs has a
superior therapeutic index than that of ibrutinib150.
PI3K inhibitors include idelalisib, duvelisib (also known as IPI-145), TGR-1022 and
ACP-319 (also known as AMG-319)151; the latter three drugs are being evaluated in clinical
trials, whereas idelalisib was approved in the United States and Europe for the treatment of
patients with relapsed CLL; this approval was based on the outcome of a clinical trial that
showed that patients treated with rituximab and idelalisib had significantly higher response
rates and a significantly longer median PFS and overall survival than patients treated with
rituximab and placebo152. As with ibrutinib, patients who initiate therapy with idelalisib can
experience a rapid reduction in lymphadenopathy that is associated with lymphocytosis.
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Similarly, this event should not be considered as a sign of disease progression.
Adverse effects of idelalisib include transaminitis (usually in the first few months of
therapy), pneumonitis and colitis; the latter usually occurs >6 months after the initiation of
therapy with this drug and is often severe enough to require cessation of therapy153.
Transaminitis seemed to be more severe in patients who received idelalisib as their initial
therapy for CLL than in patients with relapsed disease153, suggesting that transaminitis is
not directly caused by idelalisib. This is also suggested by the observations that mild
increases in the levels of serum transaminase can subside over time with continued drug
administration; furthermore, patients who have had idelalisib withheld because of
transaminitis can be restarted on this drug without experiencing apparent hepatic toxicity.
The decision to halt therapy or to re-administer the drug following resolution of
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transaminitis should consider the severity and duration of hepatic function test
abnormalities, which often do not recur upon re-institution of idelalisib therapy154.
In 2016, the FDA recommended the closure of clinical trials investigating idelalisib and
rituximab combination therapy for first-line treatment of patients with CLL, owing to a
higher number of infections and deaths in the experimental arm. As such, patients
undergoing therapy with idelalisib and rituximab should be considered for concomitant
treatment with prophylactic low-dose acyclovir to protect against reactivation of varicella
zoster virus, which causes chicken pox and shingles. Patients also should be treated with
prophylactic antibiotics to mitigate the risk for opportunistic infection, such as that caused
by Pneumocystis jiroveci. Finally, as with any patient receiving therapy with anti-CD20
monoclonal antibodies, patients should be screened for active infection with hepatitis B
virus before the initiation of therapy155, and periodically monitored for reactivation of
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cytomegalovirus, especially if they should develop unexplained symptoms of infection.
Phase I/II clinical trials of fostamatinib, an oral SYK inhibitor, caused reduction in
lymphadenopathy with concomitant lymphocytosis, an improvement in disease-related
cytopenias and relief of disease-related symptoms in most of the treated patients with
CLL156. However, dose-limiting toxicities of fostamatinib treatment include neutropenia,
thrombocytopenia and diarrhoea. Other inhibitors of SYK, such as entospletinib, are being
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evaluated in preclinical and clinical studies.
BCL-2 inhibitors—Venetoclax is a small molecule that functions as a BH3 mimetic to

inhibit BCL-2 (REF. 157). This drug is highly potent in inducing apoptosis in CLL cells,
possibly by diminishing the capacity of BCL-2 to sequester the pro-apoptotic molecule
BCL-2-interacting mediator of cell death (BIM; also known as BCL2L11)158. As such,
venetoclax is effective in patients with relapsed and/or refractory disease159 or in patients
with relapsed disease and del(17p)160. Indeed, the overall response rate for patients with
relapsed disease and del(17p) was 79%, with 8% achieving a complete response. In addition,
the estimated 12-month PFS was 72% and overall survival was 87%. On the basis of these
results, the FDA approved the use of venetoclax for patients with relapsed disease and
del(17p). Ongoing studies have shown that venetoclax can be safely combined with
rituximab or obinutuzumab. Moreover, studies are examining the use of venetoclax with or
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without an anti-CD20 monoclonal antibody, and with or without ibrutinib161,162, which

might provide higher response rates to therapy than that with venetoclax alone.
Toxicities of venetoclax include gastrointestinal disturbances, neutropenia and tumour lysis

syndrome159, which is characterized by hyperkalaemia, hyper-uricaemia and/or azotaemia.
Tumour lysis syndrome results from the rapid destruction of cancer cells and the release of
their cellular contents into the blood. Tumour lysis syndrome typically occurs when
initiating venetoclax therapy or when dosing is increased. Thus, patients start venetoclax
with a low daily dose, which is escalated each week over 5 weeks to mitigate the risk of
developing tumour lysis syndrome. Even with this strategy, patients who are at high risk for
tumour lysis syndrome because of bulky lymphadenopathy and/or lymphocytosis of >25,000
cells per µl must be hydrated and closely monitored during therapy initiation and during
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dose escalation.
Assessment of response
Historically, a favourable response to therapy has been defined as a partial remission or
complete remission. Partial remission requires a 50% reduction in tumour bulk (for example,
lymphadenopathy and splenomegaly), a 50% reduction in lymphocytosis, and platelet counts
of >100,000 cells per µl (or 50% improvement over baseline) or a haemoglobin level of >11
g per dl (or 50% improvement over baseline) without requiring transfusions or exogenous
growth factors107. Complete remission requires the normalization of blood counts,
resolution in lymphadenopathy and splenomegaly, and normal marrow function. The use of
CT to assess response in CLL is becoming more common, particularly in clinical trials.
However, the benefit of using repeated CT scans to monitor disease is uncertain, and seems
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unlikely to change patient outcome. Because of the distinct pattern of response observed
with BCR inhibitors, a new response category, namely, partial response with lymphocytosis,
has been described. Partial response with lymphocytosis is defined as a >50% reduction in
lymphadenopathy and splenomegaly, with persistent lymphocytosis; often the blood
lymphocyte counts are equal to or greater than those observed prior to therapy.
In clinical trials, it is becoming more common to evaluate for MRD with ≥0.01% of CLL
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cells among the total population of mononuclear cells in the blood or marrow. MRD can be
measured by flow cytometry or PCR with next-generation sequencing of the clonal
immunoglobulin variable region gene rearrangements163. In most clinical trials for patients
with CLL, particularly those conducted in Europe, evaluation of MRD has been performed
by flow cytometry of mononuclear cells from the marrow aspirate (the preferred method) or
from the peripheral blood. In the 6 months following anti-CD20 monoclonal antibody
treatment, the assessment of MRD is more sensitive on the mononuclear cells of the marrow
aspirate than on cells that are isolated from the blood, which will often lack detectable CLL
even when they are readily found in the marrow. Beyond a complete response, the best
predictor of long-term PFS and overall survival is the achievement of a complete remission
without evidence for MRD.
Relapsed disease
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The treatment landscape for relapsed and refractory CLL will be changing owing to the first-
line approval of ibrutinib. Currently, most patients with relapsed or refractory disease receive
chemoimmunotherapy. Standard salvage regimens include BCR inhibitors or BCL-2
inhibitors, particularly for patients with CLL and del(17p). For patients who received first-
line BTK inhibitor therapy, salvage options include chemo immunotherapy (fludarabine,
cyclophosphamide and rituximab (or bendamustine with an anti-CD20 monoclonal
antibody), PI3K inhibitor and an anti-CD20 monoclonal antibody152, high-dose
methylprednisolone and an anti-CD20 monoclonal antibody164, or lenalidomide alone or
with an anti-CD20 monoclonal antibody, although lenalidomide has not been approved for
the treatment of patients with CLL by the FDA165,166. Treatment choice depends on
individual patient characteristics and the intent of treatment. Scant data are available
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regarding the activity of small-molecule inhibitors in patients who are refractory to another
small-molecule inhibitor; better efficacy is expected for patients who discontinued use of
another small-molecule inhibitor due to intolerance167. Ibrutinib resistance is an adverse
predictor of clinical outcome, particularly for patients who were previously exposed to
chemoimmunotherapy. If a previously treated patient develops del(17p), or mutated TP53,
treatment options include ibrutinib, venetoclax, or idelalisib and an anti-CD20 monoclonal
antibody. The patient could also participate in a clinical trial. The preference for non-
chemotherapy-based treatment should be driven by prior exposure to a small-molecule
inhibitor and a review of the safety profile of the drug.
Maintenance therapy with an anti-CD20 monoclonal antibody after chemoimmunotherapy

has been shown to prolong PFS, but not overall survival, and was associated with a
significantly higher incidence of neutropenia and risk for infections168. This regimen is
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currently not considered the standard of care, but might be useful in patients with medical
comorbidities that limit other treatment options.
Quality of life
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Comorbidities
As CLL is a disease of the elderly population, assessing the effect of CLL on the patient’s
quality of life (QOL) and the coexisting comorbidities that occur in this patient population is
important169. Awareness regarding the importance of a patient’s QOL, not only during and
after treatment but also during the watch-and-wait period, is increasing.
Until recently, few clinical trials included elderly or frail patients, who account for most
patients with CLL. As such, the recommendations for therapy were largely based on results
from clinical trials that were conducted with younger patients who could better tolerate
combination drug therapies. Trials have moved away from using eligibility criteria based on
age or creatinine clearance, to using more objective measures of fitness, such as the
cumulative illness rating score, which can stratify patients for appropriate first-line or
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subsequent therapy. This has led to an increased number of published clinical trials targeting
patients who would not be deemed fit for aggressive chemoimmunotherapy approaches.
Importantly, this has also demonstrated that ‘unfit’ patients with CLL can be recruited to
clinical trials in a timely manner.
Risk of other diseases

Patients with CLL have an increased risk of other medical conditions, such as infections,
autoimmune disorders or secondary cancers, any one of which can result in substantial
morbidity and mortality.
Infections—CLL is characterized by progressive defects in both cell-mediated and

antibody-mediated immunity, including hypogammaglobulinaemia and B cell and T cell
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quantitative and functional defects170. The risk of infections increases with worsening
hypogammaglobulinaemia. The degree of immune impairment worsens with disease
progression and can be exacerbated by the immunosuppressive effects of purine analogue
chemotherapy, anti-CD20 monoclonal antibodies or drugs that inhibit kinases involved in
immune receptor signalling. Consequently, infectious complications represent a frequent
cause of morbidity and mortality in patients with CLL.
Infections are typically bacterial and frequently involve the respiratory tract. Intravenous
immunoglobulin replacement therapy can mitigate the risk of infection, particularly in
patients with hypogammaglobulinaemia who have frequent infections or a severe life-
threatening infection171. Immunoglobulin formulations that are administered subcutaneously
seem to be as effective and might be less costly172. Unfortunately, randomized studies
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assessing the relative benefit of intravenous immunoglobulin replacement therapy versus

prophylactic antibiotics in patients with CLL, hypo gammaglobulinaemia and recurrent or
serious infections have not been conducted. However, the development of another infection
soon after completing a course of antibiotics, requiring repeated antibiotic therapy, is not
uncommon in patients with CLL. These patients should be considered for immunoglobulin
replacement therapy. The use of prophylactic antimicrobial agents to prevent opportunistic
infections should be considered, particularly in patients undergoing therapy with drugs that
might worsen immune function173. Finally, because of their suppressed immune function,
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patients should avoid having live vaccines, such as those used to vaccinate against shingles.
Autoimmune complications—Autoimmune complications are common in patients with

CLL and occur in up to 25% of patients. Autoimmunity in CLL primarily targets the
haematological lineages, resulting in autoimmune haemolytic anaemia, immune
thrombocytopenic purpura, pure red cell aplasia or autoimmune granulocytopenia174.
Spontaneous or drug-related autoimmune haemolytic anaemia is the most common
autoimmune complication of CLL, the prevalence of which is related to disease stage and
progression. For example, the prevalence of autoimmune haemolytic anaemia is 2.9% in
patients with stable Binet stage A disease and >10% in patients with Binet stage B or stage
C disease. Approximately 1–5% of patients with CLL develop clinically apparent immune
thrombocytopenic purpura, which makes CLL the most common disease associated with this
disorder in adults. Pure red cell aplasia, in which the marrow ceases to produce erythrocytes
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resulting in reticulocytopenia, occurs in <1% of patients174; diagnostic evaluation requires a

marrow biopsy showing virtual absence of erythroid precursor cells without myelodysplasia,
as well as the exclusion of viral infections that can impair erythropoiesis, such Parvovirus
B19, Epstein–Barr virus, viral hepatitis B or hepatitis C and HIV infections. Even rarer is
secondary autoimmune granulocytopenia, which occurs in <0.2% of patients174; the
diagnosis requires a marrow biopsy showing maturation arrest at a late stage in granulocyte
differentiation and exclusion of other causes of isolated acquired neutropenia, such as
myelodysplasia, concomitant granular lymphocyte leukaemia or diseases that might cause
secondary immune neutropenia, such as rheumatoid arthritis, systemic lupus erythematosus,
Crohn’s disease, and related systemic autoimmune diseases.
No systematic controlled trials of treatment for autoimmune cytopenias in patients with CLL
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have been conducted. Corticosteroids remain the mainstay of initial treatment, but
mycophenolate and thrombopoietin-like agents might be helpful for patients with immune
thrombocytopenic purpura175,176. Second-line treatments include cyclosporine or rituximab.
Splenectomy can be helpful in patients with severe or recurrent immune cytopenias who are
good-risk surgical candidates, but risks further impairment of immune function. Refractory
autoimmune haemolytic anaemia or immune thrombocytopenic purpura might require
treatment of the underlying CLL, preferably with therapy that does not substantially impair
compensatory haematopoiesis.
Secondary cancers—Several large retrospective analyses have demonstrated that

patients with CLL have an increased incidence of several secondary primary malignancies
compared with an age-matched population, particularly non-melanoma skin cancers, but also
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for melanoma, sarcomas, and lung, renal and prostate cancers177. The immune deficiencies
that are associated with CLL might contribute to this increased risk, but the malignancies
observed do not mirror those in patients with other immune-deficiency diseases. Exceptions
to this observation are Merkel cell carcinoma178, which is associated with Merkel cell
polyomavirus infection, and Bowen disease, which is an aggressive form of squamous cell
carcinoma associated with human papillomavirus infection179. Although initial studies had
suggested that the risk of secondary cancers was increased following chemotherapy,
subsequent studies have suggested that the risk is similar in untreated patients who continue
on watch and wait180.
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Prolymphocytic transformation—B cell prolymphocytic transformation is a rare event,

occurring in <1% of patients. This disease is characterized by symptomatic splenomegaly,
rapidly rising numbers of leukaemia cells in the blood, >55% of which have the morphology
of a prolymphocyte on a blood smear. Diagnosis of prolymphocytic leukaemia is made by
evaluation of the blood smear, immunophenotyping and molecular genetics. The clinical
behaviour of prolymphocytic leukaemia is generally more aggressive than CLL, although
some patients still might have indolent disease. Patients with prolymphocytic leukaemia are
typically treated with combination purine analogue-based chemoimmunotherapy However,
drugs that inhibit BCR signalling, such as ibrutinib or idelalisib, might be effective in the
management of some patients, especially those with del(17p) or inactivating mutations in
TP53.
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Richter syndrome—Richter syndrome is the transformation of CLL to an aggressive

lymphoma, commonly DLBCL (BOX 2), classic Hodgkin lymphoma or an unusual
histology of Hodgkin-Reed-Sternberg-like cells surrounded by CLL cells without the
polymorphous reactive infiltrate of classic Hodgkin lymphoma181. Approximately 2–7% of
patients with CLL develop Richter syndrome, with an incidence rate of ~0.5% per year of
observation182. Richter syndrome may occur more frequently in patients with CLL cells that
harbour NOTCH1 mutations or that express certain stereotypical immunoglobulin
molecules, particularly those with a heavy-chain variable region encoded by IGHV4-39 and
a heavy-chain third complementarity-determining region (HCDR3) encoded by IGHD6-13
and IGHJ5, the so-called ‘HCDR3 subset 8’ (REF. 183).
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Clinical suspicion of Richter syndrome is raised if a patient develops new or worsening

symptoms, such as night sweats, fatigue and involuntary weight loss, a sharp increase in the
levels of serum lactic dehydrogenase, and/or a rapidly enlarging lymph node (or nodes) or an
extra-nodal lymphoid mass (or masses). PET imaging can be used to evaluate these
patients184, including directing where to perform a biopsy, which is required to establish the
diagnosis. The mainstay of treatment is chemoimmunotherapy, although newer therapies are
being investigated using some of the BCR inhibitors and/or BCL-2 inhibitors or immune
checkpoint inhibitors. Nevertheless, the prognosis of patients with Richter syndrome
generally is poor, particularly for those who are heavily pretreated for CLL and/or who have
transformation involving lymphocytes that are clonally related to the underlying CLL182.
Younger, fit patients who respond to induction therapy should be considered for allogeneic
stem cell transplantation to prolong survival.
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Acute leukaemia and myelodysplastic syndrome—Acute leukaemia and

myelodysplastic syndrome are uncommon in CLL. Overall prognosis is poor and new
treatment approaches are needed185. The rates of therapy-related acute myeloid leukaemia or
myelodysplastic syndrome following purine analogue-based chemoimmunotherapy are ~5%,
and are greatly increased in patients who undergo autologous stem cell transplantation.
Studies are underway to evaluate whether the use of novel agents, which do not expose
normal haematopoietic cells to genotoxic stress, will decrease the incidence of this serious
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complication.
Outlook
The outlook for patients with CLL has improved substantially over the past several years.
Through research on the immune biology and genetics of CLL, patients can be stratified into
subgroups with distinctive clinical features, which has improved our capacity to assess
prognosis or govern therapy. However, an understanding of the mechanisms that contribute
to immune dysfunction or how it contributes to autoimmune disease, such as autoimmune
haemolytic anaemia, therapy resistance or therapy-related complications is unknown.
Whether tyrosine kinase inhibitors can affect clonal evolution, induce and/or select for drug
resistance, or can achieve complete responses if used earlier in the course of the disease is
also unknown.
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Future treatments
Several therapies are currently under preclinical and clinical investigation for the treatment
of patients with CLL, including new drugs and treatment modalities that can modulate the
immune system, and cell transplantation.
Immune-modulatory drugs—Immune-modulatory drugs, such as thalidomide and

lenalidomide, are approved for the treatment of patients with multiple myeloma, mantle cell
lymphoma or myelodysplastic disease. Although these drugs have clinical activity in
patients with CLL, they have had limited application unless used in combination with an
anti-CD20 monoclonal antibody166,186. In CLL, lenalidomide can induce the expression of
p21WAF1/CIP1, which inhibits cyclin-dependent kinase and CLL cell proliferation187, and can
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improve immune synapse formation, potentially enhancing immune function188. In patients

with CLL, lenalidomide can mitigate the severity of hypogammaglobulinemia189, but
myelosuppression is a dose- limiting toxicity. Other dose-limiting toxicities associated with
the use of lenalidomide, particularly as a first-line therapy, include tumour flare and
tumourlysis syndrome. For unknown reasons, patients with CLL seem to be more sensitive
to lenalidomide than patients with other haematological indications, mandating the use of
low doses (for example, 2.5-5 mg per day) when initiating therapy. Low-dose aspirin is
frequently used to mitigate the risk for thromboembolic complications that are associated
with lenalidomide therapy.
Thalidomide has little activity in patients with CLL as a monotherapy, but has shown
efficacy when combined with other drugs, such as anti-CD20 monoclonal antibodies190.
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Conversely, lenalidomide monotherapy has an overall response rate of 60% (15% complete
response rate) as a first-line therapy and 40% (8% complete response rate) as a salvage
treatment165,189. However, trials assessing the use of lenalidomide as a monotherapy or
combination therapy have yielded mixed results. One phase II study reported that
lenalidomide was well tolerated as initial therapy in patients >65 years of age, with an
overall response rate of 65% and a complete response rate of 10%189. However, a
multicentre phase III trial for the same patient population had to be terminated owing to an
increased number of deaths in patients receiving lenalidomide compared with those
receiving chlorambucil191. A 7-month treatment course with lenalidomide in combination

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with rituximab was well tolerated in a multi-institutional phase II study and yielded higher
response rates; for patients <65 years of age, the overall response rate was 95%, with a 20%
complete response rate, and for patients ≥65 years of age, the overall response rate was 78%,
with an 11% complete response rate166. The PFS after completion of therapy was ~20
months for both groups. Therapy with an anti-CD20 monoclonal antibody 9 days before initi
ation of lenalidomide therapy also seems to mitig ate the risk for tumour flare
reaction192,193. The use of lenalidomide after chemoimmunotherapy has been evaluated in a
phase III maintenance trial (Continuum Trial; ClinicalTrials.gov identifier: NCT00774345)
and results are forthcoming. Ongoing trials are also examining the activity in CLL of a novel
lenalidomide analogue, CC-122 (REF. 194).
Allogeneic stem cell transplantation—Allogeneic stem cell transplantation is a

potentially curative strategy for patients with relapsed or refractory CLL, including patients
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with high-risk features such as del(17p). In two clinical studies with patients who lacked
serious medical comorbidities and had a median age of 53 or 58 years, the PFS at 3–5 years
was 40–50% and overall survival was 50–70%, but the non-relapse mortality at 3–5 years
was 25–40%195,196. Research efforts are ongoing to develop better-tolerated cell-based
therapy with a similar curative potential that can be used without the immunosuppression
and associated long-term morbidity and mortality of allogeneic stem cell transplantation.
Donor availability, advanced patient age, associated toxicities of myelosuppression, graft-

versus-host disease and impaired resistance to infections limit the application of allogeneic
stem cell transplantation in patients with CLL. In addition, the advent of BCR signalling
inhibitors and BCL-2 inhibitors provide multiple treatment options that afford well-
tolerated, long-term disease control, making allogeneic stem cell transplantation the least
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desirable option for most patients. Ongoing discussion exists around who are the appropriate
patients for allogeneic stem cell transplantation.
T cell therapy with chimeric antigen receptors—T cells can be modified ex vivo to
express new surface receptors, known as chimeric antigen receptors (CARs), which have
been engineered to target cancer cells, expanded in vitro and then reintroduced into the
patient as a treatment for CLL (BOX 3).
Both normal and malignant B cells (including CLL cells) express surface CD19, which has
been targeted with CAR technology. CD19-targeted CAR T cells have yielded long-term
PFS and relapse-free survival durations in patients with CLL; in 14 patients with relapsed or
refractory CLL, four patients achieved a complete response and four patients achieved a
partial response197. None of the complete responders had MRD and none relapsed (a median
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follow-up of 19 months). However, the efficacy of CAR T cell therapy in patients with CLL
has been modest compared with that in patients with acute lymphoblastic leukaemia; this
might be owing to qualitative defects in the T cells of patients with CLL, who are generally
older than patients with acute lymphoblastic leukaemia and already have immune
dysfunction that reflects disease-associated anergy (see BCR and B cell signalling). Ibrutinib
might partially correct some of these defects198. Larger phase II trials assessing the use of
CAR T cell therapy for CLL are in development, including the use of CAR T cells that can
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target antigens other than CD19, such as ROR1 (REFS 199,200).
The major adverse effect of CAR T cell therapy is a cytokine release syndrome, which
occurs as a result of CAR T cell activation, cytokine production and T cell expansion
following target antigen encounter. Cytokine release syndrome is characterized by fever,
hypotension and capillary leakage, but neurological toxicity, which can manifest as
confusion and seizures, has also been observed in some of the treated patients. Cytokine
release syndrome is associated with high cytokine levels, particularly IL-6, and can be
managed with an IL-6-binding factor, such as tocilizumab, supportive measures, and
glucocorticoids for severe cases. This syndrome seems to be proportionate to the antigen-
bearing tumour burden, potentially making CAR T cell therapy more amenable to treatment
of patients with MRD.
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Immune checkpoint inhibitors—Immune checkpoints are proteins that are expressed on

the surface of antigen-presenting cells that regulate the immune system by providing co-
stimulatory or co-inhibitory signals to ligands expressed on T cells and other immune
effector cells. The finding that cancer cells can evade immune detection and destruction by
inhibiting T cells has led to the development of immune checkpoint inhibitors to treat solid
tumours, Hodgkin lymphomas and non-Hodgkin lymphomas201.
The immune checkpoint receptor PD-1, and its ligands PD-L1 and PD-L2, is the most
important cognate receptor involved in the suppression of cellular immune activation. CLL
cells express high levels of PD-L1 and PD-L2 and can suppress the responses of PD-1-
expressing effector T cells106, leading to an exhausted (that is, no longer functional) T cell
phenotype. T cell exhaustion in CLL is also mediated in part by lymphocyte activation gene
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3 protein and T cell immunoglobulin mucin receptor 3 (TIM3; also known as HAVCR2).
TIM3 negatively regulates the function of type 1 helper T cells and type 1 CD8+ T cells, by
triggering cell death upon ligand binding. Other receptors on CLL cells have demonstrated
negative immune feedback, including CD276, CD200 and TNF receptor superfamily
member 14.
A partial list of immune checkpoint inhibitors that are being evaluated in the therapy of
patients with CLL or other cancers include monoclonal antibodies against PD-1, cytotoxic T
lymphocyte protein 4, B lymphocyte and T lymphocyte attenuator and its ligand TNF
superfamily member 14, the adenosine A2A receptor, indoleamine 2,3-dioxygenase, the V-
type immunoglobulin domain-containing suppressor of T cell activation, lymphocyte
activation gene 3 protein and TIM3.
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Preclinical studies in mouse models have demonstrated that checkpoint inhibitors can
reactivate immune effector cells to have anti-leukaemia activity202. However, ongoing phase
I/II trials of immune checkpoint inhibitors in patients with relapsed CLL have yet to show
much clinical activity, possibly reflecting the highly immune suppressive nature of CLL
cells and/or the ‘exhausted’ phenotype of T cells in patients with this disease.
Combination targeted therapy—We can now target the distinctive phenotypic or

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physiological features of CLL with targeted therapeutic agents, which have a higher
therapeutic index than standard chemotherapy. Through the use of combination therapy,
which targets different B cell survival signalling pathways and/or achieves better eradication
of CLL cells, we might be able to define curative treatments for most patients with this
disease.
Research on leukaemia cell survival signalling pathways, such as those governed by

interactions between leukaemia cells and cells or secreted factors within the
microenvironment (FIG. 4), might identify pathways that are not affected by BCR inhibitors.
For example, BCR inhibitors, such as ibrutinib, cannot block ROR1-dependent WNT5A
signalling, which enhances CLL cell proliferation, migration and survival93; as such,
antibodies that block ROR1-dependent signalling could potentially have synergistic activity
when used in combination with BCR inhibitors203. Furthermore, the interaction between
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CLL cells and accessory cells in the microenvironment might enhance CLL cell expression
of anti-apoptotic proteins other than BCL-2, such as MCL1, thereby contributing to therapy
resistance. As such, the therapeutic use of a selective BCL-2 antagonist, such as venetoclax,
might be more effective when used in combination with BCL-2 inhibitors161,162, which also
interfere with the homing of CLL cells to the microenvironment. Conceivably, combination
target therapy with agents that have synergistic activity will provide highly effective and
potentially curative treatment of patients with CLL.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Author Manuscript
Acknowledgments
The authors thank H.-Y. Wang and E. Broome, Department of Pathology, University of California San Diego, for
their contributions to Figures 5–7. The authors also thank A. Greaves, University of California San Diego, for help
in figure development. This Primer is supported by the US NIH PO1-CA81534 for the CLL Research Consortium;
members include K.R., W.G.W., T.J.K., C.M.C., C.J.W. and J.G. The authors also acknowledge support to F.K.S.
and G.P. from Bloodwise, Kay Kendall Leukaemia Fund, Cancer Research UK, the Southampton Experimental
Cancer Medicine Centre, and the Southampton Cancer Research UK Centre (F.K.S. and G.P.). The authors also
acknowledge support from the Leukemia & Lymphoma Society (LLS) Specialized Center of Research Program
(SCOR) grant (T.J.K.), 1R01HL116452, 1R0 1CA182461, CLL Global Reseach Foundation (W.G.W.), the Blood
Cancer Research Fund (T.J.K.), and U10CA180861.
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Box 1 | Differential diagnosis of CLL

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Small lymphocytic lymphoma

Diagnosis of small lymphocytic lymphoma is generally made following biopsy of an
enlarged lymph node, which typically has a disrupted architecture owing to the
infiltration of well-differentiated, clonal B cells with the same phenotype as chronic
lymphocytic leukaemia (CLL) cells. Patients with small lymphocytic lymphoma have
<5,000 clonal B cells per µl in the blood, but over time, patients can develop lymphocyte
counts of >5,000 cells per µl, which allows them to be reclassified as having CLL.
Monoclonal B lymphocytosis
Monoclonal B lymphocytosis is defined as <5,000 clonal B cells per µl in the blood
without other signs of lymphoma, such as enlarged lymph nodes (>1.5 cm), which would
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suggest the diagnosis of small lymphocytic lymphoma204. In most, but not all, cases, the
clonal B cells in monoclonal B lymphocytosis express CD5 and have the same immune
phenotype as CLL205. Although biopsies are not generally performed, the incidental
finding of CLL-like cells in the marrow or in normal-sized lymph nodes does not exclude
the diagnosis of monoclonal B lymphocytosis.
Cases of monoclonal B lymphocytosis are classified as being low count (<500

monoclonal B cells per µl) or high count (>500 monoclonal B cells per µl).
Approximately 5% of adults of European ancestry >40 years of age have low-count
monoclonal B lymphocytosis, as assessed via flow cytometry on blood mononuclear
cells. Although subjects with low-count monoclonal B lymphocytosis rarely progress to
CLL, 1–2% of patients with high-count monoclonal B lymphocytosis will develop CLL
per year206.
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Other lymphoproliferative diseases

Other chronic B cell lymphoproliferative diseases can present like CLL, including B cell
prolymphocytic leukaemia, follicular lymphoma, hairy cell leukaemia, mantle cell
lymphoma or marginal zone lymphoma. In addition to clinical features and pathology,
which are characteristic of these other conditions, the immune phenotype of neoplastic
lymphocytes helps to differentiate these conditions from CLL.
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Box 2 | Molecular biology of Richter syndrome

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The lymphoma cells in Richter syndrome are malignant B cells that most often resemble
those of non-germinal centre diffuse large B cell lymphoma (DLBCL), differing
morphologically from the original chronic lymphocytic leukaemia (CLL) population. In
addition, the lymphoma cells of over half the patients with Richter syndrome might not
express CD5 or CD23, which are almost invariably expressed by CLL cells. Nevertheless,
the DLBCL-like lymphoma in Richter syndrome often shares the same IGHV-DJ
rearrangement as the original CLL clone207. As such, the lymphoma cells in Richter
syndrome can express unmutated immunoglobulin heavy-chain variable region gene
(IGHV), unlike de novo DLBCL, which virtually always expresses IGHV with somatic
mutations. However, ~20% of the DLBCL-type Richter syndrome and ~50% of Hodgkin
lymphoma-type Richter syndrome have IGHV-DJ rearrangements that differ from that of
the original CLL clone, suggesting that these lymphomas might represent a de novo
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secondary malignancy; some of these seem to be associated with Epstein-Barr virus

infection and may resemble post-transplant lymphomas, particularly in patients with
severe disease-related immune dysfunction and/or treatment-related immune
suppression208.
Although the lymphoma cells of DLBCL-type Richter syndrome resemble those of de

novo DLBCL, they have distinctive genetic differences209. Of DLBCL-type Richter
syndrome lymphomas, ~60% have inactivating mutations and/or deletions in TP53, often
with deregulation of MYC, which is observed in ~40% of cases; such deregulation is
caused by translocations juxtaposing MYC to immunoglobulin loci, gene amplification of
MYC at 8q24 or somatic mutations affecting MYC trans-regulatory factors, such as
NOTCH1, which is mutated in ~30% of cases210,211. CDKN2A, which encodes p16, a
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negative regulator of cell cycle progression from G1 to S phase, is mutated and/or deleted
in ~30–50% of cases, but rarely so in CLL or de novo DLBCL209,211. Finally, Richter
syndrome lymphomas typically do not have mutations in genes encoding proteins that are
involved in nuclear factor-κB signalling or in the transcriptional repressors PRDM1/
BLIMP1 or BCL6, which are common in de novo DLBCL.
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Box 3 | Chimeric antigen receptors

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Chimeric antigen receptors (CARs) are composed of an antigen-binding domain, a stalk

and transmembrane region, an intracellular co-stimulatory signalling domain and CD3ζ
(see the figure)212. Co-stimulatory signalling domains include CD28 and CD137, which
provide the ‘second signal’ to fully activate and expand T cells upon antigen binding.
Retrovirus vectors are used to introduce the CAR gene into T cells, which integrates into
the genome of the T cell for stable expression. When the CAR T cells are exposed to the
respective antigen, antigen binding triggers activation and expansion of the CAR T cells
and eliminates the cells with target antigen. The binding domain of the CAR can be
directed against any surface antigen.
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Figure 1. Cellular origins of CLL cells

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Normal naive B cells that have undergone successful V(D)J recombination and express
functional B cell receptors that are capable of binding to antigen interact with CD4+ T cells
and accessory cells, which aggregate to form follicles that become germinal centres.
Germinal cells each have a dark zone, comprising rapidly dividing B cells, and a light zone,
comprising B cells mixed with follicular dendritic cells (FDCs), macrophages and helper T
cells (TH cells). The B cells enter the dark zone of the germinal centre where they
experience rapid proliferation and somatic hypermutation (SHM) in the genes encoding the
immunoglobulin variable regions of the heavy chain (IGHV) and the light chain (IGVL). As
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they pass through to the light zone, the B cells that express the fittest B cell receptors for
binding antigen are selected and may undergo immunoglobulin class-switch recombination.
Chronic lymphocytic leukaemia (CLL) cells that use unmutated IGHV apparently originate
from CD5+ B cells prior to experiencing SHM, whereas CLL cells that use mutated IGHV
most likely originate from CD5+ B cells that have passed through and differentiated in the
germinal centre. Some CLL cells might be derived from B cells that also have undergone
immunoglobulin class-switch recombination and express immunoglobulin isotypes other
than IgM and IgD, for example, IgG or IgA. Another subset is one with CLL cells that
express immunoglobulin with only modest somatic mutations, such as CLL cells that use
IGHV3-21 with ~97% homology to the inherited IGHV3-21 gene and an immunoglobulin
light chain encoded by an unmutated IGLV3-21; these cells might derive from a B cell that
has had constrained SHM, possibly owing to a limited need for immunoglobulin somatic
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diveresification and selection. Dashed arrows indicate speculated pathways.

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Figure 2. Range of somatic mutations in CLL

Genes that are mutated in chronic lymphocytic leukaemia (CLL) are involved in several
cellular pathways (blue boxes). As such, mutations in these genes can lead to a range of
cellular consequences, such as aberrant DNA repair and B cell receptor (BCR) signalling,
among others51,213. The minus sign from GBN1 to the MAPK–ERK pathway indicates
negative regulation. *For more detail of the BCR and its associated signalling, see FIG. 3.
ASXL1, additional sex combs-like protein 1; ATM, ataxia telangiectasia mutated; BAZ2A,
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bromodomain adjacent to zinc-finger domain protein 2A; BCOR, BCL-6 co-repressor;

BIRC3, baculoviral IAP repeat-containing protein 3; BRCC3, BRCA1/BRCA2-containing
complex subunit 3; C-NOTCH, carboxy-terminal domain of NOTCH; CARD11, caspase
recruitment domain-containing protein 11; CHD2, chromodomainhelicase-DNA-binding
protein 2; CHK2, checkpoint kinase 2; Co-A, co-activator; CSL, CBF1–Suppressor of
Hairless–LAG1 (also known as RBPJ); DDX3X, ATP-dependent RNA helicase DDX3X;
DYRK1A, dual-specificity tyrosine-phosphorylation-regulated kinase 1A; EGR2, early
growth response 2; ELF4, ETS-related transcription factor Elf-4; ERK, extracellular signal-
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regulated kinase; EWSR1, Ewing sarcoma breakpoint region 1 protein; FBXW7, F-box/WD
repeat-containing protein 7; FUBP1, far upstream element-binding protein 1; GNB1,
guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β1; H3K4, histone H3 lysine 4;
IC, intracellular domain; IKZF3, Ikaros family zinc-finger protein 3; IL-1R, IL-1 receptor;
IRF4, interferon regulatory factor 4; ITPKB, inositol-trisphosphate 3-kinase B; LRP, low-
density lipoprotein receptor-related protein; MAP2K1, dual-specificity mitogen-activated
protein kinase kinase 1; MAPK, mitogen-activated protein kinase; MED12, Mediator of
RNA polymerase II transcription subunit 12; MGA, MAX gene-associated protein; MYD88,
myeloid differentiation primary response protein MyD88; NF-κB, nuclear factor-κB; NXF1,
nuclear RNA export factor 1; P, phosphate; POT1, protection of telomeres protein 1;
PTPN11, tyrosine-protein phosphatase non-receptor type 11; RIPK1, receptor-interacting
serine/threonine-protein kinase 1; RPS15, 40S ribosomal protein S15; SAMHD1, SAM
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domain and HD domain-containing protein 1; SF3B1, splicing factor 3B subunit 1; SHP1,

Src homology region 2 domain-containing phosphatase 1 (also known as PTPN6); SYK,
spleen tyrosine kinase; TCF/LEF, T cell factor/lymphoid enhancer factor; TLR8, Toll-like
receptor 8; TNFR1, tumour necrosis factor receptor 1 (also known as TNFRSF1A); TRAF,
TNFR-associated factor; XPO, exportin; ZMYM3, zinc-finger MYM-type protein 3.
Adapted with permission from REF. 51, Macmillan Publishers Limited.
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Figure 3. B cell receptor signalling response

B cell receptor (BCR) signalling is initiated by SRC-family kinase-dependent
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phosphorylation (mainly LYN) of CD79A and CD79B that creates a docking site for the
binding and activation of spleen tyrosine kinase (SYK). SYK then triggers the formation of a
multi-component ‘signalosome’, comprising Bruton tyrosine kinase (BTK), AKT,
phosphoinositide 3-kinase (PI3K), phospholipase Cγ2 (PLCγ2) and B cell-linker protein
(BLNK), among others. CD19 is a co-receptor for BCR and is important for PI3K activation,
which recruits and activates PLCγ2, BTK and AKT. PLCγ2 generates diacylglycerol (DAG)
and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3), which triggers Ca2+ release from the
endoplasmic reticulum, leading to the activation of the MEK–extracellular signal-regulated
kinase (ERK) and nuclear factor-κB (NF-κB) signalling pathways. Other effects of BCR
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signalling include activation of mechanistic target of rapamycin complex 1 (mTORC1) and

of Rho-family GTPases, RAC1 and RHOA, which can affect the cytoskeleton. Inhibitors of
SYK, PI3K and BTK are shown. Note that this figure describes the main molecules and
interactions that are involved in positive BCR signalling, but is not an exhaustive description
of all signalling pathways or molecules activated. IKK, IκB kinase; PKC, protein kinase C.
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Figure 4. CLL microenvironment

Migration of chronic lymphocytic leukaemia (CLL) cells into the lyphoid tissu is primarily
mediated through CXC-chemokine receptor 4 (CXCR4) in response to CXC-chemokine
ligand 12 (CXCL12), which is secreted mainly by nurse-like cells (NLCs) and
mesenchymal-derived stromal cells. Migration of CLL cells into lymph nodes also occurs
via CC-chemokine receptor 7 (CCR7) in response to CC-chemokine ligand 19 (CCL19) and
CCL21, which are produced by the endothelial cells of high endothelial venules (HEVs).
HEV endothelial cells also express hyaluronan, which can interact with CD44, to facilitate B
cell signalling and might enhance the production of active matrix metalloproteinase 9
(MMP9). Once in tissues, several chemokines promote B cell survival, including CXCL12,
B cell-activating factor (BAFF; also known as TNFSF13B) and a proliferation-inducing
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ligand (APRIL; also known as TNFSF13). In addition, CLL cell survival can be promoted
through cognate interactions between CD31 and CD38, and the production by stromal cells
of WNT factors, which can interact with ROR1, ROR2 and/or various Frizzled receptors.
CLL cell contact with mesenchymal stromal cells can also be established through vascular
cell adhesion protein 1 (VCAM1)– α4β 1 integrin interactions that contribute to CLL cell
survival. In turn, CLL cells can secrete chemokines, such as CCL3 and CCL4, which can
recruit T cells and NLC-precursor cells (monocytes) to the CLL microenvironment.
Activated T cells can provide CLL cells with proliferative signals through CD40 ligand
(CD40L)-CD40 interactions and the secretion of several cytokines, such as IL-2, IL-4 and
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IL-10. In turn, activated CLL cells secrete CCL12 and CCL22, which attract more T cells
into the CLL microenvironment. In tissues, CLL cells can be exposed to environmental
and/or self-antigens that might trigger B cell activation through interactions with the surface
immunoglobulin; this could amplify the responsiveness of CLL cells to the signals and
factors that are provided by the CLL microenvironment. BAFFR, BAFF receptor (also
known as TNFRSF13C); BCMA, B cell maturation protein (also known as TNFRSF17);
BCR, B cell receptor; TACI, transmembrane activator and CAML interactor (also known as
TNFRSF13B).
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Figure 5. Blood smears from patients with CLL

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Wright–Giemsa-stained blood smears showing the typical chronic lymphocytic leukaemia

(CLL) B lymphocyte (part a), smudge cell (part b) and a prolymphocyte with a prominent
nucleolus (part c). Magnification ×500. Images courtesy of H. E. Broome, University of
California, San Diego, La Jolla, California, USA.
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Figure 6. Marrow biopsies from patients with CLL

Tissue sections of a marrow biopsy specimen stained with haemotoxylin and eosin showing
interstitial (I) or nodular (N) chronic lymphocytic leukaemia (CLL) cell involvement (part a)
or diffuse CLL cell marrow involvement (part b), which is typically associated with
advanced-stage disease (original magnification ×100). Images courtesy of H. E. Broome,
University of California, San Diego, La Jolla, California, USA.
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Figure 7. Lymph node of patients with CLL

a | Tissue sections of a lymph node stained with haemotoxylin and eosin showing numerous
pale-staining pseudofollicles, which are circled (original magnification ×20). b | Higher
(×400) magnification of a proliferation centre. Representative lymphocytes (arrows),
prolymphocytes (arrowheads) or paraimmunoblasts (circles) in a proliferation centre are
shown. Images courtesy of H.-Y. Wang, University of California, San Diego, La Jolla,
California, USA.
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Figure 8. Management algorithm for patients with CLL

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Indications for therapy of patients with chronic lymphocytic leukaemia (CLL) include late-
stage disease, evidence for rapid disease progression or disease-related symptoms. Patients
with del(17p) or mutated TP53 should be treated with therapy that does not require
functional TP53, such as ibrutinib (a Bruton tyrosine kinase (BTK) inhibitor), given the
relatively poor outcome for such patients with chemotherapy. For patients without del(17p)
or known mutations in TP53, immunoglobulin heavy-chain variable region (IGHV)
mutational status can help to define the treatment strategy; patients with unmutated IGHV
could be considered for therapy with a BTK inhibitor (such as ibrutinib) and patients with
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mutated IGHV might be good candidates for chemoimmunotherapy (CIT), if amenable.

Indeed, patients with mutated IGHV can have excellent outcomes with CIT regimens, such
as fludarabine, cyclophosphamide and rituximab, with >50% of patients having a median
progression-free survival of >10 years, including the potential for cure. If the patient is
amenable to CIT, age, medical comorbidities and myeloid reserve should be taken into
consideration. Patients >65 years of age commonly have medical comorbidities and are less
able to tolerate myelosuppressive regimens, such as fludarabine, cyclophosphamide and
rituximab. Thus, considerations should be given to using reduced dose or less
myelosuppressive chemotherapy regimens, such as chlorambucil or reduced-dose
bendamustine and an anti-CD20 monoclonal antibody for patients with limited myeloid
reserve. Patients who either do not respond, have a poor tolerance to CIT or relapse
following CIT, should be re-evaluated for del(17p) or TP53 mutations. Patients who develop
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de novo del(17p) or TP53 mutations, or have known del(17p) and/or TP53 mutations, or
who develop resistance or intolerance to ibrutinib, could be considered for therapy with
idelalisib and rituximab or the BCL-2 inhibitor venetoclax. Patients treated with CIT who do
not have del(17p) or TP53 mutations could be considered for repeat CIT if their progression-
free survival after CIT is >2 years and the patient has sufficient myeloid reserve. Such
patients also might be treated with a BTK inhibitor or a phosphoinositide 3-kinase (PI3K)
inhibitor, which also could be considered for patients who develop intolerance or resistance
to therapy with ibrutinib. Patients who develop resistance or intolerance to inhibitors of
BTK, PI3K and/or BCL-2 should be considered for clinical trials or alternative agents. LDT,
lymphocyte doubling time.
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Table 1
Rai staging system

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Risk group Clinical features Median life

expectancy*
Low risk Lymphocytosis without cytopenia, 13 years
(Rai stage 0/I) lymphadenopathy or splenomegaly
Intermediate risk Lymphocytosis, lymphadenopathy and/or 8 years

(Rai stage II) splenomegaly, but without cytopenia
High risk Lymphocytosis and cytopenia (a haemoglobin 2 years

(Rai stage III/IV) level of ≤11 g per dl and/or a platelet count of
≤100,000 cells per µl)
*
These life-expectancy estimates are increasing with the advent of newer therapies.
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Table 2
Binet staging system

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Risk group Clinical features Median life

expectancy*
Low risk Less than three palpably enlarged sites‡ without 13 years
(Binet stage A) cytopenia
Intermediate risk Three or more palpably enlarged sites‡ without 8 years

(Binet stage B) cytopenia
High risk Cytopenia (a haemoglobin level of ≤10 g per dl 2 years

(Binet stage C) and/or a platelet count of ≤100,000 cells per µl)
*
These life-expectancy estimates are increasing with the advent of newer therapies.
‡
There are five sites of lymphoid organs: cervical, axillary and inguinal nodes, the spleen and the liver.
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Chronic lymphocytic leukaemia

Medicine, University of Southampton, Southampton, UK.

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized

marrow and secondary lymphoid tissues, resulting in lymphocytosis, leukaemia cell

be associated with high expression of lymphoid enhancer-binding factor 1, which is a

CLL-linked SNP in TERT is associated with a long leukocyte telomere length31,

dietary factors or lifestyle factors increase the risk of CLL.

Chromosomal alterations—Approximately 80% of patients with CLL carry at least one

of four common chromosomal alterations: a deletion in chromosome 13q14.3 (del(13q)),

Somatic mutations—The advent of massively parallel sequencing and the application of

and identified NOTCH1 (12.6% of patients), A T M (11% of patients), BIRC3 (8.8% of

Next-generation sequencing has revealed intra-tumoural heterogeneity in CLL. Some

Epigenetic changes—The CLL epigenome shows global hypomethylation combined

with local hypermethylation, as has been observed in other cancers66–68. Indeed,

Methylation signatures can classify distinct clinical CLL subgroups69,74. As these

BCR and B cell signalling

tyrosine-protein kinase LYN and may limit B cell activation by counteracting

lymphocytosis observed in patients upon initiation of treatment with inhibitors of BTK or

Diagnosis, screening and prevention

Flow cytometric or immunohistochemical analyses of the mononuclear cells in the blood,

positive in the absence of overt autoimmune haemolytic anaemia in a large minority of

Prognostic factors and nomograms

aberrations observed on a karyotype test)125,126, or a high absolute lymphocyte count

Chemoimmunotherapy—Phase III clinical trials have validated the benefit of anti-CD20

than in patients treated for up to 48 weeks with chlorambucil146.

Upon initiation of treatment with ibrutinib, lymphadenopathy is rapidly reduced, which is

Adverse effects of ibrutinib include fatigue, diarrhoea, bleeding, ecchymoses, rash,

Similarly, this event should not be considered as a sign of disease progression.

cytomegalovirus, especially if they should develop unexplained symptoms of infection.

evaluated in preclinical and clinical studies.

BCL-2 inhibitors—Venetoclax is a small molecule that functions as a BH3 mimetic to

without an anti-CD20 monoclonal antibody, and with or without ibrutinib161,162, which

Toxicities of venetoclax include gastrointestinal disturbances, neutropenia and tumour lysis

Maintenance therapy with an anti-CD20 monoclonal antibody after chemoimmunotherapy

Risk of other diseases

Infections—CLL is characterized by progressive defects in both cell-mediated and

assessing the relative benefit of intravenous immunoglobulin replacement therapy versus

Autoimmune complications—Autoimmune complications are common in patients with

resulting in reticulocytopenia, occurs in <1% of patients174; diagnostic evaluation requires a

Secondary cancers—Several large retrospective analyses have demonstrated that

Prolymphocytic transformation—B cell prolymphocytic transformation is a rare event,

Richter syndrome—Richter syndrome is the transformation of CLL to an aggressive

Clinical suspicion of Richter syndrome is raised if a patient develops new or worsening

Acute leukaemia and myelodysplastic syndrome—Acute leukaemia and

Immune-modulatory drugs—Immune-modulatory drugs, such as thalidomide and

improve immune synapse formation, potentially enhancing immune function188. In patients

receiving chlorambucil191. A 7-month treatment course with lenalidomide in combination

Allogeneic stem cell transplantation—Allogeneic stem cell transplantation is a

Donor availability, advanced patient age, associated toxicities of myelosuppression, graft-

target antigens other than CD19, such as ROR1 (REFS 199,200).

Immune checkpoint inhibitors—Immune checkpoints are proteins that are expressed on

Combination targeted therapy—We can now target the distinctive phenotypic or

Research on leukaemia cell survival signalling pathways, such as those governed by

1854. [PubMed: 10477713]

111:5101–5108. [PubMed: 18326815]

126:2265–2273. [PubMed: 26405224]