from pathogenesis to treatment of chronic lymphocytic ...cllcanada.ca/2010/pages/from pathogenesis...

10-05-24 1:38 PMFrom Pathogenesis to Treatment of Chronic Lymphocytic Leukaemia (printer-friendly)

of 30file:///Users/dwyer/Desktop/From%20Pathogenesis%20to%20Treatment%2…0Chronic%20Lymphocytic%20Leukaemia%20(printer-friendly).webarchive

www.medscape.com

From Nature Reviews Cancer

Abstract and Introduction

Abstract

Chronic lymphocytic leukaemia (CLL) has several unique features that distinguish it from other cancers. Most CLLtumour cells are inert and arrested in G0/G1 of the cell cycle and there is only a small proliferative compartment;however, the progressive accumulation of malignant cells will ultimately lead to symptomatic disease. Pathogenicmechanisms have been elucidated that involve multiple external (for example, microenvironmental stimuli and antigenicdrive) and internal (genetic and epigenetic) events that are crucial in the transformation, progression and evolution ofCLL. Our growing understanding of CLL biology is allowing the translation of targets and biological classifiers intoclinical practice.

Introduction

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the Western world. It is characterized bythe accumulation of small B lymphocytes with a mature appearance in blood, bone marrow, lymph nodes or otherlymphoid tissues. CLL has a distinctive immunological marker repertoire and can therefore be differentiated from otherB cell lymphomas ( Box 1 ).

Authors and DisclosuresThorsten Zenz*, Daniel Mertens*†, Ralf Küppers§, Hartmut Döhner* & Stephan Stilgenbauer*

*Department of Internal Medicine III, University of Ulm, Ulm 89081, Germany.†Cooperation Unit Mechanisms of Leukemogenesis, DKFZ Heidelberg, Germany.§Institute of Cell Biology (Cancer Research), University of Duisburg-Essen Medical School, Essen, Germany.

Correspondence to S.S. Email: [email protected]

From Pathogenesis to Treatment of Chronic LymphocyticLeukaemiaThorsten Zenz; Daniel Mertens; Ralf Küppers; Hartmut Döhner; Stephan Stilgenbauer

Posted: 02/25/2010; Nat Rev Cancer. 2010;10(1):37-50. © 2010

Box 1. Definition, diagnostic criteria and cardinal features of CLL

Chronic lymphocytic leukaemia (CLL) is a malignant lymphoproliferative disorder of mature B lymphocytes.

The progressive accumulation of monoclonal B lymphocytes leads to leukocytosis, lymphadenopathy,hepatosplenomegaly, bone marrow failure, recurrent infection and is sometimes associated with autoimmune disease(for example, haemolytic anaemia).

CLL can be diagnosed if the blood clonal B lymphocyte count is >5,000/μL. Below this value monoclonal Blymphocytosis can be diagnosed in asymptomatic individuals without organ involvement.

The immunological profile of CLL cells is defined by weak surface membrane immunoglobulin (Ig) levels (most oftenIgM or both IgM and IgD) and expression of the B cell antigens CD23, CD19 and CD20 (weak), with co-expression ofCD5. CLL cells are usually negative for cyclin D1 and CD10 expression, and there is also usually no or weak

http://www.medscape.com/

http://www.medscape.com/index/list_4855_0

mailto:[email protected]



Registry data estimate the U.S. incidence of CLL to be 3.9 per 100,000 people, with a median age at diagnosis of 72years (reviewed in Ref. [1]). The incidence rates in men are nearly twice as high as in women. CLL is less commonamong people of African or Asian origin.[1] There is substantial geographic variation in CLL incidence, with highincidences in North America and Europe.[1,2] Advanced age and a family history of leukaemia or lymphoma areadditional risk factors.[1,2] Common symptoms include lymph node enlargement, constitutional symptoms and bonemarrow failure. Most patients are asymptomatic at diagnosis.[3–6]

The understanding of the pathological mechanisms involved in CLL has helped to divide the disease into subgroups,and this has had a profound impact on prognostication and treatment. Major findings have included the demonstrationthat CLL can be subdivided into cases with or without somatic mutations in the variable regions of the immunoglobulin(Ig) heavy chain (IGHV) genes.[7–9] Similarly important was the finding that CLL cells have biased IGHV usage andcarry stereotyped B cell receptors (BCRs), which suggests that antigenic drive contributes to CLL pathogenesis.[7,10–14]

The understanding of transforming events in CLL continues to evolve as genomic aberrations, epigenetic modifications,gene mutations and deregulated microRNAs have been identified (Refs [15–19]) (Fig. 1). These factors are ofpathogenic and prognostic relevance.[17,20–23] One of the most exciting aspects of this area of research is thetranslation of these findings into clinical practice. The evolving link between biology and clinical translation will be thefocus of this Review.

expression of FMC7, CD22 and CD79b.[138]

Staging systems are based on lymphadenopathy, organomegaly, platelet and haemoglobin values. The stagingsystems by Rai and Binet have important influences on treatment decisions and prognosis.[5,6]

The most important prognostic factors are clinical stage,[5,6] serum markers (for example, thymidine kinase, β2-microglobulin and soluble CD23),[124] cell marker expression (for example, CD38 and zeta-associated protein70)[8,9,125,139] and genetic parameters (for example, Ig heavy chain variable region (IGHV) mutational status,[8,9]

genomic aberrations such as 17p13 and 11q23 deletions,[22,54,55,90] TP53 (REF.[23]) and ataxia telangiectasia-mutated (ATM) mutations[20,21]). However, there is a substantial hierarchy in these prognostic factors.[22,140]

Fludarabine-refractory disease is associated with an extremely poor prognosis and is associated with TP53 mutationand/or 17p13 deletion (TABLE 2).

Young patients who have an unfavourable genetic profile (17p13 deletion) and patients with a poor response tointensive therapy are candidates for allogeneic stem cell transplantation.[126,141]



Figure 1. CLL pathogenic mechanisms and examples of targeted treatment options. a | The B cell receptor(BCR) is composed of two immunoglobulin (Ig) heavy and light chains (variable and constant regions), and CD79aand CD79b, which contain an intracellular activation motif that transmits signals to intracellular tyrosine kinases (forexample, SYK and LYN). The ability of these kinases to activate downstream pathways varies in chroniclymphocytic leukaemia (CLL) subgroups and is correlated with Ig heavy chain variable region (IGHV) mutationalstatus, zeta-associated protein 70 (ZAP70) and CD38 expression.[4,40,41] These pathways could be targeted bysmall molecule inhibitors, the most promising of which might be SYK inhibitors. b | Multiple epitopes on the CLLcell are targets for antibody (ab)-based therapies. c | The most common genetic lesions in CLL include deletion of13q14 and the downregulation of death-associated protein kinase 1 (DAPK1, a stress-activated tumoursuppressor protein[18,99]) by DNA methylation.[18] miR-15a and miR-16-1 (encoded by genes located on 13q14)have been shown to target BCL2, and may increase its expression in CLL.[143] This pathway can be targeted atmultiple levels, including through using small molecule BH3 mimetics.[136] d | Stromal and T cell interactions alsocontribute to CLL pathogenesis. Although not fully understood, some drugs (immune-modulating drugs; Imids) inuse in CLL have been shown to target the interaction with T cells.[109] The crosstalk between CLL cells andaccessory cells and soluble factors upregulates anti-apoptotic proteins, such as survivin, MCL1 and BCL2 (Refs



[103, 105, 106, 144]). Ag, antigen; BLNK, B cell linker protein; BTK, Bruton tyrosine kinase; CDK, cyclin-dependentkinase; CXCR4, chemokine receptor 4; HDAC, histone deacetylase; IL-4, interleukin 4; Me, methyl group; NF-κB,nuclear factor-κB; NFAT, nuclear factor of activated T cells; PLC-γ, phospholipase C-γ; SDF1, stromal cell-derivedfactor 1;VEGFA, vascular endothelial growth factor A.

BCR Response and IGHV Mutation

The B cell response to antigen stimulation is mediated through the BCR in normal and malignant B cells. Each B celldisplays a distinct BCR that is formed through variable combinations of V, D and J segments for the Ig heavy chain andV and J gene segments for the light chain. In addition to the combinatorial diversity of different segments, the BCRrepertoire is increased by the introduction of somatic mutations through the somatic hypermutation (SHM) processduring the germinal centre (GC) reaction ( Box 2 ) (reviewed in Ref. [24]). Functional BCRs are expressed in most Bcell lymphomas but BCR surface expression is usually weak in CLL (reviewed in Ref. [25]). In contrast to most other Bcell lymphomas, CLLs that have mutated IGHV genes can be differentiated from those that have unmutated IGHVgenes (the term IGHV is used here as usually only the heavy chain V genes are sequenced to assess the mutationstatus of a CLL, although the light chain V genes are also mutated in IGHV-mutated cases),[7–9] and the two groupsfollow a different clinical course with patients that have CLL with unmutated IGHVs showing poorer survival (Refs [8, 9,26]) (Fig. 2). The 98% sequence homology of IGHVs in CLL with the germline copies of IGHVs that is typically used todefine this separation is arbitrary, and 'grey-zone cases' (for example, those that have a IGHV homology of 97–98%)seem to have an intermediate prognosis.[27] Although the definition of CLLs with mutated and unmutated IGHVs restson the presence or absence of clonal mutations in all the CLL cells of a case, some intraclonal diversification of IGHVgenes has been reported.[28] However, the level of ongoing hypermutation is rather low, and this intraclonaldiversification may be due to the antigenic stimulation of CLL cells, which can induce activation-induced cytidinedeaminase (AID, also known as AICDA), the key enzyme for SHM. AID is indeed expressed in a subset of CLL cells,and AID-mediated DNA alterations may occur.[29]

Box 2. Germinal centre reaction and normal B cell counterpart of CLL

During T cell-dependent (TD) immune responses, antigen-activated B cells enter B cell follicles in secondarylymphoid organs and establish histological structures called germinal centres (GCs), in which these cells undergomassive clonal expansion. This proliferation takes place in the GC dark zone and is accompanied by the activation ofsomatic hypermutation (SHM), which introduces mutations at a very high rate into the immunoglobulin (Ig) variableregion genes.[24] The mutated GC B cells then migrate to the GC light zone, which is rich in CD4+ T helper (TH)cells and follicular dendritic cells (FDCs). By interacting with these cells, GC B cells that have acquired B cell receptor(BCR) affinity-increasing mutations are selected. GC B cells that have unfavourable mutations undergo apoptosis.Many GC B cells also undergo class switch recombination of their Ig heavy chain constant region genes. GC B cellsintensively migrate between and in the dark and light zones.[142] Positively selected GC B cells usually undergomultiple rounds of proliferation, mutation and selection until they finally differentiate either into memory B cells orplasma cells and leave the GC.

Which is the cellular precursor of the chronic lymphocytic leukaemia (CLL) clone? The current knowledge supports aderivation of Ig heavy chain variable region (IGHV)-mutated CLLs from post-GC memory B cells, although aderivation from B cells that accumulate mutations in a T cell-independent (TI) immune response is also discussed(see the main text). IGHV-unmutated CLLs most likely derive from antigen-activated B cells, but it is unclear whetherthese are activated conventional naive B cells, CD5+ B cells or marginal zone (MZ)-like B cells. It is also unresolvedwhether this activation takes place as part of a TI or TD immune response; the TD immune response might involvean abortive GC reaction of autoreactive B cells.



Figure 2. CLLs with unmutated or mutated IGHV genes show markedly different biological and clinicalbehaviours. Based on the degree of somatic hypermutation, chronic lymphocytic leukaemias (CLLs) can bedifferentiated into those with mutated and those with unmutated immunoglobulin heavy chain variable region(IGHV) genes.[7–9] The non-random use of IGHV genes and stereotyped B cell receptor (BCR) structures providesevidence for antigenic stimulation and drive. a | There are important biological differences between the twogroups. IGHV-unmutated CLLs have higher levels of the protein tyrosine kinase zeta-associated protein 70(ZAP70) and CD38 expression (shown in dark green) than IGHV-mutated CLLs (shown in light green). IGHV-unmutated CLLs activate key signal transduction pathways in response to BCR activation (for example, LYN, SYK,ERK and AKT) and show a different gene expression profile from IGHV-mutated CLLs.[31,32,145] Reducedsignalling in IGHV-mutated CLLs is indicated by grey text and dotted arrows. There is growing evidence thatIGHV-unmutated CLLs have significantly greater proliferative capacity than IGHV-mutated CLLs, and that this



IGHV-unmutated CLLs have significantly greater proliferative capacity than IGHV-mutated CLLs, and that thisgreater capacity is supported by different telomere lengths.[102,111,146] b | IGHV-unmutated CLLs have a greaterlikelihood of carrying and acquiring detrimental genetic lesions (11q23 and 17p13 deletion) than IGHV-mutatedCLLs.[26,30,35,147] The duration of the presence of the initial and the acquired aberrations are indicated in blue andred, respectively. Overall, 11 out of 64 patients showed clonal evolution, which was only observed in IGHV-unmutated cases. c | These biological differences may explain the different clinical courses associated with IGHV-unmutated and mutated CLLs. In a retrospective cohort from our centre (n = 365), the estimated median survivalwas not reached for the group with mutated IGHV versus 111 months for the group with unmutated IGHV (p <0.001). These survival data are unpublished but are representative of published data (for example, Refs [26, 140]).Part b is reproduced, with permission, from Ref. [35] © (2007) European Hematology Association and Ferrata StortiFoundation. BLNK, B cell linker protein; BTK, Bruton tyrosine kinase; Ca, calcium; NF-κB, nuclear factor-κB;NFAT, nuclear factor of activated T cells; PLC-γ, phospholipase C-γ; PKC, protein kinase C.

As expected based on the clinical diversity between CLLs with mutated and unmutated IGHV genes, there areimportant biological differences between the subgroups. These groups show different levels of the protein tyrosinekinase zeta-associated protein 70 (ZAP70) and CD38 expression, differential activity of key signal transductionpathways, different telomere lengths and a markedly different likelihood of carrying or acquiring detrimental geneticlesions (Refs [8, 26, 30–35] ) (Fig. 2). After BCR stimulation in CLL, key molecules of the BCR signalling cascade, suchas SYK and ZAP70 are recruited, resulting in the phosphorylation of B cell linker protein (BLNK), a central node forintracellular signalling (reviewed by Kipps[36]). This pathway is more readily activated in CLL cells that express ZAP70and have unmutated IGHVs. IGHV-mutated CLLs show weak BCR signalling and are relatively anergic (Refs [37–41])(Fig. 1, Fig. 2).

The structure of the BCR is different between IGHV-mutated and unmutated CLLs. A higher proportion of IGHV-unmutated CLL cases carry stereotyped rearrangements of the V, D and J segments that have very similarcomplementarity-determining region (CDR) 3 regions. IGHV-unmutated CLLs are also characterized by stereotyped lightchains and biased somatic mutation patterns.[11,42,43] The striking degree of structural restriction of the entire BCR inCLL suggests that common antigens are recognized by CLL cells and supports the hypothesis that an antigen-drivenprocess contributes to CLL pathogenesis.[44,45] More than 20% of CLL cases carry stereotyped BCRs andapproximately 1% carry virtually identical Igs,[12,13,44–46] which is particularly striking because it would be statisticallyunexpected to find 2 cases with such similar BCRs in 1 million patients.[4,47] In CLLs with unmutated IGHVs the BCR isusually polyreactive to autoantigens derived from endogenous or exogenous proteins or lipids generated by, forexample, oxidative stress.[4,47–49]

CLLs that have mutated IGHVs show more restricted antigen binding with oligo- or monoreactive BCRs. StereotypedBCRs are less likely to occur in mutated-IGHV CLLs. However, if BCR sequences that have IGHV mutations (which arenon-autoreactive) are reverted to their unmutated IGHV counterparts, they become auto- and polyreactive, suggestingthat IGHV-unmutated and mutated CLL cases may originate from a common precursor with autoreactivity.[48] However,other theories propose that IGHV-unmutated and mutated CLL have different cells of origin (discussed below).

The role of antigens in CLL pathogenesis is further underlined by the fact that the presence of stereotyped BCRsseems to affect prognosis. This has been most convincingly demonstrated for a specific IGHV subgroup, the IGHV3-21cases, but is likely to exist in other subgroups.[10,12,13,46,50,51]

IGHV1-69 was among the first genes that were shown to be overrepresented in patients that have CLL with unmutatedIGHV genes. In these patients there was the use of specific diversity and joining gene segments, a longer than averageCDR3 and certain common amino acid motifs.[7,52,53] The use of the IGHV3-21 gene segment was associated withhighly restricted Ig heavy and light chain use (IGKV/IGLV) and an aggressive clinical course that was independent ofthe IGHV mutation status.[10,50] Both IGHV1-69 and IGHV3-21 have the highest frequencies (>40%) of stereotypedVHDHJH rearrangements.[13] The correlations between clinical course and IGHV gene mutation and/or other biologicalparameters are not absolute, and antigens seem to have a crucial role.



parameters are not absolute, and antigens seem to have a crucial role.

The role of IGHV mutation status in guiding therapy is currently unresolved and treatment decisions should not bebased on it outside of clinical trials.[3,54,55] Future treatment strategies may target deregulated signalling pathways(such as SYK, nuclear factor-κB (NF-κB) or ERK) specifically in IGHV-unmutated or mutated CLLs.[36] An attractivetranslation of the above findings would also be to target antigenic drive; for example, by reducing antigen exposure ashas been documented for mucosa-associated lymphoid tissue lymphomas. However, this strategy will be difficult, giventhe frequent autoreactivity of BCRs with the constant presence of autoantigens.[49,56,57]

Normal B Cell Counterpart

The cellular origin of CLL cells is still debated. Originally, it was thought that CLL derives from CD5 + B cells becauseCLL cells invariably express the CD5 antigen and certain mouse strains regularly develop CLL-like malignancies froman accumulation of CD5+ B cells with increasing age.[58] Although murine CD5+ B cells are established as a distinct Bcell subset (B1a B cells) that has self-replenishing features, a propensity to produce autoreactive antibodies andfunctions mainly in T-independent (TI) immune responses,[59] it is unclear whether they are analogous to human CD5+

B cells. Indeed, human CD5+ B cells are mostly unresponsive to TI antigens and rarely produce autoreactiveantibodies.[47,48] A CD5+ B cell origin of CLL was further questioned when it was shown that approximately half of CLLcases carry somatically mutated IGHV genes,[8,9] whereas normal CD5+ B cells rarely have IGHV mutations.[60]

As SHM was thought to be strictly linked to GC B cells, CLLs that have mutated IGHV genes were proposed to bederived from B cells that had undergone a GC reaction; that is, post-GC memory B cells. Because CLLs are mostlyderived from non-class switched B cells, IGHV-mutated CLLs would derive from IgM+IgD+CD27+ B cells that carrymutated IGHV genes and may be non-class switched memory B cells.[61] However, it is controversial whetherIgM+IgD+CD27+ B cells are post-GC B cells, or whether they acquire their IGHV mutations during TI immuneresponses that do not involve the GC or during a primary, antigen-independent BCR diversification process.[62]

IgM+IgD+CD27+ peripheral blood B cells seem to be closely related to splenic marginal zone B cells, and these cellsplay an important part in such TI immune responses.[62] Consequently, IGHV-mutated CLLs were proposed to derivefrom marginal zone-like B cells that underwent SHM during TI immune responses.[47] It is, however, still unclear wherethis process happens as AID, which is essential for SHM, is not expressed in marginal zone B cells.[63]

Several findings argue that IGHV-mutated CLLs are derived from post-GC B cells. First, when global gene expressionprofiles of CLL cells were compared to naive, GC, memory and cord blood CD5+ B cells, it was shown that IGHV-mutated CLLs are most similar in their gene expression patterns to memory B cells[31] (however, a caveat of thisanalysis is that a mixture of class-switched memory B cells and IgM+IgD+/lowCD27+ B cells were used as memory Bcells[61]). Second, approximately one-third of IGHV-mutated CLL cases carry mutations in BCL6 , which is the masterregulator of the GC B cell differentiation programme.[64,65] BCL6 is targeted by the SHM machinery, although at anapproximately 50-fold lower level than IGHV genes.[66] As SHM is strictly dependent on high levels of target genetranscription[67] and, as high-level BCL6 transcription is restricted to GC B cells,[68] BCL6 should remain unmutated inB cells that are undergoing IgV gene hypermutation in a TI immune response. Therefore, CLL cases with BCL6mutations are most likely derived from B cells that have experienced the GC processes. Moreover, as the frequency ofIGHV gene-mutated CLLs that have mutated BCL6 is the same as the frequency of classical post-GC B cells withBCL6 mutations (30%),[64–66] most, if not all, CLLs that have mutated IGHV genes may derive from precursors thatunderwent SHM in the GC. Third, a subset of CLLs expresses IgG, indicating that these cases derive from class-switched B cells. These CLLs mostly express IgG1 or IgG3 subclasses, which are typical of GC-associated classswitching, whereas they rarely express IgG2 (which is typical of class switching in TI immune responses).[69,70] Fourth,we recently showed that IgM+IgD+CD27+ B cells are often clonally related to classical class-switched memory B cellsand derive from common GC B cell clones, further reinforcing the notion that at least a large fraction of these IGHV-mutated IgM+IgD+ B cells descend from GC B cells.[71]



CLLs that have unmutated IGHVs also surprisingly showed a gene expression pattern that was more similar to post-GCmemory B cells than to naive or CD5+ B cells, so it was speculated that IGHV-unmutated CLLs may also derive frommemory B cells.[31] It has, however, recently been shown that a substantial fraction of human CD5+ B cells aretransitional B cells.[72] Consequently, the potential similarities between CLL cells and mature CD5+ B cells might havebeen blurred in the gene expression studies by using a mixture of transitional and mature CD5+ B cells. Nevertheless,there is evidence that GC reactions generate some memory B cells that lack somatic mutations.[71] Importantly, manyIGHV-unmutated CLLs express poly- and autoreactive antibodies,[49,56,57] and the CLL cells show an activatedphenotype.[33] This evidence strongly argues that IGHV-unmutated CLLs are derived from antigen-experienced B cells.The antigenic specificities of these B cells seem to include both TI and T-dependent (TD) (auto) antigens.[49,56,57]

Concerning TD antigens, there are some indications that autoreactive B cells are prevented from undergoing full GCreactions.[73] Therefore, one may speculate that IGHV-unmutated CLLs derive from antigen-stimulated B cells, includingboth TI and TD stimulations (see the figure in Box 2 ). As these B cells are chronically stimulated because of theirautoreactivity, but may be prevented from undergoing apoptosis owing to their acquisition of primary transformingevents, the B cells might acquire features of antigen-experienced memory B cells without undergoing a GC reaction.

Box 2. Germinal centre reaction and normal B cell counterpart of CLL

During T cell-dependent (TD) immune responses, antigen-activated B cells enter B cell follicles in secondarylymphoid organs and establish histological structures called germinal centres (GCs), in which these cells undergomassive clonal expansion. This proliferation takes place in the GC dark zone and is accompanied by the activation ofsomatic hypermutation (SHM), which introduces mutations at a very high rate into the immunoglobulin (Ig) variableregion genes.[24] The mutated GC B cells then migrate to the GC light zone, which is rich in CD4+ T helper (TH)cells and follicular dendritic cells (FDCs). By interacting with these cells, GC B cells that have acquired B cell receptor(BCR) affinity-increasing mutations are selected. GC B cells that have unfavourable mutations undergo apoptosis.Many GC B cells also undergo class switch recombination of their Ig heavy chain constant region genes. GC B cellsintensively migrate between and in the dark and light zones.[142] Positively selected GC B cells usually undergomultiple rounds of proliferation, mutation and selection until they finally differentiate either into memory B cells orplasma cells and leave the GC.

Which is the cellular precursor of the chronic lymphocytic leukaemia (CLL) clone? The current knowledge supports aderivation of Ig heavy chain variable region (IGHV)-mutated CLLs from post-GC memory B cells, although aderivation from B cells that accumulate mutations in a T cell-independent (TI) immune response is also discussed(see the main text). IGHV-unmutated CLLs most likely derive from antigen-activated B cells, but it is unclear whetherthese are activated conventional naive B cells, CD5+ B cells or marginal zone (MZ)-like B cells. It is also unresolvedwhether this activation takes place as part of a TI or TD immune response; the TD immune response might involvean abortive GC reaction of autoreactive B cells.


of 30file:///Users/dwyer/Desktop/From%20Pathogenesis%20to%20Treatment%2…Chronic%20Lymphocytic%20Leukaemia%20(printer-friendly).webarchive

Taken together, although the cellular origin of CLL cells has not been finally clarified, there is strong evidence thatIGHV-mutated CLLs (mostly) stem from antigen-experienced post-GC memory B cells. IGHV-unmutated CLLs alsooriginate from antigen-experienced B cells that acquire features of memory B cells. Whether CLL cells derive from aparticular subset of B cells (naive, marginal zone or CD5+) is less clear. As some features of CLL cells resemble thestill enigmatic regulatory B cells (for example, the expression of CD25 and the secretion of interleukin 10 (IL-10)), thepossibility that CLL cells are derived from such regulatory B cells is also currently being discussed.[74]

MBL, a CLL Precursor State?

Clonal B cell populations with a CLL immunophenotype have been detected in approximately 3.5% of healthy individualswho have normal or slightly increased lymphocyte counts (monoclonal B cell lymphocytosis; MBL).[75] The occurrenceof such clones increases with age, (8% aged >70).[75] Using more sensitive multicolour flow cytometry, MBL wasdetectable in 12% of healthy individuals (aged >40).[76] In most instances the CLL-like clones carry somatically mutatedIGHV genes, which suggests that MBL is predominantly a precursor of IGHV-mutated CLL.

Is MBL a precursor state of CLL? In favour of this idea is the fact that CLL-like B cells appear at an increasedfrequency in relatives of patients with CLL, indicating a common genetic predisposition for such clonal expansions.[77]

Furthermore, a recent study suggested that virtually all cases with CLL are preceded by MBL.[78] Most important is thefinding that MBL clones often carry genetic lesions that are typical for CLL, such as the 13q14 deletion[79] (discussedbelow). This shows that MBL is often not simply an (age-related) expansion of normal B cells, but that the clones havealready acquired initial genetic lesions, which are a hallmark of a pre-malignant tumour precursor cell. Regarding otherfeatures, however, at first glance MBL may not resemble CLL. MBL clones carry somatically mutated IGHV genes muchmore frequently than CLL,[78,79] and it has been reported that the IGHV gene use in MBL is untypical for CLL and thatstereotyped BCRs are rare.[80] However, other studies have reported the frequent use of IGHV genes that are typical ofCLL in MBL.[78,79] The overrepresentation of B cell clones that have mutated IGHV genes in MBL might be related tothe more aggressive behaviour of IGHV-unmutated CLLs, which might not persist as long in a stable MBL stage.



the more aggressive behaviour of IGHV-unmutated CLLs, which might not persist as long in a stable MBL stage.

In conclusion, although there are still unresolved issues about MBL, several key features of MBL support the idea that itis a precursor stage of CLL (with mutated IGHVs). It must, however, be stressed that only a small fraction of MBL willever progress to CLL, as the frequency of MBL is approximately 100-fold higher than the frequency of CLL.[81]

Genomic Aberrations and Gene Mutations

Approximately 80% of CLL cases show aberrations in a few recurrently affected chromosomal regions (Ref. [22]) (Table 1 ). IGHV-mutated and unmutated CLLs share genomic aberrations, but the incidence of high-risk aberrations ishigher in CLLs that have unmutated IGHVs.

Deletions in Band 13q14

Deletion of 13q14 is the structural aberration most frequently found in CLL.[22] CLLs that have a 13q14 deletion as thesole abnormality have a favourable course of disease (Ref. [22]) ( Table 2 ). Various groups have attempted to identifya tumour suppressor gene in this region. No inactivation of candidate genes by mutation has been demonstrated, but acomplex epigenetic regulatory tumour suppressor mechanism has been described that controls the expression of the

Table 1. Summary of genetic profiles: different clinical CLL cohorts and MBL

IGHV mutation statusor genetic aberration

MBL(%)[79]

MBL orlymphocytosis(%)[79]

Early-stageCLL (%)§[150]

Unselected(%)[22,26]

At first-linetreatment(%)[150]

RefractoryCLL(%)[149,151]

IGHV mutations

Mutated IGHV 85 90 59 44 31 24

Unmutated IGHV 15 10 41 56 69 76

Copy number changes

Deletion 13q14 39 58 40* 36* 34* 22*

Trisomy 12q13 18 21 13 16 11 12

Deletion 11q23‡ 0 6 10 18 21 25

Deletion 17p13‡ 0 3 4 7 3 31

Mutation of genes other than IGHV

TP53 mutation‡ ND ND ND8–10(REFS[23,129])

8-12|| 37

*Deletion of 13q14 that occurs as the sole abnormality in CLL or MBL when assessed byfluorescence in situ hybridization. ‡17p13 and11q23 deletions and mutation of TP53 occur at anincreased incidence as disease advances. Even if these occur as secondary genetic lesions,they have a profound effect on the clinical course and outcome of disease. §High-risk geneticaberrations can also be found in early-stage and untreated patients. ||Unpublished data from ourgroup (the German CLL Study Group (GCLLSG) CLL4 and CLL8 trials). CLL, chroniclymphocytic leukaemia; IGHV, immunoglobulin heavy chain variable region genes; MBL,monoclonal B cell lymphocytosis; ND, not determined.



whole region (also in cases without deletion of 13q14).[82]

Two microRNA genes, mir-15a and mir-16-1 , located in the crucial 13q14 region have been implicated in CLLpathogenesis (Refs [15, 16, 83]) (Fig. 1; Table 2 ). A mouse model with a targeted deletion of the mir-15a–mir-16-1locus recapitulates many features of CLL (U. Klein and R. Dalla-Favera, personal communication). This suggests thatmiR-15a and miR-16-1 have a direct pathogenetic role in CLL.

Table 2. Biological and clinical impact of common genomic aberrations in CLL

Aberration Incidence(%)[22] Genes involved‡ PFS (months)[22]

§OS (months)[22]§

No aberration 18 ? 49 111

Deletion13q14 55 (36)* mir-15a, mir-16-1 and non-coding

RNAs[15,82,143] 92* 133*

Deletion11q23 18 ATM, ?[19–21,89,132] 13 79

Trisomy12q13 16 Gene dosage of unknown genes,[88] ? 33 114

Deletion17p13 7 TP53(REFS[17, 23]) 9 32

t14q32 4 IgH, ? ND ND

*Deletion of 13q14 as the sole abnormality when assessed by fluorescence in situhybridization.‡Confirmed or potentially important pathways that are targeted by theseaberrations are included.§The clinical impact of these aberrations is shown by the progression-free and overall survival data from a previously published series.[22] ATM, ataxia telangiectasia-mutated; CLL, chronic lymphocytic leukaemia; IgH, immunoglobulin heavy locus; ND, notdetermined; OS, overall survival; PFS, progression-free survival (time to first treatment).



§OS (months)[22]§



RNAs[15,82,143] 92* 133*

Deletion11q23 18 ATM, ?[19–21,89,132] 13 79






Deletions at 13q14 occur at high frequencies in other lymphomas and solid tumours, and a recent study has implicatedmiR-15a and miR-16-1 in the pathogenesis of prostate cancer through their targeting of cyclin D1 and WNT3A, whichpromote survival and proliferation.[84]

Deletions of ATM (11q22–q23)

Although they are rarely found in early-stage disease, approximately one-quarter of patients with advanced CLL have11q23 deletions ( Table 1 ). Correspondingly, patients who have 11q23 deletions have a more rapid diseaseprogression and extensive lymphadenopathy.[22,54,55,85] In studies that aimed to delineate the 11q23 deletions in CLL,all aberrations affected a minimal consensus region in chromosome bands 11q22.3–q23.1. This region harbours theataxia telangiectasia-mutated ( ATM ) gene in almost all cases. ATM mutations have been shown to be present in 12%of all patients with CLL and in approximately one-third of the cases with a 11q23 deletion.[20] The ATM protein kinaseis a central component of the DNA damage pathway and mediates cellular responses to DNA double-strand breaks(DSBs). ATM deficiency leads to ataxia–telangiectasia, which is characterized by extreme sensitivity to irradiation,genomic instability and a predisposition to lymphoid malignancies.[86] ATM activates cell cycle checkpoints, can induceapoptosis in response to DNA breaks and functions directly in the repair of DNA DSBs by maintaining DNA ends inrepair complexes.[87] Other disease-associated genes in 11q22–q23 have been investigated, and a gene dosage effecthas been documented. The pathogenic role of other genes in the region remains unresolved (Refs [88, 89]) ( Table 2 ).




MBL(%)[79]






IGHV mutations

Mutated IGHV 85 90 59 44 31 24


Copy number changes

Deletion 13q14 39 58 40* 36* 34* 22*

Trisomy 12q13 18 21 13 16 11 12

Deletion 11q23‡ 0 6 10 18 21 25

Deletion 17p13‡ 0 3 4 7 3 31



8-12|| 37

*Deletion of 13q14 that occurs as the sole abnormality in CLL or MBL when assessed by



Trisomy 12

Trisomy 12 is among the most frequent aberrations in CLL (10–20%) ( Table 1 ), but the genes involved in thepathogenesis of CLL with trisomy 12 are unknown. A previously described association with poor outcome has not beenconfirmed.[54,55] Consistent with these observations, the incidence of trisomy 12 does not increase with advanced stageor progression to refractory disease ( Table 1 ).

fluorescence in situ hybridization. ‡17p13 and11q23 deletions and mutation of TP53 occur at anincreased incidence as disease advances. Even if these occur as secondary genetic lesions,they have a profound effect on the clinical course and outcome of disease. §High-risk geneticaberrations can also be found in early-stage and untreated patients. ||Unpublished data from ourgroup (the German CLL Study Group (GCLLSG) CLL4 and CLL8 trials). CLL, chroniclymphocytic leukaemia; IGHV, immunoglobulin heavy chain variable region genes; MBL,monoclonal B cell lymphocytosis; ND, not determined.



§OS (months)[22]§



RNAs[15,82,143] 92* 133*

Deletion11q23 18 ATM, ?[19–21,89,132] 13 79







MBL(%)[79]






IGHV mutations

Mutated IGHV 85 90 59 44 31 24




Copy number changes

Deletion 13q14 39 58 40* 36* 34* 22*

Trisomy 12q13 18 21 13 16 11 12

Deletion 11q23‡ 0 6 10 18 21 25

Deletion 17p13‡ 0 3 4 7 3 31



8-12|| 37

*Deletion of 13q14 that occurs as the sole abnormality in CLL or MBL when assessed byfluorescence in situ hybridization. ‡17p13 and11q23 deletions and mutation of TP53 occur at anincreased incidence as disease advances. Even if these occur as secondary genetic lesions,they have a profound effect on the clinical course and outcome of disease. §High-risk geneticaberrations can also be found in early-stage and untreated patients. ||Unpublished data from ourgroup (the German CLL Study Group (GCLLSG) CLL4 and CLL8 trials). CLL, chroniclymphocytic leukaemia; IGHV, immunoglobulin heavy chain variable region genes; MBL,monoclonal B cell lymphocytosis; ND, not determined.



MBL(%)[79]






IGHV mutations

Mutated IGHV 85 90 59 44 31 24


Copy number changes

Deletion 13q14 39 58 40* 36* 34* 22*

Trisomy 12q13 18 21 13 16 11 12

Deletion 11q23‡ 0 6 10 18 21 25

Deletion 17p13‡ 0 3 4 7 3 31



8-12|| 37

*Deletion of 13q14 that occurs as the sole abnormality in CLL or MBL when assessed byfluorescence in situ hybridization. ‡17p13 and11q23 deletions and mutation of TP53 occur at anincreased incidence as disease advances. Even if these occur as secondary genetic lesions,they have a profound effect on the clinical course and outcome of disease. §High-risk geneticaberrations can also be found in early-stage and untreated patients. ||Unpublished data from ourgroup (the German CLL Study Group (GCLLSG) CLL4 and CLL8 trials). CLL, chronic



Deletions in Band 17p13 or TP53 Mutations

Deletion of 17p13 is found in 4–9% of CLLs at diagnosis or at initiation of the first treatment.[17,22,54,55] Although the17p13 deletion usually encompasses most of the short arm of chromosome 17p, the deletion always includes band17p13, where the tumour suppressor TP53 (which encodes p53) is located (Fig. 3). Among CLL cases that havemonoallelic 17p13 deletions, the majority show mutations in the remaining TP53 allele (>80%).[23,90] Among caseswithout 17p13 deletion, TP53 mutations are much rarer[23] (see below).

group (the German CLL Study Group (GCLLSG) CLL4 and CLL8 trials). CLL, chroniclymphocytic leukaemia; IGHV, immunoglobulin heavy chain variable region genes; MBL,monoclonal B cell lymphocytosis; ND, not determined.



Figure 3. TP53 mutations in CLL. Comparison of overall survival stratified by the absence or presence of17p13 deletions and TP53 mutations. Patients with a 17p13 deletion or TP53 mutation have a poor outcome andare therefore prime candidates for experimental strategies with novel agents and stem cell transplantation.[126] b |17p13 deletion is invariably associated with loss of TP53 as confirmed by fluorescence in situ hybridization (FISH)('+' denotes a TP53 deletion). The red bars indicate the extent of the 17p13 deletion, which often includes most ofthe chromosome 17 short arm (data are unpublished but are representative of published results[148]). c | Multiplegenomic aberrations target the p53 pathway in chronic lymphocytic leukaemia (CLL). The figure shows a simplifiedschematic of the p53 pathway and response to DNA damage. In the absence of stress, p53 levels are low as p53levels are partly mediated through the ubiquitin ligase MDM2, which binds to p53 and promotes its degradation.MDM2 is also induced by p53 in a regulatory loop. MDM2 is located on chromosome 12, so CLL cases withtrisomy 12 have low p53 levels and favourable overall survival. In response to stress (for example, DNA damage),



trisomy 12 have low p53 levels and favourable overall survival. In response to stress (for example, DNA damage),p53 is activated through various pathways, including ataxia telangiectasia-mutated (ATM). ATM is deleted inapproximately 20% of CLL cases and is associated with a poor outcome. TP53 loss or mutation is central torefractory CLL.[149] The microRNA miR-34a is associated with p53 defects and refractory CLL.[130] Part a isreproduced, with permission, from Ref. [23] © (2008) American Society of Hematology. PUMA, p53 upregulatedmodulator of apoptosis (also known as BBC3).

Recurrent Translocations

In contrast to other types of leukaemia or B cell lymphoma in which specific and recurrent Ig locus-associatedtranslocations deregulate known oncogenes, recurrent balanced translocations are rare in CLL.[22,91,92] As many Ig-associated chromosomal translocations happen as errors during SHM or class-switch recombination,[93] the lack ofsuch translocations may be easily understood in non-class-switched IGHV-unmutated CLLs, in which the precursorshave apparently not been exposed to these Ig gene-remodelling processes. However, this argument does not hold truefor IGHV-mutated CLLs, in which the precursors were exposed to SHM and, in a fraction of cases, also had undergoneclass switching. The lack of Ig-associated translocations may support the notion that CLL is derived from post-GC Bcells, in which SHM and class switching are silenced.[94] However, most CLLs also lack translocations that areassociated with errors during V(D)J recombination — a process that is active in the CLL precursor cells. Therefore, thelack of frequent Ig locus-associated translocations may also reflect the fact that the specific combination of transformingevents that are involved in CLL pathogenesis does not include an oncogene that is a typical target for Ig-associatedchromosomal translocations.

A recent study of metaphases from CLL cells suggested that (mostly unbalanced) translocations occurred in 34% ofCLLs.[91] In a larger series of over 500 patients, genomic aberrations (primarily genomic imbalances) were detected in83% of cases by chromosome banding and in 78% of cases by fluorescence in situ hybridization, by screening for alimited number of aberrations. Recurrent reciprocal translocations were rare and mostly targeted the IgH locus in band14q32 or the 13q14 region with the concomitant loss of genomic material.[92]

Genetic Susceptibility to CLL

Approximately 5–10% of patients with CLL report a family history of leukaemia and lymphoma. [1,2] The relative risk offirst-degree relatives with CLL has been estimated to be 7.5-fold in the United States and Europe.[2] MBL has beenfound in 13.5–18% of first-degree relatives.[77,81] Recently, a genome-wide association study identified 6 previouslyunreported 'CLL risk loci' at bands 2q13, 2q37.1, 6p25.3, 11q24.1, 15q23 and 19q13.32 (Ref. [95]). These data providethe first evidence for the existence of common, low-penetrance susceptibility loci for CLL. The strongest evidence foran association between CLL and a particular gene was found with linkage disequilibrium at 6p25.3, whichencompasses interferon regulatory factor 4 ( IRF4 ).[95] IRF4 is particularly interesting in the context of B celllymphomas because it is a key regulator of B lymphocyte development and proliferation and has important functions inthe GC reaction.[24,96]

Epigenetic Alterations in CLL Pathogenesis

Global and gene-specific aberrant DNA methylation has been detected in CLL. An analysis of the global methylationprofile in CLL found that 2.5–8.1% of the CpG islands in CLL samples were aberrantly methylated.[97] Aberrantmethylation has been described for genes that are specifically deregulated in CLL (for example, hypomethylation ofBCL2 and T cell leukaemia/lymphoma 1 ( TCL1 )), and the expression levels of ZAP70 correlate closely with themethylation of specific CpG islands in ZAP70 (Ref. [98]).

The loss or reduced expression of death-associated protein kinase 1 (DAPK1) owing to a rare mutation was reported ina large CLL kindred.[18] However, almost all sporadic CLL cases also show epigenetic silencing of DAPK1 by promotermethylation, suggesting that DAPK1 deregulation is a key pathogenic event in CLL (Ref. [18]) (Fig. 1). Nevertheless, theprecise function of DAPK1 remains unclear. DAPK1 links cellular stresses, such as starvation or growth factor



precise function of DAPK1 remains unclear. DAPK1 links cellular stresses, such as starvation or growth factordeprivation, to the induction of either apoptosis or autophagy, depending on the cellular context and the externalstimuli.[99]

Epigenetic modifications are an attractive therapeutic target because they are reversible. Several compounds, includinghistone deacetylase inhibitors, have been tested on CLL cells in vitro and in clinical studies. Although these compoundshave been shown to be biologically and clinically active, the results in CLL have been less encouraging compared withother leukaemias.[100]

Microenvironment

CLL cells interact with and seem to shape their microenvironment, which consists of T cells, stromal cells and solublefactors.[101] Some of these interactions are located in specific compartments[102] (for example, proliferation centres,discussed below). The importance of the microenvironment in CLL is easily appreciated by the fact that CLL cellsrapidly undergo apoptosis when they are removed from patients.[103] This process can be prevented by adding anumber of cytokines or other cell types to the CLL cell culture.

In the lymph node, the microenvironment provides anti-apoptotic signals and proliferative stimuli, resulting in theformation of proliferation centres of CLL cells (pseudofollicles) that are not found in other lymphomas. CLL cells seem torecruit accessory cells[104,105] and thereby create a microenvironment that supports their own survival. There is anincrease of CD3+ T cells, most of which are CD40L + CD4 +, which cluster in and around pseudofollicles.[106] Thesecells can stimulate CLL cells through the interaction of CD40 and CD40L, and this stimulus synergizes with BCRsignalling.[107] Several anti-apoptotic signalling pathways are induced, including the caspase inhibitor survivin (alsoknown as BIRC5), which is specifically expressed at high levels in proliferation centres,[106] and NF-κB, the activationstatus of which was found to be a prognostic marker in CLL.[108] The CD40–CD40L interaction or NF-κB may be suitedto therapeutic intervention (for example, using monoclonal antibodies, CD40 ligation or specific inhibitors) (Fig. 1). CLLcells also induce phenotypical changes in T cells, the most prominent of which is a defective and reduced formation ofthe immunological synapse.[109]

CLL cells that are in close proximity and in contact with activated CD4+ T cells show expression of the cell surfacemarker CD38 (Ref. [110]). This is of interest because CD38 has been linked to the proliferation of CLL cells. Thepresence of high numbers of CD38+ CLL cells in the blood is associated with a poor prognosis.[8,26,111] In addition,CD38 is an interaction partner for CD31 expressed on 'nurse-like cells' (NLCs).[112] NLCs can develop in vitro fromCD14 + monocytes by interaction with CLL cells,[113] and secrete stromal cell-derived factor 1 (SDF1) and the TNFfamily ligands a proliferation-inducing ligand (APRIL; also known as TNFSF13) and B cell activating factor (BAFF; alsoknown as TNFSF13B), which protect CLL cells from apoptosis.[114–116] A potential role of APRIL in the pathology ofCLL was suggested by the demonstration that transgenic mice overexpressing APRIL develop a CLL-like disease.[117]

The dependency of CLL cells on their microenvironment is exploited by novel therapeutic compounds that target thesupporting non-malignant T and natural killer cells and show efficacy in CLL.[118,119]

Clinical Management, Therapy and Prognosis

In contrast to most other leukaemias, CLL is not necessarily treated at diagnosis, but rather after symptomaticdisease.[3] This strategy is mainly based on the results of randomized trials that compared early and late treatment thatshowed no benefit of early treatment.[120] However, early intervention trials are currently selecting high-risk subgroupsof CLL based on biological and clinical criteria to reassess early intervention.

Over the past decades, there has been a transition from single-agent alkylator-based therapies to nucleosideanalogues, combinations of both alkylators and nucleoside analogues and, most recently, chemo-immunotherapy.Complete response rates have improved from 7% to a maximum of 70%.[54,121] Historical comparisons suggest that the



overall survival has improved with the application of chemo-immunotherapy.[122] However, to date none of therandomized trials of combination chemotherapy showed a survival benefit for a specific treatment in CLL when only thetreatment arms were compared.[54,123] Data from our group published in abstract form suggests that 'modern' therapiesare changing the outcome of CLL. Multivariate analysis in the prospective German CLL Study Group (GCLLSG) CLL4trial suggests that overall survival may be altered when the genetic profile is considered in addition to treatmentarms.[90] The logical approach to improve outcome is therefore the development of prognostic and predictive models,which would lead to stratified treatment strategies for specific disease subgroups. It may also be helpful to identifybiological low-risk groups to decrease their treatment intensity.

Genotype-specific Therapy

The ultimate goal of this approach would be a genotype- or risk factor-adapted therapy in all patients. This depends onthe identification of prognostic and predictive factors that have the strongest clinical impact. The most powerfulprognostic factors in CLL include age, Binet stage or Rai classification, serum markers[124] and genetic factors (such asgenomic aberrations,[22] IGHV mutation status,[8] ZAP70 (Ref. [125]) and TP53 mutations[23]). Few studies haveaddressed potential predictive factors that optimize treatment.

The first risk-adapted treatment for patients with CLL has been developed for patients with 17p13 deletions who have avery poor prognosis with alkylator- and purine analogue-based chemo-immunotherapy (Refs [54, 55, 90]) (Fig. 3). Asthere is evidence that several 'biological' agents, such as alemtuzumab, corticosteroids, lenalidomide and flavopiridolact independently of functional p53 in CLL, current treatment approaches in clinical trials use these agents upfront withearly allogeneic stem cell transplantation.[126]

Mechanisms of Treatment Resistance

In spite of highly effective treatment options, all patients with CLL will eventually relapse after conventional therapy.Patients relapsing within 2 years after intensive first-line therapy have a poor response to salvage treatment and shortoverall survival. It is important to address both the time to relapse and the intensity of preceding treatment. Obviousobstacles to the investigation of mechanisms that underlie early relapse and refractory disease lie in the clonalheterogeneity of samples before treatment. The existence of subclones is best documented for cases of 17p13 deletionand TP53 mutation; however, they are also likely to exist for other unidentified mutations or epimutations, leading tochemoresistance.[127]

p53 Pathway

TP53 plays a central part in our current understanding of why some patients fail to respond to chemotherapy in CLL.Since the early 1990s, TP53 mutations and 17p13 deletions have been associated with a poor response to alkylatingagents and short survival.[17,128] Data from prospective trials confirm these results.[54,55] Mutations of TP53 have beenfound in 4–10% of patients with untreated CLL.[17,23,128] TP53 mutations are associated with higher geneticcomplexity, although the precise effect of complex genetic aberrations in CLL — especially independently of TP53mutations — are not well defined.[23] Recent data suggest that the clinical behaviour of cases that have only a TP53mutation is similar to cases that have deletion of one TP53 allele and mutation of the remaining allele (Refs [23, 129])(Fig. 3). Although the overall incidence of TP53 mutations is lower than that of most other cancers, the clinicalconsequences of the mutation are striking. However, it is important to appreciate that TP53 mutation or 17p13 deletionwill only explain a proportion of refractory cases (25–50%). Other components of the p53 pathway, including ATM(discussed below) and miR-34a (a microRNA component of the p53 pathway), seem to contribute to drugresistance.[21,130]

ATM and DNA Repair

Although ATM inactivation has been associated with refractoriness to chemotherapy through failure to activate p53 andp21, data from larger randomized trials indicate that the primary response is independent of the presence of 11q23



deletion.[54,55,131,132] There is evidence from our group published in abstract form that more intensive combinationchemotherapy may be particularly beneficial compared to fludarabine alone in patients who have 11q23 deletions, andthe addition of the anti-CD20 antibody rituximab may further enhance efficacy.[90,133] This observation is consistentwith the concept that inactivation of ATM is more likely to lead to genomic instability and secondary resistance due toan impaired DNA damage response, which may be particularly detrimental in sublethally exposed cells.[134] Smallmolecules that inhibit the MDM2–p53 interaction and thereby increase p53 levels have been shown to induce apoptosisin CLL cases with deletion of ATM,[135] and similar approaches may be useful therapeutically.

Treatment Resistance to Other Agents

Treatment resistance has mostly been studied in relation to chemotherapy. With the advent of small-molecule inhibitorsand antibody-based treatments in CLL, the near future will see a wealth of emerging information on specific resistancemechanisms for numerous novel drug classes. Notably, the cellular microenvironment may contribute to drug resistanceas CLL cells with stromal support developed 1,000-fold higher resistance to inhibitors of BCL2 and BCL-XL than cellscultured without cellular support.[136]

Translating Biological Insights into Treatment

The understanding of biological mechanisms in CLL has dramatically developed over recent years, but we are onlybeginning to use our growing understanding of biological subgroups to alter the clinical management of CLL. Onedilemma is that the clinical course is generally benign in the majority of patients and therefore clinical endpoints areslowly reached. This slows down the translation of biological insights into clinical practice. In spite of this, the growingnumber of approaches that act differently from classical chemotherapy show great promise and some of them areparticularly promising for CLL (for example, SYK inhibition).[137] Increasing emphasis is being put on CLL subgroupsthat are defined by specific biological characteristics with the goal of improving both response rates and overall survival.

Conclusions and Perspectives

Unique discoveries have been made that set CLL apart from other cancers. Based on immunological and geneticassessment of BCR function, it is possible to divide CLL into subtypes (such as IGHV-unmutated and mutated, IGHV3-21 and potentially other IGHV gene-defined subsets) that have distinct biological and clinical characteristics. Theprecise cellular origin of CLL remains a matter of debate, but there is evidence that IGHV-mutated CLLs derive frompost-GC B cells, and that IGHV-unmutated CLLs stem from B cells that have been activated by antigens. Thedependence and interaction of CLL cells with the microenvironment is of pivotal importance and is beginning to be usedas a drug target. The most common genetic lesion — deletion 13q14 — may serve as a model of how deregulated non-coding RNAs contribute to cancer initiation, and this may become a 'druggable' target in the future. Although epigeneticmodification generally leads to downregulation of DAPK1 in CLL, specific aberrations (11q23 and 17p13 deletions) andgene mutations (TP53 and ATM) help to define distinct biological and clinical subgroups of CLL.

Overall, CLL may serve as a model for how microenvironmental stimuli, antigenic drive and epigenetic, as well asgenetic, deregulation are combined in cancer pathogenesis. Importantly, there are a growing number of agents that acton specific biological targets and therefore open new therapeutic horizons.

Sidebar 1

At a Glance

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the Western world. It is characterized bythe accumulation of small B lymphocytes that have a mature appearance.Two subsets of CLL cases can be differentiated by the degree of somatic hypermutation (mutated and unmutatedimmunoglobulin heavy chain variable region (IGHV) genes) that have distinct clinical and biological behaviours.



Overall, more than 20% of CLL cases carry stereotyped B cell receptors, suggesting that common antigen(s) arerecognized by CLL cells.Clonal B cell populations with a CLL immunophenotype have been detected in 3.5% of healthy individuals(monoclonal B cell lymphocytosis; MBL). MBL is often a CLL precursor.Approximately 80% of CLLs show aberrations in a few frequently affected chromosomal regions, including13q14 (mir.15a and mir16.1), 11q23 (ataxia telangiectasia-mutated; ATM), trisomy 12 and 17p13 (TP53).Recurrent translocations are rare in CLL.Global and gene-specific aberrant DNA methylation has been detected in CLL. Almost all sporadic CLL casesalso show epigenetic silencing of death-associated protein kinase 1.In lymphoid organs, CLL cells interact with and seem to shape their microenvironment, which consists of T cells,stromal cells and soluble factors. This interaction is emerging as a therapeutic target.p53 plays a central part in our current understanding of why some patients fail to respond to chemotherapy.The most powerful prognostic factors include 17p13 deletion, TP53 mutation, 11q23 deletion, IGHV mutationstatus, serum markers, clinical stage and age.CLL may serve as a model of how microenvironmental stimuli, antigenic drive and epigenetic, as well as genetic,deregulation are combined in cancer pathogenesis.

Sidebar 2

Stereotyped B cell receptors Strikingly similar B cell receptors, which often arise from the use of common H and L chain V region gene segmentsthat share CDR3 structural features (such as their length, amino acid composition and unique amino acid residues atrecombination junctions).

Antigenic drive CLL cells seem to be selected by a limited set of antigenic epitopes at some point in their development. CLL cells arestimulated by the binding of these antigens to the BCR.

Somatic hypermutation A process that modifies the immunoglobulin variable region genes by introducing mutations into them at a high rate.

Anergic A state in which B or T cells are unresponsive and cannot be activated by antigen.

IGHV1-69 A specific IGHV gene found at a high frequency in CLLs with unmutated IGHV.

CpG island A region of DNA with a high density of cytosine–phosphoguanine dinucleotides, which are near the transcriptional startsites of 40% of all mammalian genes. Cytosine methylation in CpG islands is generally associated with stable silencingof the associated gene.

Immunological synapse The supramolecular structure that is established between a T cell and an antigen-presenting cell or B cell.

Binet stage A clinical staging system most commonly used in Europe based on lymphadenopathy, spleen and liver size and bloodcount (red cells and platelets).

Rai classification A clinical staging system most commonly used in the United States.



A clinical staging system most commonly used in the United States.

References

1. Dores, G. M. et al. Chronic lymphocytic leukaemia and small lymphocytic lymphoma: overview of the descriptiveepidemiology. Br. J. Haematol. 139, 809–819 (2007).

2. Goldin, L. R., Pfeiffer, R. M., Li, X. & Hemminki, K. Familial risk of lymphoproliferative tumors in families ofpatients with chronic lymphocytic leukemia: results from the Swedish Family-Cancer Database. Blood 104,1850–1854 (2004).

3. Hallek, M. et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from theInternational Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group1996 guidelines. Blood 111, 5446–5456 (2008).

4. Chiorazzi, N., Rai, K. R. & Ferrarini, M. Chronic lymphocytic leukemia. N. Engl. J. Med. 352, 804–815 (2005).5. Binet, J. L. et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate

survival analysis. Cancer 48, 198–206 (1981).6. Rai, K. R. et al. Clinical staging of chronic lymphocytic leukemia. Blood 46, 219–234 (1975).7. Fais, F. et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen

receptors. J. Clin. Invest. 102, 1515–1525 (1998).8. Damle, R. N. et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic

lymphocytic leukemia. Blood 94, 1840–1847 (1999).9. Hamblin, T. J., Davis, Z., Gardiner, A., Oscier, D. G. & Stevenson, F. K. Unmutated Ig VH genes are associated

with a more aggressive form of chronic lymphocytic leukemia. Blood 94, 1848–1854 (1999).The two pivotal studies by Hamblin et al. and Damle et al. establish the prognostic impact of IGHV mutationalstatus in CLL.

10. Tobin, G. et al. Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vλ2-14 geneuse and homologous CDR3s: implicating recognition of a common antigen epitope. Blood 101, 4952–4957(2003).

11. Messmer, B. T., Albesiano, E., Messmer, D. & Chiorazzi, N. The pattern and distribution of immunoglobulin VHgene mutations in chronic lymphocytic leukemia B cells are consistent with the canonical somatic hypermutationprocess. Blood 103, 3490–3495 (2004).

12. Ghia, P. et al. Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocyticleukemia: the lesson of the IGHV3-21 gene. Blood 105, 1678–1685 (2005).

13. Stamatopoulos, K. et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors:pathogenetic implications and clinical correlations. Blood 109, 259–270 (2007).

14. Schroeder, H. W. Jr & Dighiero, G. The pathogenesis of chronic lymphocytic leukemia: analysis of the antibodyrepertoire. Immunol. Today 15, 288–294 (1994).An analysis indicating that CLL cells use a IGHV repertoire that is characteristic of mature B cells, and suggeststhat antigens may play a part in the pathogenesis of this disease.

15. Calin, G. A. et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 inchronic lymphocytic leukemia. Proc. Natl Acad. Sci USA 99, 15524–15529 (2002).This study establishes the link between 13q14 deletion and downregulation of miR-15a and miR-16-1 in CLL.

16. Cimmino, A. et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl Acad. Sci. USA 102,13944–13949 (2005).

17. Döhner, H. et al. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogsin chronic B-cell leukemias. Blood 85, 1580–1589 (1995).

18. Raval, A. et al. Downregulation of death-associated protein kinase 1 (DAPK1) in chronic lymphocytic leukemia.Cell 129, 879–890 (2007).This study describes a pathogenetic link between downregulation of DAPK1 (by methylation) and CLL.

19. Schaffner, C., Stilgenbauer, S., Rappold, G. A., Döhner, H. & Lichter, P. Somatic ATM mutations indicate apathogenic role of ATM in B-cell chronic lymphocytic leukemia. Blood 94, 748–753 (1999).



20. Austen, B. et al. Mutations in the ATM gene lead to impaired overall and treatment-free survival that isindependent of IGVH mutation status in patients with B-CLL. Blood 106, 3175–3182 (2005).

21. Austen, B. et al. Mutation status of the residual ATM allele is an important determinant of the cellular responseto chemotherapy and survival in patients with chronic lymphocytic leukemia containing an 11q deletion. J. Clin.Oncol. 25, 5448–5457 (2007).

22. Döhner, H. et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N. Engl. J. Med. 343, 1910–1916 (2000).Development of the hierarchical model of CLL prognosis based on recurrent genomic aberrations.

23. Zenz, T. et al. Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia:results from a detailed genetic characterization with long-term follow-up. Blood 112, 3322–3329 (2008).

24. Klein, U. & Dalla-Favera, R. Germinal centres: role in B-cell physiology and malignancy. Nature Rev. Immunol.8, 22–33 (2008).

25. Küppers, R. Mechanisms of B-cell lymphoma pathogenesis. Nature Rev. Cancer 5, 251–262 (2005).26. Kröber, A. et al. VH mutation status, CD38 expression level, genomic aberrations, and survival in chronic

lymphocytic leukemia. Blood 100, 1410–1416 (2002).27. Hamblin, T. J., Davis, Z. A. & Oscier, D. G. Determination of how many immunoglobulin variable region heavy

chain mutations are allowable in unmutated chronic lymphocytic leukaemia — long-term follow up of patientswith different percentages of mutations. Br. J. Haematol. 140, 320–323 (2008).

28. Gurrieri, C. et al. Chronic lymphocytic leukemia B cells can undergo somatic hypermutation and intraclonalimmunoglobulin VHDJH gene diversification. J. Exp. Med. 196, 629–639 (2002).

29. Albesiano, E. et al. Activation-induced cytidine deaminase in chronic lymphocytic leukemia B cells: expression asmultiple forms in a dynamic, variably sized fraction of the clone. Blood 102, 3333–3339 (2003).

30. Oscier, D. G., Thompsett, A., Zhu, D. & Stevenson, F. K. Differential rates of somatic hypermutation in VH genesamong subsets of chronic lymphocytic leukemia defined by chromosomal abnormalities. Blood 89, 4153–4160(1997).

31. Klein, U. et al. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneousphenotype related to memory B cells. J. Exp. Med. 194, 1625–1638 (2001).This study demonstrates that the global gene expression of IGHV-mutated and unmutated CLLs are more similarto memory B cells than to naive or CD5+ B cells.

32. Rosenwald, A. et al. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cellchronic lymphocytic leukemia. J. Exp. Med. 194, 1639–1647 (2001).This study compared gene expression profiles of CLL samples with unmutated and mutated IGHV anddiscovered that ZAP70 was differentially expressed in these subgroups.

33. Damle, R. N. et al. B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype ofactivated, antigen-experienced B lymphocytes. Blood 99, 4087–4093 (2002).

34. Damle, R. N. et al. Telomere length and telomerase activity delineate distinctive replicative features of the B-CLLsubgroups defined by immunoglobulin V gene mutations. Blood 103, 375–382 (2004).

35. Stilgenbauer, S. et al. Clonal evolution in chronic lymphocytic leukemia: acquisition of high-risk genomicaberrations associated with unmutated VH, resistance to therapy, and short survival. Haematologica 92, 1242–1245 (2007).

36. Kipps, T. J. The B-cell receptor and ZAP-70 in chronic lymphocytic leukemia. Best. Pract. Res. Clin. Haematol.20, 415–424 (2007).

37. Chen, L. et al. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia. Blood 105, 2036–2041(2005).

38. Lanham, S. et al. Differential signaling via surface IgM is associated with VH gene mutational status and CD38expression in chronic lymphocytic leukemia. Blood 101, 1087–1093 (2003).

39. Mockridge, C. I. et al. Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status. Blood 109, 4424–4431 (2007).

40. Guarini, A. et al. BCR-ligation induced by IgM stimulation results in gene expression and functional changes onlyin IgVH unmutated chronic lymphocytic leukemia (CLL) cells. Blood 112, 782–792 (2008).



in IgVH unmutated chronic lymphocytic leukemia (CLL) cells. Blood 112, 782–792 (2008).41. Muzio, M. et al. Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a

molecular signature of anergy. Blood 112, 188–195 (2008).42. Hadzidimitriou, A. et al. Evidence for the significant role of immunoglobulin light chains in antigen recognition and

selection in chronic lymphocytic leukemia. Blood 113, 403–411 (2009).43. Widhopf, G. F. et al. Nonstochastic pairing of immunoglobulin heavy and light chains expressed by chronic

lymphocytic leukemia B cells is predicated on the heavy chain CDR3. Blood 111, 3137–3144 (2008).44. Messmer, B. T. et al. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in

promoting chronic lymphocytic leukemia. J. Exp. Med. 200, 519–525 (2004).The demonstration of a striking degree of structural restriction of the entire BCR in CLL, which suggested thatcommon antigens could be recognized by CLL cells.

45. Widhopf, G. F. et al. Chronic lymphocytic leukemia B cells of more than 1% of patients express virtually identicalimmunoglobulins. Blood 104, 2499–2504 (2004).The findings of virtually identical Igs in 1.3% of CLLs provided compelling evidence that the Igs expressed byCLL B cells are highly selected and unlike the Igs expressed by naive B cells.

46. Murray, F. et al. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocyticleukemia: implications for the role of antigen selection in leukemogenesis. Blood 111, 1524–1533 (2008).

47. Chiorazzi, N. & Ferrarini, M. B cell chronic lymphocytic leukemia: lessons learned from studies of the B cellantigen receptor. Annu. Rev. Immunol. 21, 841–894 (2003).

48. Herve, M. et al. Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cellprecursors despite expressing different antibody reactivity. J. Clin. Invest. 115, 1636–1643 (2005).

49. Lanemo Myhrinder, A. et al. A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteriaare targets for chronic lymphocytic leukemia antibodies. Blood 111, 3838–3848 (2008).

50. Ghia, E. M. et al. Use of IGHV3-21 in chronic lymphocytic leukemia is associated with high-risk disease andreflects antigen-driven, post-germinal center leukemogenic selection. Blood 111, 5101–5108 (2008).

51. Tobin, G. et al. Somatically mutated Ig VH3-21 genes characterize a new subset of chronic lymphocyticleukemia. Blood 99, 2262–2264 (2002).

52. Potter, K. N. et al. Features of the overexpressed V1-69 genes in the unmutated subset of chronic lymphocyticleukemia are distinct from those in the healthy elderly repertoire. Blood 101, 3082–3084 (2003).

53. Widhopf, G. F. & Kipps, T. J. Normal B cells express 51p1-encoded Ig heavy chains that are distinct from thoseexpressed by chronic lymphocytic leukemia B cells. J. Immunol. 166, 95–102 (2001).

54. Catovsky, D. et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocyticleukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet 370, 230–239 (2007).

55. Grever, M. R. et al. Comprehensive assessment of genetic and molecular features predicting outcome in patientswith chronic lymphocytic leukemia: results from the US Intergroup Phase III Trial E2997. J. Clin. Oncol. 25, 799–804 (2007).

56. Catera, R. et al. Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis andoxidation. Mol. Med. 14, 665–674 (2008).

57. Chu, C. C. et al. Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognizenonmuscle myosin heavy chain IIA. Blood 112, 5122–5129 (2008).

58. Förster, I., Gu, H. & Rajewsky, K. Germline antibody V regions as determinants of clonal persistence andmalignant growth in the B cell compartment. EMBO J. 7, 3693–3703 (1988).

59. Montecino-Rodriguez, E. & Dorshkind, K. New perspectives in B-1 B cell development and function. TrendsImmunol. 27, 428–433 (2006).

60. Fischer, M., Klein, U. & Küppers, R. Molecular single-cell analysis reveals that CD5-positive peripheral blood Bcells in healthy humans are characterized by rearranged Vκ genes lacking somatic mutation. J. Clin. Invest. 100,1667–1676 (1997).

61. Klein, U., Rajewsky, K. & Küppers, R. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressingthe CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker forsomatically mutated (memory) B cells. J. Exp. Med. 188, 1679–1689 (1998).



somatically mutated (memory) B cells. J. Exp. Med. 188, 1679–1689 (1998).62. Weill, J. C., Weller, S. & Reynaud, C. A. Human marginal zone B cells. Annu. Rev. Immunol. 27, 267–285

(2009).63. Willenbrock, K., Jungnickel, B., Hansmann, M. L. & Küppers, R. Human splenic marginal zone B cells lack

expression of activation-induced cytidine deaminase. Eur. J. Immunol. 35, 3002–3007 (2005).64. Capello, D. et al. Identification of three subgroups of B cell chronic lymphocytic leukemia based upon mutations

of BCL-6 and IgV genes. Leukemia 14, 811–815 (2000).65. Pasqualucci, L., Neri, A., Baldini, L., Dalla-Favera, R. & Migliazza, A. BCL-6 mutations are associated with

immunoglobulin variable heavy chain mutations in B-cell chronic lymphocytic leukemia. Cancer Res. 60, 5644–5648 (2000).

66. Pasqualucci, L. et al. BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutationacting outside Ig loci. Proc. Natl Acad. Sci. USA 95, 11816–11821 (1998).

67. Fukita, Y., Jacobs, H. & Rajewsky, K. Somatic hypermutation in the heavy chain locus correlates withtranscription. Immunity 9, 105–114 (1998).

68. Allman, D. et al. BCL-6 expression during B-cell activation. Blood 87, 5257–5268 (1996).69. Hashimoto, S. et al. Somatic diversification and selection of immunoglobulin heavy and light chain variable region

genes in IgG+ CD5+ chronic lymphocytic leukemia B cells. J. Exp. Med. 181, 1507–1517 (1995).70. Wakai, M. et al. IgG+, CD5+ human chronic lymphocytic leukemia B cells. Production of IgG antibodies that

exhibit diminished autoreactivity and IgG subclass skewing. Autoimmunity 19, 39–48 (1994).71. Seifert M, Küppers R. Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells

and the dynamics of memory B cell generation. J. Exp. Med. 16 November 2009 (doi: 10.1084/jem.20091087)72. Sims, G. P. et al. Identification and characterization of circulating human transitional B cells. Blood 105, 4390–

4398 (2005).73. Matejuk, A. et al. Exclusion of natural autoantibody-producing B cells from IgG memory B cell compartment

during T cell-dependent immune responses. J. Immunol. 182, 7634–7643 (2009).74. Stevenson, F. K. & Caligaris-Cappio, F. Chronic lymphocytic leukemia: revelations from the B-cell receptor.

Blood 103, 4389–4395 (2004).75. Rawstron, A. C. et al. Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic

leukemia are present in 3.5% of adults with normal blood counts. Blood 100, 635–639 (2002).76. Nieto, W. G. et al. Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in

healthy subjects using a highly sensitive multicolor flow cytometry approach. Blood 114, 33–37 (2009).77. Rawstron, A. C. et al. Inherited predisposition to CLL is detectable as subclinical monoclonal B-lymphocyte

expansion. Blood 100, 2289–2290 (2002).78. Landgren, O. et al. B-cell clones as early markers for chronic lymphocytic leukemia. N. Engl. J. Med. 360, 659–

667 (2009).79. Rawstron, A. C. et al. Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N. Engl. J. Med. 359,

575–583 (2008).The first detailed study on the incidence, genetic profile and clinical course of MBL.

80. Dagklis, A. et al. The immunoglobulin gene repertoire of low-count CLL-like MBL is different from CLL: diagnosticimplications for clinical monitoring. Blood 114, 26–32 (2009).

81. Marti, G. et al. Overview of monoclonal B-cell lymphocytosis. Br. J. Haematol. 139, 701–708 (2007).82. Mertens, D. et al. Allelic silencing at the tumor-suppressor locus 13q14.3 suggests an epigenetic tumor-

suppressor mechanism. Proc. Natl Acad. Sci. USA 103, 7741–7746 (2006).83. Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel genes coding for small

expressed RNAs. Science 294, 853–858 (2001).84. Bonci, D. et al. The miR-15a–miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic

activities. Nature Med. 14, 1271–1277 (2008).85. Döhner, H. et al. 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by

extensive nodal involvement and inferior prognosis. Blood 89, 2516–2522 (1997).86. Lavin, M. F. Ataxia–telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nature Rev.

Mol. Cell Biol. 9, 759–769 (2008).



Mol. Cell Biol. 9, 759–769 (2008).87. Bredemeyer, A. L. et al. ATM stabilizes DNA double-strand-break complexes during V(D)J recombination.

Nature 442, 466–470 (2006).88. Kienle, D. L. et al. Evidence for distinct pathomechanisms in genetic subgroups of chronic lymphocytic leukemia

revealed by quantitative expression analysis of cell cycle, activation, and apoptosis-associated genes. J. Clin.Oncol. 23, 3780–3792 (2005).

89. Kalla, C. et al. Analysis of 11q22–q23 deletion target genes in B-cell chronic lymphocytic leukaemia: evidencefor a pathogenic role of NPAT, CUL5, and PPP2R1B. Eur. J. Cancer 43, 1328–1335 (2007).

90. Stilgenbauer, S. et al. Biologic and clinical markers for outcome after fludarabine (F) or F plus cyclophosphamide(FC) — comprehensive analysis of the CLL4 trial of the GCLLSG. Blood (ASH Annual Meeting Abstracts) 112,2089 (2008).

91. Mayr, C. et al. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.Blood 107, 742–751 (2006).

92. Haferlach, C., Dicker, F., Schnittger, S., Kern, W. & Haferlach, T. Comprehensive genetic characterization ofCLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgVH status andimmunophenotyping. Leukemia 21, 2442–2451 (2007).

93. Küppers, R. & Dalla-Favera, R. Mechanisms of chromosomal translocations in B cell lymphomas. Oncogene 20,5580–5594 (2001).

94. Klein, U. & Dalla-Favera, R. New insights into the phenotype and cell derivation of B cell chronic lymphocyticleukemia. Curr. Top. Microbiol. Immunol. 294, 31–49 (2005).

95. Di Bernardo, M. C. et al. A genome-wide association study identifies six susceptibility loci for chronic lymphocyticleukemia. Nature Genet. 40, 1204–1210 (2008).These data provide evidence for common, low-penetrance susceptibility loci for CLL.

96. Lu, R. Interferon regulatory factor 4 and 8 in B-cell development. Trends Immunol. 29, 487–492 (2008).97. Rush, L. J. et al. Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets. Cancer

Res. 64, 2424–2433 (2004).98. Corcoran, M. et al. ZAP-70 methylation status is associated with ZAP-70 expression status in chronic

lymphocytic leukemia. Haematologica 90, 1078–1088 (2005).99. Bialik, S. & Kimchi, A. The death-associated protein kinases: structure, function, and beyond. Annu. Rev.

Biochem. 75, 189–210 (2006).100. Byrd, J. C. et al. Depsipeptide (FR901228): a novel therapeutic agent with selective, in vitro activity against

human B-cell chronic lymphocytic leukemia cells. Blood 94, 1401–1408 (1999).101. Caligaris-Cappio, F. & Ghia, P. Novel insights in chronic lymphocytic leukemia: are we getting closer to

understanding the pathogenesis of the disease? J. Clin. Oncol. 26, 4497–4503 (2008).102. Messmer, B. T. et al. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic

leukemia B cells. J. Clin. Invest. 115, 755–764 (2005).This elegant study demonstrated that CLL cells had definable and often substantial birth rates. CLL is not astatic disease but a dynamic process.

103. Lagneaux, L., Delforge, A., Bron, D., De Bruyn, C. & Stryckmans, P. Chronic lymphocytic leukemic B cells butnot normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood 91,2387–2396 (1998).

104. Ghia, P. et al. Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4+, CD40L+ Tcells by producing CCL22. Eur. J. Immunol. 32, 1403–1413 (2002).

105. Burger, J. A. et al. High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocyticleukemia B cells in nurselike cell cocultures and after BCR stimulation. Blood 113, 3050–3058 (2009).

106. Granziero, L. et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cellchronic lymphocytic leukemia. Blood 97, 2777–2783 (2001).

107. Ghia, P. et al. Differential effects on CLL cell survival exerted by different microenvironmental elements. Curr.Top. Microbiol. Immunol. 294, 135–145 (2005).



108. Hewamana, S. et al. Rel a is an independent biomarker of clinical outcome in chronic lymphocytic leukemia. J.Clin. Oncol. 27, 763–769 (2009).

109. Ramsay, A. G. et al. Chronic lymphocytic leukemia T cells show impaired immunological synapse formation thatcan be reversed with an immunomodulating drug. J. Clin. Invest. 118, 2427–2437 (2008).The demonstration of impaired immunological synapse formation and immune dysfunction in T cells from patientswith CLL that can be reversed by immune-modulating drugs.

110. Patten, P. E. et al. CD38 expression in chronic lymphocytic leukemia is regulated by the tumormicroenvironment. Blood 111, 5173–5181 (2008).

111. Damle, R. N. et al. CD38 expression labels an activated subset within chronic lymphocytic leukemia clonesenriched in proliferating B cells. Blood 110, 3352–3359 (2007).

112. Deaglio, S. et al. CD38 and CD100 lead a network of surface receptors relaying positive signals for B-CLLgrowth and survival. Blood 105, 3042–3050 (2005).

113. Burger, J. A. et al. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneousapoptosis through stromal cell-derived factor-1. Blood 96, 2655–2663 (2000).

114. Kern, C. et al. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrinepathway. Blood 103, 679–688 (2004).

115. Novak, A. J., Bram, R. J., Kay, N. E. & Jelinek, D. F. Aberrant expression of B-lymphocyte stimulator by Bchronic lymphocytic leukemia cells: a mechanism for survival. Blood 100, 2973–2979 (2002).

116. Tsukada, N., Burger, J. A., Zvaifler, N. J. & Kipps, T. J. Distinctive features of "nurselike" cells that differentiatein the context of chronic lymphocytic leukemia. Blood 99, 1030–1037 (2002).

117. Planelles, L. et al. APRIL promotes B-1 cell-associated neoplasm. Cancer Cell 6, 399–408 (2004).118. Chanan-Khan, A. et al. Results of a Phase 1 clinical trial of thalidomide in combination with fludarabine as initial

therapy for patients with treatment-requiring chronic lymphocytic leukemia (CLL). Blood 106, 3348–3352 (2005).119. Ferrajoli, A. et al. Lenalidomide induces complete and partial remissions in patients with relapsed and refractory

chronic lymphocytic leukemia. Blood 111, 5291–5297 (2008).120. Dighiero, G. et al. Chlorambucil in indolent chronic lymphocytic leukemia. French Cooperative Group on Chronic

Lymphocytic Leukemia. N. Engl. J. Med. 338, 1506–1514 (1998).121. Keating, M. J. et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and

rituximab as initial therapy for chronic lymphocytic leukemia. J. Clin. Oncol. 23, 4079–4088 (2005).122. Abrisqueta, P. et al. Improving survival in patients with chronic lymphocytic leukemia (1980–2008): the Hospital

Clinic of Barcelona experience. Blood 114, 2044–2050 (2009).123. Eichhorst, B. F. et al. Fludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy of

younger patients with chronic lymphocytic leukemia. Blood 107, 885–891 (2006).124. Wierda, W. G. et al. Characteristics associated with important clinical end points in patients with chronic

lymphocytic leukemia at initial treatment. J. Clin. Oncol. 27, 1637–1643 (2009).125. Crespo, M. et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic

lymphocytic leukemia. N. Engl. J. Med. 348, 1764–1775 (2003).The first demonstration that ZAP70, as detected by flow-cytometric analysis, correlates with IGHV mutationalstatus, disease progression and survival in CLL.

126. Dreger, P. et al. Indications for allogeneic stem cell transplantation in chronic lymphocytic leukemia: the EBMTtransplant consensus. Leukemia 21, 12–17 (2007).

127. Zenz, T. et al. How little is too much? p53 inactivation: from laboratory cutoff to biological basis of chemotherapyresistance. Leukemia 22, 2257–2258 (2008).

128. el Rouby, S. et al. p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistanceand is independent of MDR1/MDR3 gene expression. Blood 82, 3452–3459 (1993).

129. Rossi, D. et al. The prognostic value of TP53 mutations in chronic lymphocytic leukemia is independent ofdel17p13: implications for overall survival and chemorefractoriness. Clin. Cancer Res. 15, 995–1004 (2009).

130. Zenz, T. et al. miR-34a as part of the resistance network in chronic lymphocytic leukemia. Blood 113, 3801–3808 (2009).

131. Pettitt, A. R. et al. p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternativeto TP53 mutation. Blood 98, 814–822 (2001).



Acknowledgements We acknowledge the important contributions of the numerous researchers whose work could not be cited due to space restrictions. Theauthors are supported in part by the German José Carreras Leukemia Foundation (R06/28v, R06/13 and R08/26f), Else Kröner-Fresenius-

to TP53 mutation. Blood 98, 814–822 (2001).132. Stankovic, T. et al. Inactivation of ataxia telangiectasia mutated gene in B-cell chronic lymphocytic leukaemia.

Lancet 353, 26–29 (1999).133. Stilgenbauer, S. et al. Genomic aberrations, VH mutation status and outcome after fludarabine and

cyclophosphamide (FC) or FC plus Rituximab (FCR) in the CLL8 trial. Blood (ASH Annual Meeting Abstracts)112, 781 (2008).

134. Callen, E., Nussenzweig, M. C. & Nussenzweig, A. Breaking down cell cycle checkpoints and DNA repair duringantigen receptor gene assembly. Oncogene 26, 7759–7764 (2007).

135. Kojima, K. et al. Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent andtranscription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chroniclymphocytic leukemia. Blood 108, 993–1000 (2006).

136. Vogler, M. et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance toABT-737 in chronic lymphocytic leukemia. Blood 113, 4403–4413 (2009).

137. Buchner, M. et al. Spleen tyrosine kinase is overexpressed and represents a potential therapeutic target inchronic lymphocytic leukemia. Cancer Res. 69, 5424–5432 (2009).

138. Matutes, E. et al. The immunological profile of B-cell disorders and proposal of a scoring system for thediagnosis of CLL. Leukemia 8, 1640–1645 (1994).

139. Rassenti, L. Z. et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor ofdisease progression in chronic lymphocytic leukemia. N. Engl. J. Med. 351, 893–901 (2004).

140. Kröber, A. et al. Additional genetic high-risk features such as 11q deletion, 17p deletion, and V3-21 usagecharacterize discordance of ZAP-70 and VH mutation status in chronic lymphocytic leukemia. J. Clin. Oncol. 24,969–975 (2006).

141. Montserrat, E. et al. How I treat refractory CLL. Blood 107, 1276–1283 (2006).142. Allen, C. D., Okada, T., Tang, H. L. & Cyster, J. G. Imaging of germinal center selection events during affinity

maturation. Science 315, 528–531 (2007).143. Calin, G. A. et al. MiR-15a and miR-16-1 cluster functions in human leukemia. Proc. Natl Acad. Sci. USA 105,

5166–5171 (2008).144. Pedersen, I. M. et al. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-

1. Blood 100, 1795–1801 (2002).145. Wiestner, A. et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated

immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 101, 4944–4951(2003).

146. Roos, G. et al. Short telomeres are associated with genetic complexity, high-risk genomic aberrations, and shortsurvival in chronic lymphocytic leukemia. Blood 111, 2246–2252 (2008).

147. Shanafelt, T. D. et al. Prospective evaluation of clonal evolution during long-term follow-up of patients withuntreated early-stage chronic lymphocytic leukemia. J. Clin. Oncol. 24, 4634–4641 (2006).

148. Fabris, S. et al. Molecular and transcriptional characterization of 17p loss in B-cell chronic lymphocytic leukemia.Genes Chromosomes Cancer 47, 781–793 (2008).

149. Zenz, T. et al. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia(CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53–p21 dysfunction, and miR34a in aprospective clinical trial. Blood 114, 2589–2597 (2009).

150. Zenz, T., Dohner, H. & Stilgenbauer, S. Genetics and risk-stratified approach to therapy in chronic lymphocyticleukemia. Best Pract. Res. Clin. Haematol. 20, 439–453 (2007).

151. Stilgenbauer, S. et al. Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia:clinical results and prognostic marker analyses from the CLL2H study of the German Chronic LymphocyticLeukemia Study Group. J. Clin. Oncol. 27, 3994–4001 (2009).



Stiftung (P20/07//A11/07), Deutsche Krebshilfe (108,355, 106,142 and 107,239), SBCancer Helmholtz Alliance on Systems Biology and theGlobal CLL Research Foundation. We thank Antonio Sarno and John Byrd for critical reading of the manuscript. We thank the GCLLSG andthe chairman Michael Hallek for long-standing cooperation and support. We thank the European Research Initiative on CLL (ERIC) forongoing discussions.

Competing interests statement The authors declare competing financial interests.

Nat Rev Cancer. 2010;10(1):37-50. © 2010

from pathogenesis to treatment of chronic lymphocytic ...cllcanada.ca/2010/pages/from pathogenesis...

Documents