Blood Reviews 24 (2010) 135–141

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Blood Reviews

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REVIEW

Biologic and clinical significance of molecular profiling in ChronicLymphocytic Leukemia

Tom Butler, J.G. Gribben ⁎Medical Oncology, Institute of Cancer, Barts and the London School of Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom

⁎ Corresponding author. Tel.: +44 207 882 3804; faxE-mail addresses: t.butler@qmul.ac.uk (T. Butler), j.g

(J.G. Gribben).

0268-960X/$ – see front matter © 2010 Elsevier Ltd. Adoi:10.1016/j.blre.2010.03.004

a b s t r a c t

a r t i c l e i n f o

Keywords:

B-cell Chronic Lymphocytic Leukemia (CLL)Prognostic markers17p deletion

CLL is extremely heterogeneous in its clinical course, with some patients living decades with no need fortreatment whilst others have a rapidly aggressive clinical course. A major focus of research has been to try toidentify those biological factors that influence this heterogeneity. The goal of therapy has been to maintainthe best quality of life and treat only when patients become symptomatic from their disease. For the majorityof patients this means following a “watch and wait” approach to determine the rate of progression of thedisease and assess for development of symptoms. Any alteration to this approach will require identificationof criteria that define patients sufficiently “high-risk” that they gain benefit by introduction of early therapy.The use of molecular profiling to suggest particular therapies is currently appropriate only in defining thetreatment of the minority of patients with 17p deletions or p53 mutations and in all other circumstancesremains a clinical trial question.

: +44 207 882 3891.ribben@qmul.ac.uk

ll rights reserved.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemiain thewesternworld1 and affects predominantly elderly individualswithonly one third of patients being under the age of 60 at presentation. CLLfollows a highly variable clinical course. Approximately 25% of patientsrequire therapy at diagnosis due to bonemarrow failure or to symptomssuch as bulky adenopathy, organomegaly, fatigue or B-symptoms such asfevers, night sweats,weight loss or extreme fatigue. There is awide rangeof initial presenting features, most commonly painless lymphadenopa-thy, followed by splenomegaly and or hepatomegaly. Only 5% of patientspresent with lymphadenopathy without evidence of leukemic infiltra-tion, and in this situation the disease is known as small lymphocyticlymphoma (SLL).

CLL cells aremonomorphic small roundB lymphocytes,with only rareprolymphocytes seen and the diagnostic criteria are shown in Table 1.The diagnosis is made by the detection of a clonal population of small Blymphocytes in PB or BM, or by LN biopsy showing cells expressing thecharacteristic morphology and immunophenotype. CLL cells expressCD19, dim CD20, dim CD5, CD23, CD79a andweakly express surface IgMand IgD. Expression of CD38 is variable and has prognostic significance inthis disease,2,3 and for this reason CD38 should be included in theimmunophenotypic panel in this disease. A scoring system has beenproposed,4 and in difficult cases, particularly those in which there is anatypical immunophenotype, the detection of specific cytogenetic and

molecular features can be helpful inmaking the definitive diagnosis. Theinternational workshop on Chronic Lymphocytic Leukemia (iwCLL) re-vised guidelines require a lymphocytosis of greater than 5000/µL main-tained for more than 3 months with the cells expressing the diagnosticimmunophenotype for the diagnosis of CLL.5 CLL is increasingly diag-nosed in asymptomatic patients when a lymphocytosis is found at thetimeof a routineblood count. Intriguingly, the increasinguse of immuno-phenotyping led to the identification of individuals with circulatingclonal B cells, often with the characteristic phenotype of CLL, but belowthe 5000/µL threshold demanded by CLL guidelines. These cases havebeen catered for with the creation of a new entity, monoclonal B-celllymphocytosis (MBL). The prevalence of MBL in the population is high,with estimates of 3–5%.6 It was noted that these patients often had a veryindolent disease course, and the obvious hypothesis was that a minorityof these patients would go on to develop full-blown CLL (though notnecessarily need treatment), analogous to the link between monoclonalgammopathy of undetermined significance (MGUS) and myeloma. Thishypothesis seems to have been supported by the finding of precedingMBL in the stored samples of patients latterly diagnosed with CLL.6

2. Treatment

Some individuals diagnosed at an early stage may remain asymp-tomatic for the rest of their lives, with their lifespan unaffected by CLL,while others may progress to aggressive disease over time. Treatmentguidelines state that therapy should be reserved for those withadvanced, symptomatic or progressive disease with treatment beingconsidered palliative due to the incurable nature of the disease withconventional chemotherapeutic agents and the often advanced age of

Table 1Diagnosis of CLL.

Clonal expansion of abnormal B lymphocytes in peripheral blood

At least 5×109 B lymphocytes/L (5000/µL)b55% atypical/immature lymphoid cellsLow density of surface Ig (IgM or IgD) with κ or λ light chainsB-cell surface antigens (CD19, CD20 [dim], CD23)CD5 surface antigen

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the patient. Several trials involving more than 2000 patients withearly disease have shown no survival benefit to treating patients withalkylating agents with early versus deferred therapy.7

More recently, there has been improvement in the overall survivalof CLL, particularly since the 1980s and in older age groups. This is notexplained by lead-time bias due to the greater use of automated bloodcounters picking up early stage disease.1 There has been significantimprovement over the past decade in the results of treatment of CLLwith the use of combination chemotherapy and chemo-immunother-apy. This has resulted in improvements in response rates, completeremission (CR) rates and in progression free survival (PFS), but hasnot yet been demonstrated to result in improved overall survival (OS).

Chlorambucilwas the standard of care formany years, and can inducepartial remissions (PR) but few CRs. Survival with this agent has been3–5 years, with no gains in OS with treatment.8 The subsequentintroduction of purine analogues such as Fludarabine in the 1980simproved response rates and PFS, particularly in combination with otheragents such as cyclophosphamide andmitoxantrone. However, improve-ments in OS using these newer agents have proven more difficult toestablish. The use of monoclonal antibodies such as alemtuzumab andrituximab is improving response rates further. Early analysis of theGerman CLL Study Group (GCLLSG)-led CLL8 study comparing the use offludarabine and cyclophosphamide with rituximab, fludarabine andcyclophosphamide has shown that patients treated with chemo-immunotherapy achieved a significant improvement in PFS comparedto patients treated with chemotherapy using fludarabine and cyclophos-phamide alone. Newer agents are in development, and have shownpromise in the laboratory, and in initial trials. These new treatments arebeing driven by an increasing understanding of the biology of the disease.The use of allogeneic stem cell transplantation has led to cure in theminority of patients for whom this approach is indicated. The efficacy,toxicity and cost of the various treatment regimes are highly variable.

3. Assessing response to treatment

The assessment of response to treatment in CLL has becomesomewhat more complicated than in other hematological diseases.Clinical experience in acute leukemia and lymphoma indicated thevalueof CR to chemotherapy as a significant surrogate marker of long-termsurvival. In these diseases, CR can be relatively simply assessed by theeradication of leukaemic blasts from bone marrow or resolution oflymphomatous masses as assessed radiologically. In CLL, the criteria forCRandpartial response (PR) are based on similar criteria. CR requires nodisease on physical examination and microscopic examination of bloodand bone marrow, and recovery of peripheral blood counts. Additionalgroupings of nodular PR (as CR, butwith residual nodules of CLL in bonemarrow) and PR exist.9 Recently, more sensitive tests to detect residualdisease have become available, specifically multicolor flow cytometry10

and polymerase chain reaction (PCR) for clones defined by immuno-globulin variable gene sequences. The two techniques have comparablesensitivities. In some patients achieving conventional CR, one or both ofthese methods of detecting minimal residual disease (MRD) can bepositive. Patients who have no MRD demonstrated by these methodshave a longer remission duration and survival.11,12 In contrast, differ-ences in PFS between nodular PR and conventional PR appear to beminimal, questioning the value of morphological criteria. The hypoth-

esis is now that if we could improve the proportion of patients attaininga CR, and improving the quality of the CR by eliminating MRD, then wecould improve the survival of patients with CLL.13 As yet, there is noevidence that altering therapy on the basis of MRD results has a clearrole in guiding therapy, though the strategy is attractive, and has pre-cedents in other diseases. This is being investigated in a number ofongoing clinical trials.

Whilst improvements in CR rates between fludarabine (20–30%)versus chlorambucil (5–10%) and other alkylator-based regimens havetranslated into longer PFS, it has been difficult or impossible to detectimprovements inOS.8 The heterogeneity of patient groups, the commonuse offludarabine for relapsedpatients post alkylator treatment, and thelong periods of remission associatedwith CLL havemade interpretationof trial outcomes far from straightforward. Improvements in CR and OScontinue to be made by the use of combination chemotherapy usingfludarabine and cyclophosphamide (FC), immunotherapy (rituximaband alemtuzumab), and stem cell transplantation (both autologous andallogeneic).

Inparallelwithnewtherapeuticmaneuvers, therehas beendramaticprogress in the understanding of the basic biology of CLL, and thedevelopment of a panoply of prognostic factors. The knowledge gainedwithuse of theseprognostic factors has not directly led to clarification ofthe wisest plan for treatment of individual patients. Here, we shall firstdiscuss the various markers of prognosis and subsequently examinetheir role in management of the disease. Prognostic markers may beclinical characteristics of the patient, features of the leukemic cellsthemselves, or other biological variables.

4. Conventional prognostic factors

The Rai and Binet staging systemswere based upon their prognosticsignificance and the stage of disease remains perhaps the most usefulprognostic factor in CLL. They are based on readily available parametersthat seem to reflect the biology of the disease. The staging systemdeveloped by Rai assumes that CLL cells first proliferate in the bonemarrow and blood, followed by the lymph nodes, then spleen and liver,and finally anaemia and thrombocytopenia develops, caused by hightumor burden in the bone marrow. Binet modified his system based onfurther multivariate analyses of patients (Table 2).

These staging systems define early (Rai 0,Binet A), intermediate (Rai I/II, Binet B) and advanced (Rai III/IV, Binet C) stage disease with medianestimated survival times of greater than 10, 5 to 7, and 1 to 3 years,respectively. The prognostic value of these stages based only on clinicalexamination and a simple blood count has been confirmed by numerousstudies. However, there is heterogeneity within a single group, particu-larly early stage disease.

Binet stage A disease comprises 60–80% of cases, has a median age atdiagnosis of 64 years, and an expected survival of N10 years, close to thelife expectancy of an agematchednon-CLL population. However over 25%of these ‘indolent’ cases die of causes related to CLL, 40% progress toadvanced stages, and 50% require treatment.14 Therefore the stagingsystems of Rai and Binet are unable to predict accurately which patientsamong the majority early stage group will develop progressive disease.They also do not predict the likelihood of response to treatment in anindividualwith advanced disease. Furthermore, in amultivariate analysis,clinical staging was not retained as an independent prognostic markerwhenother prognosticmarkerswere included in themodel.15 To developrisk-adapted therapies in this heterogeneous disease, better informationthatpredicts diseasebehavior atdiagnosis is required.Avarietyof biologicmarkers has been evaluated and have shown mixed utility.

5. Markers of tumor burden and serum markers in the routineclinical laboratory

As well as clinical staging, other parameters accessible to thetypical clinical laboratory have been evaluated that reflect tumor

Table 2Staging systems in CLL.

Rai stage Characteristics Treat in practice?

0 Lymphocytosis N1.5×109/L NoI Lymphocytosis+lymphadenopathy Symptomatic patients onlyII As above±splenomegaly±hepatomegaly Symptomatic patients onlyIII As above+anaemia Hb b11 g/dL YesIV Thrombocytopenia b100×109/L Yes

Binet stage Characteristics Treat in practice?

A Fewer than 3 areas of lymphadenopathy Symptomatic patients onlyB 3 or more areas of lymphadenopathy Symptomatic patients onlyC Anaemia Hb b11 g/dL and/or thrombocytopenia b100×109/L Yes

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burden. To be useful in clinical practice, a test has to be routinelyavailable and be validated (reproducible, etc). These laboratory testsare presumed to reflect aspects of disease biology, and some are easilyemployed for day-to-day use. Absolute lymphocyte count is anindependent risk factor,15 as is the lymphocyte doubling time. Thoughcorrelated with stage, the doubling time has independent prognosticvalue: a doubling time of less than 12 months identified a populationof patients with poor prognosis, even among early stage diseasepatients.16,17 Another measure of disease burden is the histologicalpattern of bone marrow infiltration by the disease. Cases with diffuseinfiltration have a poorer prognosis than those with a nodularpattern.17 Bone marrow histology is also used as a criterion ofresponse to treatment in most studies, though there is an increasinglyprevalent view that assessment of minimal residual disease (MRD)may provide a more useful guide to treatment than histologicalevaluations (see above). Lactate dehydrogenase (LDH) is an intracel-lular enzyme present in all tissues and is commonly elevated in CLLand other lymphomas, reflecting the high rates of lymphocyteturnover. It can also reflect the hemolysis that can accompany CLLand its treatment. Elevated levels have been shown to have prognosticvalue.18 Thymidine kinase (TK) is a cellular enzyme involved in DNAsynthesis, and serum levels are probably related to the number ofdividing leukemia cells, again reflecting tumor burden. TK has beenshown to correlate with the proliferative activity of CLL cells,19 withprognostic utility in early stage disease.20 Beta-2-microglobulin (β2M)is an extracellular protein noncovalently associated with the class Imajor histocompatibility complex (MHC), and expressed by nucleatedcells. Serum levels show positive correlation with clinical stagingsystems, adverse prognostic features, and short survival.21 β2M maybe a more powerful predictor of survival than clinical staging, and canpredict response to chemotherapy.12

6. Cytogenetics

Unlike many of the other low-grade B-cell malignancies, non-random reciprocal chromosomal translocations occur rarely in CLL/SLL, including translocations involving the immunoglobulin loci.However, a number of recurrent abnormalities have been identified.The poorly dividing CLL cells are reluctant to provide metaphasespreads for classical karyotyping, and the use of agents such as4-phorbol-l2-myristate-l3-acetate (PMA) to stimulate mitosis canincrease metaphase yields. These studies identified recurrent abnor-malities that were used to design fluorescence in situ hybridization(FISH) probes, which further increased sensitivity. Aberrationsdetected by FISH have been shown to have strong prognosticsignificance and can be detected in most cases of CLL. In acomprehensive study chromosomal abnormalities were detected byFISH in 268 of 325 CLL patients studied (82%).18 This study demon-strated convincingly that genomic aberrations in CLL are importantindependent predictors of disease progression and survival. The mostcommon recurrent chromosomal abnormalities observed include

deletion of the long arm of chromosome 13 (del 13q), del 11q,trisomy 12, del 17p and del 6q.15,18

The nature of these recurrent abnormalities points to the loci ofcandidate genes involved in pathogenesis. The most commonabnormality is del 13q14, which occurs in 55% of cases, and hasbeen associated with a favorable prognosis. The first report linkingmicroRNAs to cancer was in CLL, where it was demonstrated that twomicroRNA clusters, mir-15a and mir16-1, were located within thedeleted region at 13q14.22 The next most common cytogeneticabnormality is del 11q, seen in approximately 20% of cases of CLL.This deletion is associated with a distinct clinical presentationincluding younger age, male sex, bulky lymphadenopathy and poorprognosis. The Ataxia Telangiectasia Mutated (ATM) gene is locatedwithin theminimal region of loss at 11q23, suggesting that alterationsin DNA repair pathways may be involved in the pathogenesis of thedisease. This is further supported by the finding that mutations in theATM gene are associated with poor prognosis, and may not alwayscorrelate with deletions of 11q.23 Trisomy 12 occurs in up to 20% ofcases of CLL/SLL, but the molecular mechanism by which this geneticabnormality contributes to leukemogenesis is unknown. Althoughless common and occurring in less than 10% of patients at diagnosis,del 17p is associated with rapid progression of disease, poor responseto therapy and short survival. The deletion involves the p53 locus at17p13, and it is clear that mutations in the p53 gene can contribute todisease progression and alter the sensitivity of CLL cells to chemo-therapy agents. Mutations in p53 may be detected in up to 23% ofpatients without 17p deletion as assessed by FISH, and confer asimilarly poor prognostic impact.24 Ongoing studies are assessing theimpact of specific cytogenetic abnormalities in response to particulartherapeutic approaches.

7. Immunoglobulin heavy chain (IgHV) rearrangements and IgHVusage

An important advance in the understanding of CLL was made withthe demonstration that 50% of CLL cases have somatic hypermutation(SHM) in the variable regions of the immunoglobulin heavy chain(IgHV) genes and that this has prognostic significance, with casesexhibiting SHM (M-CLL) having a more indolent clinical course andlonger survival than those without SHM (UM-CLL). 2,25The levels ofsomatic hypermutation in particular B cells is evaluated by compar-ison of the sequence of the rearranged variable region gene withgermline sequences, and guidelines have been reported for analysis ofIgHV rearrangements from the working group of European ResearchInitiative in CLL guidelines.26 Sequences with less than 98% homologyto germline are considered to have undergone somatic hypermuta-tion. UM-CLL cells are presumed to have not passed through agerminal center in the generation of an antibody response (naïve), oralternatively to have responded to T cell independent antigens. Thefinding that CLL cases can be divided into mutated (M) andunmutated (UM) groups implied that the two groups may be diseases

Table 3Impact of molecular markers on prognosis in chronic lymphocytic leukemia (CLL).

Marker Frequency, % TTT, mo OS, mo Reference

Cytogenetics del 13p 55 92 133 Dohner et al 200018

Normal 18 49 111Trisomy 12 16 33 114del 11q 13 13 –

del 17p 9 9 –

IgVH Mutated 47 110 300* Rassenti et al 200444

Unmutated 53 42 115* Hamblin et al 199925

ZAP70 Negative 54 110 NS Rassenti et al 200444

Positive 46 35 NSCD38 Negative 67 94 193a Rassenti et al 200859

Positive 33 40 109a Hamblin et al 20023

Abbreviations: TTT, time to treatment; OS, overall survival.a Hamblin et al 1999,25 2002.3

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that arise from different normal cellular counterparts.27 Cases of CLLthat have undergone SHM are presumed to have experienced this inthe germinal center, and perhaps therefore arise from antigenexperienced memory B cells, although this suggestion was notsupported by gene expression profiling studies.28

As well as informing us about the possible pathophysiology of thedisease, it has become clear that the mutation status of a particularcase of CLL is one of the most powerful predictors of prognosis, withUM-CLL cases typically progressing rapidly and having shortersurvival times than M-CLL.2,25 There is an association betweenunfavorable cytogenetic aberrations (del 17p and del 11q) andUM-CLL, whilst del 13q is more frequent in M-CLL. Studies haveshown that VH status and cytogenetics are independent prognosticfactors, and when incorporated into models, clinical staging (Rai andBinet) loses independent prognostic value.29

Other data indicate the centrality of the immunoglobulin B CellReceptor (BCR) to the biology of CLL. UM-CLL appears to expresshigher levels of surface immunoglobulin, and displays a greater abilityto respond to stimulation of the BCR, with M-CLL tending to resembleanergic cells in producing lack of response.30,31 Analysis of variableregion sequences demonstrated that CLL cells utilize a biasedrepertoire of V genes characterized by over-representation of selectedIg gene segments, in particular IGHV1-69, IGHV4-34, IGHV3-7, andIGHV3-21.32 SHM does not occur uniformly among IGHV genes:IGHV1-69 is more prevalent than expected in UM-CLL, with IGHV4-34increased in M-CLL. There is also bias of use of IGHV3-21 in bothUM-CLL and M-CLL,33 and also bias in the use of particular genes inlight chains. An apparent exception to the generalization thatmutatedCLL cases have good prognosis is in the subgroup of patients with CLLcells that use IGHV3-21 since these patients have relatively aggressivedisease even when the expressed IGHV3-21 is mutated.34

Not only is the immunoglobulin gene repertoire expressed by CLLcell biased, it is also notable for the existence of subsets with nearidentical (stereotyped) CDR3 sequences of their BCRs, whichconstitute the main antigen-binding region and therefore imply therecognition of structurally similar epitopes is likely to be positivelyselecting the leukemic clones. The presence of such stereotypicrearrangements may also have prognostic significance.35,36

These findings have led some authors to suggest a microbialantigen or autoantigen as a stimulus to CLL growth, and the immuno-globulin expressed by some CLL clones has been shown to haveautoantibody activity.37,38

8. Surrogates for mutation status

It is not possible to perform IgHV mutational status on a routinebasis in clinical laboratories and attempts have therefore been madeto identify surrogate markers for mutational status and its powerfulrole as a prognostic tool. In particular, expressions of two proteins,ZAP70 and CD38, which both have prognostic significance, have beenexamined. CLL cells demonstrate a continuum of expression of theseproteins, and it is necessary to determine a cut-off point at which acase is deemed to be positive or negative, leading to difficulties instandardization since different laboratories assess individual cases asbeing positive or negative for expression.

In a study comparing the gene expression of UM-CLLwithM-CLL, itwas found that only a small number of differentially expressed genesseparate the two groups,39 the most specific being a gene encoding a70 kDa zeta-associated protein (ZAP-70).40 Most mutated cases areZAP-70 negative and unmutated cases ZAP70 positive. ZAP70expression can be measured by number of conventional methodsincluding western blotting, reverse transcriptase-PCR, immunohisto-chemistry, and flow cytometry.40–43 Levels of expression are higher inT cells and NK cells than in CLL cells, and it is important that effectivemultiparametric gating strategies are used to ensure that expressionis being measured in the CLL cells. A variety of cut-offs for expression

level have been adopted, with a consensus that 20% threshold mayprove the most useful.44 ZAP-70 expression is also stable over time.45

The relationship of ZAP-70 positivity and UM-CLL is not absolute,however, with discrepant cases ranging from 8 to 25%.44,45 Thesediscordant cases may have other biological features with poorprognostic implications such as del 17p, del 11q or use of IGHV3-21.46 Some studies have suggested that ZAP70 status is more useful asa predictor of time to progression than mutation status, but thisremains controversial. 47Difficulty with standardization has plaguedstudies measuring ZAP-70, and impaired its translation into astandard clinical tool.

CD38 is a surface marker associated with CLL, and easilydetermined using standard flow cytometric methods. It was initiallyfound to correlate with IgHV mutation status,2 however therelationship is not absolute, and there is a concern that CD38expression may vary over time.3 Also, it has become clear that CD38and VH mutation status are independent prognostic variables.2Thefield is somewhat confused by a variety of cut-offs being used todefine a case as being CD38 positive (5%, 7%, 20% or 30% in differentseries),48–50 and it has been suggested that CD38 should be evaluatedby its modal expression by flow cytometry, or by antigen density. Itmay be that CD38 and ZAP-70 provide complementary prognosticinformation, with expression of both markers conferring a poorprognosis, lack of both a good prognosis, and an intermediate groupwith discordant expression.51 Originally valued asmere surrogates formutation status, it may be that ZAP-70 and CD38 status are ultimatelyaccepted as complementary prognostic markers with independentvalue.

Other surrogates of mutation status have been suggested includingexpression of thymidine kinase, activation-induced cytidine deami-nase, lipoprotein lipase A and ADAM29.52–54 MicroRNA arrays haverevealed a 13 gene signature found to correlate with ZAP-70 status,UM-CLL and disease progression,22 and two of the most differentiallyexpressed microRNAs (miR15a and miR 16) are located at the 13qlocus commonly deleted in CLL. Recent work has suggested that thisalteredmicroRNA expression regulates expression of genes regulatingapoptosis and cell-cycle progression.55 Epigenetic alterations incancer are well described, and aberrant methylation has beendemonstrated in CLL, both globally56 and for specific genes.57,58 Theuse of drugs as epigenetic therapy in CLL is being evaluated in ongoingtrials.Whilst array-based analyses of different subgroupsmywell leadto novel insights into biology, their routine use in the clinical setting iscurrently not possible (Table 3).

9. Molecular profiling in clinical practice

It is obvious that the diagnostic methods and prognostic toolsrelating to CLL have expanded in accompaniment to knowledge of theunderlying pathophysiology of the disease. However, this has not

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translated into straightforward recommendations as to when andhow to treat an individual patient. What we do know is that themolecular profile of CLL provides insight into the underlyingpathogenesis of the disease and provides predictors of time toprogression, response to therapy and overall survival. High-riskfeatures include the high-risk cytogenetic features del 11q and del17p, IgHV unmutated status, use of the IGHV3-21 gene segment,expression of ZAP70 and expression of CD38. It is tempting tospeculate that thesemarkers can now be useful in clinical practice, buta number of questions remain. There is no evidence to date that if apatient presents with “high-risk” disease features that there is anybenefit in offering early therapy. This question is now being addressedin ongoing and planned clinical trials; until the results of these studiesare available, patients should not be offered treatment on the basis ofany molecular marker until the standard criteria for treatment arereached.5

With the finding that a number of molecular markers haveprognostic significance it is not surprising that many of these factorsare correlated. However, there are still discrepancies, withmany caseshaving some high-risk and other low-risk molecular features, andmore than 50% of IgHV unmutated cases have no unfavorablecytogenetics.46 There is also an association between unfavorablecytogenetic aberrations (del 17p and del 11q) and unmutated CLL,while 13q deletion is more frequent in mutated CLL. A recent studyinvolving more than 1000 CLL patients examined the relative value ofZAP70, CD38 and IgHV mutation status and found that ZAP70expression was the strongest predictor of time from diagnosis torequirement for treatment.59 It may be that high-risk cytogenetics,IgHV mutational status, ZAP70 and CD38 provide complementaryprognostic information, with expression of both markers conferring apoor prognosis, lack of both a good prognosis, and discordantexpression an intermediate prognosis.44,46,59

Most of the modern prognostic markers were validated byretrospective analysis, often from single-center studies, but havenow been applied to prospective randomized clinical trials. Thesestudies suggest that the samemolecularmarkers that identify patientswithmore aggressive disease also impact on outcome after treatment.This finding is not surprising since these same factors have beenpredictive of overall survival in retrospective studies, where it wouldhave been expected that the same treatment optionswould have beenoffered to patients regardless of risk factors, known and unknown.Predicting subsets of patients who will respond to a given therapymay be useful to avoid unnecessary toxicity and avoid the develop-ment of resistance. Deletions of 11q correlate with a lower response tofludarabine and early relapse after autologous stem cell transplanta-tion,60 whilst high serum β2M levels are correlated with inferiorresponse to chemo-immunotherapy.12 Studies have been publishedexamining the impact of these factors on response in prospectiverandomized trials in previously untreated patients with CLL, 61–63andthese results have been confirmed in a number of other studies thathave been reported in abstract format only. These findings suggestpoor-risk features for CLL are largely also predictive of poor response.

Clinical trials are underway to evaluate the utility of usingmolecular profiling. The German and French CLL Study Groups CLL7trial is randomizing patients in Binet stage Awith high-riskmarkers toeither watch and wait or FCR therapy. A UK-based trial (RESPeCT) isevaluating the use of lenalidomide in a similar high-risk patient group,and similar trials are recruiting or planned. Outside the setting ofclinical trials, there is not yet any evidence to change recommenda-tions as to when treatment should be initiated. Once patients becomesymptomatic or fulfill the criteria for the need for treatment there isnot yet sufficient data to suggest that the detection of any of thesemarkers should alter which therapy is offered with the exception ofthose patients who present with del 17p or mutations of p53. Again itshould be stressed that even the detection of this poorest of theprognostic markers is not an indication for earlier treatment in

asymptomatic patients. Treatment should be initiated only whenindicated by standard criteria, i.e. when they are symptomatic.However, we know that these abnormalities are unique in guidingclinicians as to which therapies are indicated (or rather which are notindicated due to lack of efficacy). Deletions of 17p and/or p53 genemutations have been associated with failure after treatment withalkylating agents, purine analogues, and rituximab.46,64 Evidence isaccumulating on the efficacy of anti-CD52 monoclonal antibodytherapy (alemtuzumab) in CLL with p53 gene abnormalities.65–67

Although this represents only a small group of previously untreatedpatients, these patients should be treated preferably in clinical trials,examining agents that have efficacy in patients without functionalp53. Since responses are likely to be of short duration, and patientswho are young enough to consider allogeneic stem cell transplant andin whom suitable donor can be found are then eligible for clinicalstudies examining the role of reduced intensity conditioningallogeneic stem cell transplants in first response. There is insufficientdata available presently to alter therapy for any other group ofpatients. Nonetheless, the ongoing characterization of novel prognos-tic factors provides an insight into the biology of this disease and mayidentify new therapeutic targets.

So, does the clinician benefit from obtaining these tests in routineclinical practice? Several of the factors cannot readily be obtainedincluding IgHV mutational status and the current assays forexpression of ZAP70 are unreliable, with no clear guidelines on theestablished methodology or what expression level designates ZAP70positive or negative. There is considerable ongoing debate regardingthe clinical utility of CD38 expression and its stability over time.Cytogenetics assessed by FISH appears to be robust and reproducibleand provides prognostic information for the clinician and patients.Since cytogenetic abnormalities also change over time with theevolution of new genetic changes, it maymakemore sense to performthis analysis at the time of institution of therapy. Clinicians may bebetter served using cheaper and more established markers of diseaseincluding lactate dehydrogenase and beta-2 microglobulin, which canbe incorporated into nomograms to assess risk of progression.68

Therefore, decision making in clinical practice should be made onthe basis of clinical features of the disease, and the use of molecularprofiles in the management of CLL remains a clinical trial questiononly. There are two important questions that molecular profilingmight plausibly provide an answer to: When should we initiatetherapy, and with what regimen? There is not yet any evidence thatearly treatment is beneficial in patients that are defined as high-risk.Ongoing and future trials will hopefully provide some answers to thisquestion. Until we have the results of these trials, treatment should beinitiated only when guidelines indicate, generally speaking when anindividual patient is symptomatic of disease. This advice also appliesto those with p53 abnormalities. As to the second question, there iscurrently a dearth of evidence to suggest a change of first-lineregimen based on molecular markers of disease. The only clearexception to this at present is in symptomatic patients with 17pdeletion or p53 abnormalities, for whom efforts should be made totreat these patients with agents that may act independently of p53.

10. Practice points

• The treatment and survival of CLL has advanced in parallel to ourunderstanding of the biology of the disease.

• Many new prognostic markers have been identified, some of whichare powerful predictors of survival and response to therapy.

• There is insufficient evidence to advise departures from current NCIguidelines based on these prognostic factors. Patients shouldgenerally be treated only when symptomatic.

• The only prognostic marker that should influence the therapeuticregimen is the presence/absence of 17p deletions or p53 mutations.

140 T. Butler, J.G. Gribben / Blood Reviews 24 (2010) 135–141

11. Research agenda

• Prospective, randomized clinical trials are ongoing, and are the onlyway to establish the true utility of molecular profiling.

• The identification of prognostic markers gives us clues to biology,and the further understanding of pathophysiology should provideus with new targets for therapy.

Conflicts of interest

T. Butler has no declared conflicts of interest.J.G. Gribben has received honoraria for advisory boards from

Roche, Celgene, Genzyme, and Mundipharma.

Funding sources

T. Butler is funded by a Cancer Research UK Molecular Pathology ofCancer grant. The sponsor had no role in the writing of the manuscript.

J.G. Gribben is funded by Program Grants from The National CancerInstitute and from the Cancer Research UK and from CLL Global Alliance.

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