Minimal residual disease assessment in chronic lymphocytic leukaemia

Best Practice & Research Clinical Haematology Vol. 20, No. 3, pp. 499–512, 2007 doi:10.1016/j.beha.2007.03.004 available online at http://www.sciencedirect.com

11 Minimal residual disease assessment in chronic lymphocytic leukaemia Hazem A. Sayala *

MRCP(UK)

Clinical Reasearch Fellow

Andy C. Rawstron

PhD

Principal Clinical Scientist

Peter Hillmen

MD, PhD

Consultant Haematologist Haematological Malignancy Diagnostic Service, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK

The concept of minimal residual disease (MRD) eradication in chronic lymphocytic leukaemia (CLL) is a relatively new one, as conventional therapy with alkylating agents is relatively ineffective and responding patients almost always have a significant tumour burden remaining at the end of treatment. However, a variety of novel therapies is now yielding higher response rates, and responses of better quality are now routinely achieved. This progress in therapy has been paralleled by an improvement in laboratory assays, allowing detection of CLL cells to levels as low as ten CLL cells in a million leukocytes. In this chapter we briefly review the existing methods for MRD assessment, the clinical relevance of MRD eradication in CLL, and the therapies available to attain this endpoint. Key words: chronic lymphocytic leukaemia; minimal residual disease.

Despite improvements in the treatment of haematological malignancies over the last few decades, many patients relapse after an initial response to treatment. Such relapses are derived from the same clone detected at the time of diagnosis. Minimal residual disease (MRD) can be defined as the level of disease detectable by the most sensitive technique available. In general, for haematological malignancies the persistence of detectable MRD at the end of therapy predicts relapse and is associated with an inferior outcome. This is because the residual disease persisting at the end of therapy has * Corresponding author. Tel.: þ44 113 3926285; Fax: þ44 113 3926286. E-mail address: [email protected] (H.A. Sayala). 1521-6926/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.

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survived exposure to chemotherapeutic agents, implying that these tumour cells are likely to be relatively resistant to further therapy. In effect the treatment has potentially selected for resistant sub-clones. This presumably explains the fact that responses to second-line therapy are almost always inferior to the initial response. MRD assessment in a certain disorder requires the existence of a disease-specific characteristic permitting the development of a sensitive assay. This can be a chromosomal translocation, such as the t(9;22) of chronic myeloid leukaemia or the t(15;17) of acute promyelocytic leukaemia, or a disease-specific immunophenotype allowing the development of sensitive flow-cytometric assays such as that observed in multiple myeloma. In addition, an even distribution of tumour cells in blood or marrow is essential, and this appears to be generally observed in the blood- or marrow-derived malignancies. Therefore the eradication of the malignant clone is increasingly becoming an important therapeutic endpoint if relapses, and the occurrence of resistant disease, are to be prevented. Many clinical trials are investigating risk stratification of patients based on MRD quantification at various time points, and treatment strategies are designed accordingly. In acute myeloid leukaemia, MRD level at the end of induction and consolidation therapy is a powerful predictor of relapse-free survival and overall survival.1,2 Similar data are available for paediatric patients with acute lymphoblastic leukaemia (ALL)3,4, adult patients with standard-risk ALL5, patients with chronic myeloid leukaemia6, and patients with multiple myeloma.7 For most of these disorders MRD measurement is carried out as a routine part of response assessment following therapy. It is often the case that patients with persistent or higher levels of MRD are treated differently to patients who achieve MRD negativity. Chronic lymphocytic leukaemia (CLL) has somewhat lagged behind these other disorders in terms of MRD assessment, but with the recent advent of more effective therapies and the development of techniques to identify MRD in CLL, this is now becoming true for CLL. The evidence supporting this apparently bold statement is reviewed below. RESPONSE ASSESSMENT IN CLL In order to compare the outcomes of various therapeutic interventions and the results of clinical trials conducted by different groups, standardized criteria for response assessment are necessary. The National Cancer Institute-sponsored Working Group (NCI-WG) on CLL published the first set of guidelines in 1988.8 These guidelines were adopted by most collaborative groups in the field of CLL. Revised guidelines were subsequently published in 1996 to reflect the change in practice in CLL treatment.9 A complete response as defined by the NCI-WG criteria is defined by resolution of symptoms, disappearance of palpable disease, and the absence of morphological evidence of CLL in the marrow together with a relative normalization of peripheralblood parameters. In cases that attain a CR as judged by National Cancer Institute (NCI-WG) criteria, there often remains a considerable infiltrate of CLL in the marrow when tested by highly sensitive techniques such as flow cytometry. CLL cells were detected by MRD flow (i.e. >0.05% of leukocytes) in 32% (8/25) of samples with no morphological evidence of tumour.10 This approach to response assessment reflects the lack of efficacy of early treatment regimens based on alkylating agents where complete responses are rarely achieved and MRD assessment was not applicable. Newer, more effective therapeutic drugs, such as the purine analogues and monoclonal antibodies, have become available since the 1996 criteria were published and are now

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being combined to significantly improve their efficacy. In this way deeper responses are commonly achieved, and more sensitive assays have been developed allowing the detection of CLL cells at a very low level. This improvement in diagnostic and therapeutic strategies calls for a revision of the current response assessment criteria to mirror the rapidly changing practices in CLL treatment. METHODS OF DETECTION OF MRD IN CLL The assessment of marrow morphology is the approach for response assessment utilized in the 1996 NCI-WG criteria after therapy for CLL. This approach has a low sensitivity and allows patients with a significant tumour burden to be classified as complete responders. The general principle of MRD detection assays is to identify malignant cells on the basis of a unique profile that is not shared by their normal counterparts. Highly sensitive assays are now available which can detect single CLL cells in 104–106 leukocytes.10,11 The two techniques that are used for MRD detection in CLL are the polymerase chain reaction (PCR), which identifies CLL cells on the basis of their immunoglobulin heavy chain (IgH) gene rearrangement, and multi-parameter flow cytometry identifying residual CLL cells on the basis of their surface expression of unique combinations of cell markers. In contrast to many other haematological malignancies, CLL does not have a specific structural chromosomal abnormality that can be used for MRD monitoring. On the other hand, and in common with other B-cell malignancies, the malignant clone can be identified by its unique IgH gene rearrangement.12 Two PCR strategies can be used to detect MRD in CLL: consensus primer PCR and allelespecific oligonucleotide PCR (ASO-PCR). Both are based on the detection of immunoglobulin gene rearrangement in the CLL clone. Highly sensitive MRD assessment by flow cytometry has become a reality due to developments in flow-cytometry technology, such as cytometers capable of analysing four or more colours and that can process cells extremely rapidly. In addition, a much more detailed understanding of the normal maturation of B cells from progenitors to mature B cells has permitted the separation of CLL cells from their normal counterparts. Therefore, multi-parameter flow cytometry is likely to become the mainstay for MRD assessment in CLL for routine use. Consensus primer PCR – low sensitivity The principle of this technique is to amplify the third complementarity-determining region (CDRIII) of the IgH gene using a standard set of universal oligonucleotide primers, a primer that recognizes a consensus sequence in the JH region, and a primer to family-specific framework regions.13,14 Consensus PCR is a qualitative method with variable sensitivity (1 CLL cell in 102–104 normal cells).15,16 Binding of consensus primers may fail because of mutations in the IgH gene, limiting the applicability of the assay to 70–80% of patients.10 The assay’s sensitivity is reduced by the presence of normal polyclonal B cells14 and by binding of consensus primers to germ-line DNA in normal leukocytes.10 The assay tends to be limited to detecting CLL cells when they represent more than 2% of total B cells17,18, and is even less sensitive in the presence of polyclonal mature or progenitor B cells. Allele-specific oligonucleotide PCR (ASO-PCR) – high sensitivity This technique involves the design of oligonucleotide primers specific for an individual patient’s malignant clone and the use of these primers for MRD detection in follow-up

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samples. This approach overcomes the interference of normal B cells seen with consensus primers and significantly improves the assay’s sensitivity. ASO- PCR is currently the most sensitive technique for MRD detection in CLL, with a reported sensitivity of as low as 1 CLL cell in 106 leukocytes.11 The use of ASO primers or combinations of ASO and consensus primers in a real-time PCR assay allows quantification of MRD, although the sensitivity is slightly lower at 1 CLL cell in 104–105 cells.19–21 However, the design of ASO primers is time-consuming, labour-intensive, expensive, and not readily available in most routine laboratories. Studies directly comparing the results of MRD flow cytometry versus consensus PCR versus ASO-PCR show that MRD flow has a superior sensitivity to consensus PCR and a sensitivity approaching that of ASO-PCR.16 The results of quantitative PCR and MRD flow were concordant in the majority of samples and patients in a more recent series comparing the three techniques in the same group of patients.22 CD5/19 D flow cytometry – low sensitivity Early flow-cytometric assays designed for response assessment were effectively extensions of diagnostic flow cytometry; they were usually two-colour approaches and relied on detection of co-expression of CD5 and CD19 or CD20 together with monoclonality of light-chain expression. Cut-off points for normal CD5 expression were used to identify patients with residual CLL. The general principle was to consider the presence of >25% of CD19þ cells co-expressing CD5 as a positive MRD test.23,24 Slight variations to this approach were also used, including reducing the cut-off point to 15% if the assay was performed on marrow25, or the use of co-expression of CD5 and CD20 with a cut-off point of 10% to identify residual CLL.26 The presence of normal polyclonal B cells, and of B-cell progenitors in the marrow, interferes with the test sensitivity, especially when CLL cells are present at low levels following therapy. These conventional tests can detect 1 CLL cell in 200 normal cells.27 However, a fundamental problem with this approach is that the phenotype used to isolate CLL cells is not unique for CLL cells. In particular, when there are either high levels of mature polyclonal B cells or B-cell progenitors, then conventional flow cytometry becomes very insensitive. Unfortunately, at times of maximum response it would be expected that the patient would have a recovery of the normal B-cell compartment, and hence conventional flow cytometry becomes less sensitive. MRD flow cytometry – high sensitivity In 2001 a highly sensitive flow-cytometry technique (MRD flow) was introduced with a sensitivity of one CLL cell in 104–105.10 The initial MRD flow assay was designed using a combination of four antibodies (CD5/19/20/79b) and a sequential gating strategy to effectively separate CLL cells form normal B cells (Figure 1). The addition of CD38 helps to discriminate CLL from B-cell progenitors in marrow samples. The sensitivity of this assay is superior to that of consensus primer PCR, and its applicability is wider. In a recent series of 40 CLL patients who underwent stem-cell transplantation (25 autologous and 15 allogeneic), MRD flow cytometry and allele-specific PCR yielded concordant results in the majority of patients and samples.22 Other antibody combinations have been proposed, and the results of these are being validated in an effort to standardize the process of MRD testing and facilitate comparison of results between various groups.28 Combinations containing CD20 cannot be used for MRD

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Figure 1. Co-expression of CD5 and CD20 distinguishes chronic lymphocytic leukaemia (CLL) cells from B-cell progenitors and mature B cells.

assessment in rituximab-treated patients where normal B cells lack CD20 expression. The combination of CD81/CD22/CD19/CD5 (Figure 2) has recently been reported as being as sensitive and as specific as previously reported assays, with the additional advantage of being applicable to rituximab-treated patients.29 A good correlation between MRD level in paired peripheral-blood and marrow samples has been demonstrated. However, in the case of monoclonal antibodies, such as alemtuzumab, there is clearance of CLL in peripheral blood prior to any significant clearance of the marrow in most cases. This correlation is useful in deciding the timing of MRD assessment and the type of sample used, as peripheral-blood assessment is more convenient.22 Overall, flow cytometry yields quick results and is readily available in most laboratories, making it the method of choice for MRD assessment where results can be used to inform treatment decisions. Standardization of MRD assessment in CLL Recently a group of laboratories with an interest in MRD detection in CLL have collaborated under the auspices of the European Research Initiative on CLL (ERIC) to develop a consensus for MRD assessment. This group has recently reported their findings, and these will be adopted in the new version of the NCI response criteria that will be published in 2007. The consensus from this work is that a level of a single CLL cell in 10,000 leukocytes can be reliably detected by either MRD flow cytometry or allele-specific PCR. Therefore MRD negativity in CLL will be defined as less than a single CLL cell in 10,000 leukocytes. It also appears that the analysis of peripheral

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Figure 2. Chronic lymphocytic leukaemia (CLL) cells show weaker expression of CD81 and CD22 compared to B-cell progenitors and mature B cells, respectively.

blood is appropriate for most therapies. The exception is when patients are receiving monoclonal antibodies, such as alemtuzumab or rituximab, in which case marrow should be studied. This is because with antibody therapy there is a discrepancy between the peripheral blood, which is rapidly cleared, and the marrow, which takes longer to clear. Allele-specific PCR is approximately a log more sensitive than MRD flow, but in view of its expense and relative inconvenience is unlikely to become standard in routine practice. However, in research protocols where MRD eradication is a desired end-point then allele-specific PCR is likely to have a role. In routine use MRD flow cytometry is recommended.30 CLINICAL RELEVANCE OF MRD ERADICATION IN CLL Patients who achieve more profound remissions, regardless of the therapy used, have a better survival compared to those with poorer responses. Among patients treated with fludarabine as front-line therapy, the survival of patients achieving a complete remission (CR) or nodular partial remission (nPR) is significantly superior to that of patients in PR who in turn had a significantly longer survival than resistant patients.31 More recent series reported the results of a variety of combination chemotherapy regimens, monoclonal antibodies or high-dose procedures with stem-cell rescue. Examples of these include fludarabine plus cyclophosphamide (FC)32, fludarabine, cyclophosphamide plus mitoxanthrone (FCM)33, fludarabine, cyclophosphamide and rituximab34,35, the monoclonal antibody alemtuzumab36, and autologous and allogeneic

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stem-cell transplantation.22,37–40 Assessment of MRD status was part of the analysis in some of these series. Patients who achieved an MRD-negative remission in most of these series had significantly longer progression-free survival and, in some series, better overall survival. The major flaw in the analysis of the data in this way is that MRD eradication was the aim of therapy for all patients but was achieved in only a variable proportion, often a relatively small minority. It is possible that the patients who achieved MRD-negativity were going to survive longer regardless of the therapy given, and that effectively MRD eradication is only a prognostic marker. This seems an implausible explanation given the excellent survival of MRD-negative patients, even in relapsed and fludarabine-refractory patients. In addition there is a single small randomized trial of alemtuzumab consolidation compared to observation alone after initial therapy with fludarabine-based regimens which demonstrated a significantly prolonged progression-free survival for the alemtuzumab patients, among whom five of 11 achieved MRD negativity.41 It therefore seems likely that eradication of detectable MRD in this context results in at least prolonged progression-free survival, although larger randomized trials are required. Therefore the correlation between MRD-negative status and improved survival requires validation prospectively in randomized clinical trials. THERAPEUTIC MODALITIES ACHIEVING MRD NEGATIVITY MRD eradication in CLL can be achieved with a variety of therapeutic interventions. These include combination cytotoxic chemotherapy, monoclonal antibodies, combinations of monoclonal antibodies and cytotoxic agents, and autologous or allogeneic stem-cell transplantation. MRD eradication after cytotoxic chemotherapy Conventional therapy for CLL with cytotoxic agents only rarely achieves a CR. CR rates of 10% and 25% are quoted after first-line therapy with chlorambucil and single-agent fludarabine respectively.42 MRD assessment was not carried out in most early series. With the introduction of newer, more effective agents – namely purine-analoguecontaining combinations – higher CR rates are achieved, and a proportion of these patients achieve MRD negativity. In a series of 34 previously untreated patients treated with fludarabine and cyclophosphamide, the CR rate was 35%, with 8% of the complete responders showing persistence of CD5-positive lymphocytes in the marrow.32 The combination of fludarabine, cyclophosphamide and mitoxanthrone achieved MRD eradication in 10/60 (17%) of patients with relapsed CLL33 and 17/64 (27%) of previously untreated patients.43 The relapsed/refractory patients and the previously untreated patients who achieve MRD eradication following FCM have a better overall survival than those patients who have detectable MRD at the end of therapy. Monoclonal antibodies and MRD eradication The uses of monoclonal antibodies, in particular rituximab and alemtuzumab, have been reported extensively in the treatment of CLL. Rituximab used as a single agent at the conventional dose (375 mg/m2 weekly for four doses) in the treatment of CLL achieves only partial responses of short duration.44,45 Even when higher doses of rituximab are employed, such as 375 mg/m2 three times a week for 12 doses46, or with doses of up to 2250 mg/m2,47 complete responses are still

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extremely uncommon. It appears that the main role of rituximab will be in combination with cytotoxic chemotherapy. Rituximab, fludarabine and cyclophosphamide (FCR) combination used in relapsed CLL led to molecular remissions in 32% of complete responders.35The same regimen used in previously untreated CLL produced better results, with two thirds of responding patients attaining MRD eradication as assessed by twocolour flow cytometry.34 Follow-up in these series is short, and a correlation between MRD eradication and better survival has not been reported. In contrast to rituximab, the monoclonal antibody alemtuzumab (Campath or antiCD52) used as a single agent is more effective in achieving responses of better quality, including MRD eradication. Alemtuzumab is currently licensed for the treatment of refractory CLL, and most of the published series describe outcomes in heavily pretreated patients. In the majority of these studies formal MRD assessment was not carried out. The two largest groups with available MRD data are the series reported by Keating et al in 2002 and Moreton et al in 2005. In the former series 93 relapsed patients were treated with alemtuzumab with an overall response rate of 33% and a CR rate of 2%. Peripheral-blood flow cytometry showed clearance of CD19/CD5 co-expressing cells. Marrow biopsies were examined, but MRD status was not assessed.48 The series reported by Moreton et al36 described 91 previously treated patients who received alemtuzumab to maximum response. The overall response rate was 55% with a CR rate of 36%. MRD assessment of the marrow was carried out for the patients in this series using a highly sensitive four-colour flow-cytometry assay (MRD flow);18/91 patients achieved MRD eradication with alemtuzumab, including six patients who were classified as partial responders by NCI-WG criteria due to persisting therapy-induced cytopenias. Overall survival and treatment-free survival were significantly longer for MRD-negative patients compared to MRD-positive responders and non-responders (Figure 3). The reports of eradication of detectable MRD with alemtuzumab as a single agent in relapsed/refractory CLL led to the use of alemtuzumab where MRD eradication was more likely. The observation that patients with bulky lymph-node disease do not respond well to alemtuzumab suggests that one role for the drug may well be in the consolidation of remissions following conventional chemotherapy in an attempt to achieve MRD eradication. Several groups have studied alemtuzumab in this setting (Table 1). In the series by Montillo et al, 34 patients received alemtuzumab consolidation after treatment with fludarabine-based regimens. The CR rate improved from 35% to 79.4%. MRD eradication as assessed by consensus PCR was achieved in 56% of this group.49 In the series reported by O’Brien et al, MRD eradication was achieved in 38% of patients treated with alemtuzumab following chemotherapy.50 In the series reported by Wendtner et al, 11 patients were treated with alemtuzumab consolidation following fludarabine alone or in combination with cyclophosphamide. The trial accrued a small number of patients and had to be terminated prematurely due to excessive toxicity. MRD status was evaluable for six of the 11 patients; five of the six patients showed MRD negativity as measured by ASO-PCR. The numbers are small, but there was a significant difference in progression-free survival in favour of the alemtuzumab-treated group.41 MRD eradication following stem cell transplantation There have been numerous reports of patients receiving high-dose therapy and rescue with either autologous or allogeneic stem cells to treat CLL. It is clear that the patients who have been treated with stem-cell transplantation (SCT) are highly selected in that

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Figure 3. The median survival for minimal residual disease (MRD) negative responders has not been reached. MRD-positive patients with complete response (CR) had a median survival of 41 months (95% CI, 15.94–66.06 months). Patients with partial response (PR) had a median survival of 30 months (95% CI, 0–62.89 months). Non-responders (NR) had a median survival of 15 months (95% CI, 9.73–20.27 months). Reprinted from Moreton et al (Journal of Clinical Oncology 2005; 23: 2971–2979) with permission the American Society of Clinical Oncology.

they are usually younger and previously fit. MRD assessment using a variety of methods has been reported in a few of these series. Autologous SCT An important factor to consider when analysing the results of autologous SCT in CLL is that only a proportion of patients will respond adequately and then have sufficient

Table 1. Summary of studies assessing the role of alemtuzumab consolidation after chemotherapy. Route

Dose

Study

Median interval from chemotherapy to maintenance

O’Brien et al50 (n ¼ 24) O’Brien et al50 (n ¼ 17) Rai et al55 Rai et al56 Wendtner et al41

6 (1e40) months 6 (1e40) months w2 months w2 months 67 (45e90) days

IV IV IV SQ IV

10 mg 30 mg 30 mg 30 mg 30 mg

Montillo et al49 (n ¼ 35)

At least 8 weeks after fludarabine

SQ

10 mg TIW

TIW TIW TIW TIW TIW

Response

39% improved response 56% improved response (92% OR; 42% CR) 44% improved response 45% improved response; Improved PFS 44% improved response

IV, intravenous; SQ, subcutaneous; TIW, thrice weekly; OR, overall response; CR, complete remission.

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stem cells harvested to proceed with the transplant. In the MRC CLL pilot study of autologous SCT as front-line therapy after initial therapy with fludarabine, only 65 out of 115 patients (56.5%) who commenced fludarabine within the study proceeded to SCT.40 Relapse after autologous SCT appears to be inevitable51, and the detection of MRD strongly predicts impending clinical relapse.10,15,16,38 The series reported by Milligan et al showed that 16/20 (80%) evaluable patients achieved MRD negativity in the first 6 months following transplantation, as assessed by low sensitivity (consensus primer) PCR. The presence of detectable disease was highly predictive of eventual clinical disease recurrence in this series.40 If a patient is destined to become MRDnegative after autologous SCT this occurs immediately after the SCT, and there is no further fall in the level of detectable disease thereafter.52 Earlier series of CLL patients receiving autologous SCT showed MRD eradication rates of 64–75%.37,53 Lower MRD eradication rates after autologous transplantation have been described for highrisk unmutated CLL – 23% in one series – and these responses were not durable.52 More recently Moreno et al22 reported a series of 40 patients (25 autografts and 15 allografts) with evaluation of MRD at several time points using four-colour flow cytometry, consensus primer PCR and quantitative PCR; 15/22 evaluable patients were MRD-negative at 3–6 months following autograft. There was a significantly lower risk of relapse and a trend towards better survival for MRD-negative patients. In this same series and among MRD-positive patients the level of MRD as measured by qPCR and MRD flow cytometry showed a statistically significant survival benefit for those patients who had lower levels of MRD. Allogeneic SCT While autologous transplantation in CLL is usually performed with the intention of disease control, allogeneic SCT is the only treatment option with curative potential. MRD eradication following allogeneic SCT is well documented. The kinetics of MRD eradication after allo-SCT follows a different course from that following autologous SCT. Persistence of MRD early in the post-transplant period is not associated with an increase in the risk of relapse, and delayed clearance of MRD has been described.38,52 Complete remissions have been described up to 2 years after alloSCT.54 In the series reported by Ritgen et al, seven of nine patients achieved durable MRD eradication after non-myeloablative SCT from HLA-identical donors.52 In the series reported by Moreno et al, MRD detection early (3–6 months) after transplant did not predict clinical relapse. Six patients showed a progressive fall in MRD levels, with five of these becoming MRD-negative within the first year and the remaining patient showing intermittent MRD negativity. Of the six patients who remained MRD-positive after allograft, four had stable MRD levels on long-term follow-up (6–84 months) with no clinical relapses. The remaining two patients showed a progressive rise in MRD level, and one of these relapsed clinically at 53 months post-transplant, while the other patient was in clinical CR more than 60 months after transplant.22 MAINTAINING MRD-NEGATIVE REMISSIONS It is clear that, with the exception of allogeneic SCT, when MRD is detectable following therapy the level of disease will inexorably increase. The time from MRD relapse to clinical relapse is directly related to the rate at which the detectable CLL burden increases with time. This can be used to predict when a patient might progress to further therapy. The kinetics of relapse suggest that if a tolerable and effective strategy

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can be devised, then CLL may be a good candidate for maintenance therapy. At present no such approach has been developed, but there are several groups working on such strategies.

CONCLUSION A substantial body of evidence is currently available to support the concept that MRD eradication in CLL is associated with a longer progression-free survival and possibly better overall survival. This finding has frequently been reported irrespective of the treatment used or the method utilized for MRD assessment. The combination of chemotherapy with rituximab or alemtuzumab, alemtuzumab in consolidation following conventional therapy, and the safer application of allogeneic SCT make MRD eradication in CLL a reasonable goal of therapy for many patients. It is now necessary to confirm the apparent dramatic effect of MRD eradication on survival in CLL in prospective randomized clinical trials.

Practice points  criteria for response assessment need to be reviewed to incorporate MRD status  standardization of MRD detection techniques and reporting is crucial to allow comparison of results of future clinical trials

Research agenda  clinical trials are needed to answer the questions of whether MRD eradication is a desirable endpoint at front-line therapy and what the ideal treatment is for attaining this endpoint  what is the role of consolidation therapy to convert MRD-positive remissions to MRD-negative remissions?  is relapse at MRD level an indication for re-treatment?  Is there a role for maintenance therapy?

CONFLICT OF INTEREST STATEMENT Dr Hazem A. Sayala: research supported by unrestricted educational grant from Schering Healthcare Ltd. Dr Andy C. Rawstron: no relevant conflict of interest. Dr Peter Hillmen: unrestricted educational research grant from Schering AG. Speaker’s fees Berlex and Schering AG.

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