Remission maintenance treatment options in chronic lymphocytic leukemia

Accepted Manuscript Anti-Tumour Treatment Remission Maintenance treatment options in chronic lymphocytic leukemia Alexander Egle, Lisa Pleyer, Thomas Melchardt, Tanja Nicole Hartmann, Richard Greil PII: DOI: Reference:

S0305-7372(18)30138-5 https://doi.org/10.1016/j.ctrv.2018.08.003 YCTRV 1807

To appear in:

Cancer Treatment Reviews Cancer Treatment Reviews

Received Date: Revised Date: Accepted Date:

4 June 2018 2 August 2018 7 August 2018

Please cite this article as: Egle, A., Pleyer, L., Melchardt, T., Nicole Hartmann, T., Greil, R., Remission Maintenance treatment options in chronic lymphocytic leukemia, Cancer Treatment Reviews Cancer Treatment Reviews (2018), doi: https://doi.org/10.1016/j.ctrv.2018.08.003

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Remission Maintenance treatment options in chronic lymphocytic leukemia

Alexander Egle, Lisa Pleyer, Thomas Melchardt, Tanja Nicole Hartmann, and Richard Greil Department of Internal Medicine III with Hematology, Medical Oncology, Hemostaseology, Infectious Disease, Rheumatology, Oncologic Center, Laboratory for Immunological and Molecular Cancer Research, Paracelsus Medical University Salzburg Salzburg Cancer Research Institute and Cancer Cluster Salzburg

Corresponding Author: Prof. Dr. Richard Greil.

Prof Richard Greil, Department of Internal Medicine III with Hematology, Medical Oncology, Hemostaseology, Infectious Disease, Rheumatology, Oncologic Center, Laboratory for Immunological and Molecular Cancer Research, Paracelsus Medical University Salzburg Salzburg Cancer Research Institute and Cancer Cluster Salzburg Müllner Hauptstraße 48 A-5020 Salzburg, Austria [email protected]

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Remission Maintenance treatment options in chronic lymphocytic leukemia

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Abstract Chronic lymphocytic leukemia (CLL) treatment has come a long way in the last two decades, producing increases in tumor control to the point of generating sizeable numbers of patients with undetectable minimal residual disease and creating overall survival benefits in randomized comparisons. Most of this has been achieved by limited-term treatment approaches including chemotherapeutic and immune-therapeutic drugs. More recently, novel therapies targeting signaling pathways essential for the survival of the neoplastic clones have opened avenues that provide disease control in long-term treatment designs, mostly without producing deep remissions. In this disease, where current treatments are largely unable to effect a cure, prolonged therapy designs using maintenance approaches are explored and 5 randomized studies of maintenance have recently been published. This review shall summarize available results from a systematic literature review in a clinical context and outline basic biology principles that should be heeded in this regard. Keywords Chronic lymphocytic leukemia, maintenance, anti-CD20 antibodies, kinase-inhibitors, lenalidomide, long-term treatment Current state of CLL treatment The introduction of combination chemotherapy via the addition of antibody strategies and the advent of novel targeted drugs, recently licensed (ofatumumab, obinutuzumab, ibrutinib, Idelalisib and venetoclax - in order of licensing), have significantly changed the landscape in CLL. Several of these approaches have yielded overall survival benefits in randomized trials in different settings [1-3], suggesting real progress. Adding to the results from randomized trials, the community has tried to determine the effect on the improvements for the patients by comparing outcomes between different generations of trials or by using cohort designs. Indeed, there is some evidence that the recent intensification of therapy has changed the natural course of CLL [4], reporting a significant 60 to 67% decrease in mortality of patients with advanced CLL stages and age <70a, when comparing a patient cohort from 1980 to 1996 with one from 1997 to 2004. However, this improvement was not observed for older CLL patients and early stages and at the time included only 13% of patients getting rituximab-containing treatment. Clearly, anti-CD20 antibody containing therapies have improved survival when combined with intensive chemotherapy backbones in young and fit patients [3] or with weak chemotherapy in unfit or elderly patients [2]. Progression-free and overall survival benefits observed with novel drugs (unfortunately often against very weak comparators) have been reported in frontline treatment and in relapsed/refractory cohorts [1, 5, 6]. Collectively, the data suggest that important advances in the treatment of CLL have

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materialized. However, chemoimmunotherapy approaches still do not result in cure in the overwhelming majority of patients, with steady rates of relapses observed, and thus far, novel drugs do not appear to solve the problem of steady rates of relapse, at least not in relapsed/refractory populations, as evidenced by the necessity for treatment options after exhaustion of kinase inhibitors, a situation that remains a challenge [7-9]. Due to the limited follow up of frontline therapy trials using novel drugs, it is not yet clear, whether cure may be available in such settings. Conclusively, with the notable exception of CLL with mutated IgVH and functional p53 treated with FCR, where there is a valid argument that there may be a fraction that might be cured [10], CLL remains an incurable disease. Even when accounting for the discussion of curability of IgVH mutated CLL[10-12], incurability is indeed currently true for a majority of CLL patients since, 1) only a minority of patients is eligible for FCR treatment (e.g. only 10% of patients in the CLL10 trial[13] were over 70, which is the current median age of CLL patients at diagnosis[14]), 2) of these only a fraction of about 40% is expected to have a mutated IgVH gene[15] and 3) a potential cure rate in the mutated cohort would be estimated around 70%[10, 12]. Thus, a provisional cure would be attainable in less than 10% of CLL patients with a conservative estimate of around 30-35% being eligible for FCR treatment. With an incurable disease at hand the question remains whether an early treatment may improve outcomes. Next to the fact that a relevant number of CLL patients managed in an expecting fashion will never need treatment, the historic observation was that early treatment did not change overall survival times[16] and this is reflected in current treatment guidelines. With the advent of tangible overall survival benefits from intensive chemoimmunotherapy (with FCR) this concept was revisited for a high-risk population with low probability of long term treatment-free survival in the German/French CLL7 trial [17]. However, with the currently available follow up there is no overall survival (OS) difference associated with early FCR treatment of high risk CLL patients compared to the randomized controls that were treated at the time of standard iwCLL/NCI WG treatment indications. Therefore, even though follow up for this trial is short, the current standard of defining treatment indications remains in place. Once a treatment indication is established, current guidelines [18-20] suggest a stratification of patients according to genetic risk profiles (mainly regarding p53 dysfunction) and fitness for intensive chemoimmunotherapy. Notably, the current guidelines have very limited information on the role of maintenance strategies, ranging from the option to use ofatumumab maintenance after 2nd line treatment [20] to the statement that maintenance cannot be generally recommended, but may have some benefit for patients with a high risk of relapse [18, 19]. Given, that a number of randomized trials addressing maintenance strategies have recently been reported, this review aims to summarize the available data and to provide discussion as

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to what the role of maintenance may be in the current treatment algorithms and what the biologic basis for such strategies could be.

Goals of treatment The ultimate goal of cancer treatment is cure, and in the absence of that option, a prolonged life in high quality is the next reasonable goal. Thus, overall survival (OS) is a very important endpoint. However, in chronic disease with long OS and availability of effective treatments, OS may turn out to be an impossible goal to test due to crossover effects and/or the required long follow up time. In an environment where the options change much quicker than even the most optimistic turn-around times for OS results, OS becomes a highly impractical endpoint. Thus, surrogates have been developed to more quickly assess the impact of a therapy for patients. In a meta-analysis, progression free survival (PFS) has been shown to be a surrogate for OS in the era of chemotherapy and chemoimmunotherapy [21], but this was only true for relapsed/refractory disease and not in 1 st line trials (likely due to effective salvage options and limited follow-up). With novel drugs on the market, the performance of PFS to predict OS after 1st line treatment can only have worsened. However, it remains clear that a very short PFS after 1 st line treatment points to a severe prognosis [22], and this is reflected in current guidelines that advise to select different approaches for treatment of relapse according to the primary PFS (greater than 24 months or not). A more immediate parameter that has shown excellent power to predict PFS outcomes after chemo-, immuneand chemoimmunotherapy regimens is the measurement of minimal residual disease (MRD). MRD outperformed clinical response assessment across a large number of trials [10, 23-27] and was on its way to be recognized as an accepted surrogate marker by registration authorities [28, 29]. As such, residual tumor mass is a very powerful parameter when assessing the potential for tumor control of a certain treatment approach (for an outline see Figure 1 A) and this is mirrored across many trials. From this perspective, the different rates of MRD control achievable by different treatments allows for a rapid comparison of their potency (see below). However, more recently, the advent of the novel kinase inhibitors with their unique phenotype of response (i.e. redistribution of CLL cells from nodal compartments into the peripheral blood [30, 31]) means that for these drugs the response depth as measured by classical clinical staging or MRD measurements have relatively effectively been uncoupled from the observed PFS. Thus, to date there is no single optimal clinical or laboratory endpoint with which to judge the success of treatment approaches in CLL. For the comparison of drugs with different effects on CLL distribution and usability of MRD assessments, trials will largely have to rely on PFS measurements (or novel concepts that are yet to be developed). Importantly, for the comparison of different treatment approaches when at least one of them is given over a prolonged period of time it may be necessary to

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develop some mode of assessing “resistance-free survival”, i.e. the time from starting one type of therapy until a resistance to that treatment is observed. This can either result from multiple lines of the same therapy or from continuous treatment with one treatment concept (i.e. continuous treatment or an induction/maintenance approach). Such a parameter may ideally be connected to a measurement of clonal evolution – even though that type of parameter may be very difficult to define and standardize. Also, both clinical long-term followup designs to include second failure and molecular definitions of failure may also meet with financial limitations in trial sponsoring that may be prohibitive. Remission depth from different regimens When approaching remission maintenance after any limited-term induction therapy, it is important to assess the quality of remission achieved before starting the maintenance approach. Concentrating on first line treatment and on chemoimmunotherapy approaches, where valid MRD data were presented [2, 13] and data from novel drugs [1], where practically no MRD control is reported from first line experiences (Table 1), it is clear that induction results vary widely in terms of efficacy between regimens and, more indirectly between different patient cohorts with different fitness. Moving to pretreated patient sets, the homogeneity of datasets is even smaller and the influence of patient selection becomes as large as that of treatments. We thus abstain from listing those values and only mention that, next to datasets from chemoimmunotherapy (e.g. [32, 33]), there was a report of meaningful rates of MRD control in a group of relapsed/refractory CLL patients treated with venetoclax plus rituximab and this trial also suggested that relapse-free intervals could be observed in MRD-negative patients that had discontinued treatment [34]. However, follow-up for these patients is still relatively short and it is not completely clear whether venetoclax derived MRD negative states are similar in their power to predict relapse outcomes to those observed after chemoimmunotherapy. One important question is whether a maintenance approach should be considered in patients with high quality remissions. Looking at the data from FCR treated patients, however, there seems to be a relatively steady rate of relapses even in the group of patients with the best quality of response (i.e. an MRD-“negative” state at 10-4) [24]. However, follow-up is limited and a different view may develop for the mentioned exception of patients with mutated IgVH genes. On the other hand, very deep remission after induction may indicate a specific sensitivity of the disease to components of the induction regimen and encourage the use of such components in a maintenance approach. Such a situation of deep remission may even be most conductive to therapeutically maintain CLL cells below a certain threshold for longer and delay relapse even more than would be possible in lesser remission states (as

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conceptually outlined in Figure 1B). However, very long follow up may be necessary to prove such a hypothesis.

Maintenance treatment in CLL

In a majority of treatment situations therapeutic interventions aiming at an improvement of response after an induction treatment (a situation that may be called “consolidation treatment”) or a stabilization of response (“remission maintenance treatment”) are strategies worth contemplating. In practice, these two options can rarely be separated effectively in a situation where neither approach creates cures for conceptual reasons. We will thus refer to all sorts of post-remission treatments as “maintenance” for the purpose of this review. An ideal maintenance approach follows a non-curative treatment with measured or suspected residual disease and fulfils a number of criteria that will be discussed throughout the further review: 1) efficacy regarding tumor control, 2) a high resistance barrier, 3) a low rate of severe side-effects, and 4) an ease of application The biology underlying these restraints will be revisited towards the end of this review, but first we want to use the clinical database to illustrate some points regarding the development of maintenance approaches in a clinical context. To this end, we performed a systematic review of the literature using the following search terms: “chronic lymphocytic leukemia”, “chronic lymphocytic leukemia”, CLL, and “maintenance”. All randomized clinical trials presenting results were included in the presentation (for an overview of maintenance trial results and reported effects of subgroups consult Tables 2 and 3). Regarding Phase 1 and 2 clinical experiences with maintenance strategies, a choice of trials is presented, to generate perspective “Failed attempts” Early on, chlorambucil was used as a long-term treatment with trials using up to 12 months of chlorambucil treatment [35-39]. While the toxicity was reported to be relatively low and the oral regimen was relatively easy to apply, the efficacy was limited generating a very low number of complete remissions (CRs) even with prolonged treatment and close to 50% of patients were progressive within the 1st year (i.e. during projected treatment), suggesting a relatively rapid development of resistance. In general, prolonged chemotherapy, similar to the “treat until progression” strategies prevalent in medical oncology, has not been explored very much, due to the necessity of frequent i.v. application, side effects and the notion that chemotherapy selects for high-risk features such as dysfunctions along the DNA damage pathways.

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As antibody strategies became available, these were explored to counteract these problems. Alemtuzumab was used as a consolidation/maintenance approach and showed a number of interesting features: there was a clear advantage of the treatment with regards to disease control for patients with del17p high risk cytogenetics and the subcutaneous formulation made for a relatively easy administration [40, 41]. Indeed, long-term analysis of a phase 3 Alemtuzumab maintenance trial showed significant improvement of survival endpoints [42]. However, severe toxicities (specifically opportunistic infections) rendered these advantages moot [43-46]. Trials exploring lower dose of MRD-targeted Alemtuzumab schedules, promising better tolerability, were initiated by some groups (e.g. [47]), but did not lead to large phase 3 trials. The largest experience with antibody maintenance, however, stems from anti-CD20 antibody treatment and is outlined in the following paragraphs. CD20 Antibodies in Maintenance treatment Following the establishment of a maintenance paradigm in other indolent lymphoma entities [48], we have now seen full publication of four major randomized trials exploring anti-CD20 antibodies[49-53]. All four trials reported on maintenance treatment after chemoimmunotherapy remission induction in CLL and prompted this review. Initial observations of prolonged use of rituximab in CLL stems from a phase 2 trial by Hainsworth and colleagues [54]. Rituximab induction and four weekly courses of rituximab in 6-monthly intervals as maintenance for 2 years resulted in a median PFS of 18.6 months in this first-line population. However, induction therapy using rituximab as a monotherapy has never been established as frontline standard and may not be sufficient to induce remission qualities that are “worth maintaining”. Rituximab maintenance after R-fludarabine induction [55] significantly improved response duration of MRD positive patients with four monthly rituximab cycles and maintenance for 12 monthly doses at a low rituximab dose (150mg/m2). However, patient numbers were very small and the comparator was not randomized, resulting in detectable imbalances between treatment groups. A Spanish report on rituximab maintenance after intense chemoimmunotherapy induction with R-FMC [56] suggested a potential for improvement of clinical and MRD responses by addition of maintenance rituximab, but toxicities observed during the maintenance phase were significant. It has to be kept in mind, however, that chemoimmunotherapy induction alone leads to a significant rate of late infections due to the sometimes prolonged and severe immunosuppression. Indeed, during the observation phase of late events after FCR induction in the MD Anderson cohort, severe infections and death in up to 15% of patients were observed in the 2 years following remission induction [57-59]. Thus, randomized trials with the capacity to differentiate maintenance-related toxicities and toxicities from the “hangover” from the chemoimmunotherapy induction are clearly required (see also Tables 2 and 3). A better

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understanding of a trade-off between remission depth after an induction treatment and the associated toxicities, together with a clear knowledge of the potency of potential maintenance regimens, may allow to tailor the toxicity of the induction regimen to the needs of the specific patient population. This may decrease toxicities, while preserving longer-term disease control by relying on the maintenance approach. To this end, an Italian trial reported results with rituximab maintenance after chlorambucil and rituximab induction [60]. In this randomized phase 2 trial the primary endpoint was overall response rates after induction, but the trial also reported on 66 patients with response after induction treatment that had been randomized to either observation or rituximab maintenance (365mg/m2 every 8 weeks for 12 doses). With an observation time of 34.9 months the study reported a trend for longer PFS in the R maintenance arm (p=0.07) and a similar trend for an increase in adverse events, but the low number of patients in each arm precluded firm conclusions on these secondary endpoints.

We recently reported our experience with rituximab maintenance treatment after chemoimmunotherapy induction in a randomized trial powered to detect a PFS benefit in our AGMT CLL8a Mabtenance trial [51]. Patients were recruited after achieving any response to least 4 cycles of rituximab-containing induction in 1st or 2nd line of treatment and entered the study after complete analysis of induction response including MRD assessments. The starting point for the randomized maintenance treatment in our trial was an overall relatively high quality of remissions, which could be expected from the relatively intense induction regimens (mostly FCR and BR) used in 1st or 2nd line. Overall 56% of patients had a clinical CR or CRi and the same rate of 56% of evaluable patients had an MRD-negative state from peripheral blood (available in 92% of participants). Notably, patients’ responses can fall into discordant groups - i.e. MRD-positive CRs as well as MRD-negative PRs - strongly suggesting that the two parameters MRD and CR are linked, but not identical [16]. Overall, MRD appeared to have a higher potential of predicting outcome than clinical staging. In comparison with other trials form 1 st line cohorts, and considering the presence of 20% second-line patients in the cohort, the induction results seem to indicate a high degree of tumor control before start of the maintenance in our trial. Still, with regards to the primary endpoint of PFS rituximab maintenance led to a 50% risk reduction for relapse across the trial, with no significant difference between subgroups when analyzed by Forest plot (see also Table 3 for subgroup comparisons between trials). The only two groups with relevantly lower risk reduction were the patients that were MRD-negative in PB or BM before maintenance. However, in these two groups the confidence intervals were very high as expected from the scarcity of progression events in these groups with the currently limited follow up. It thus remains unclear whether rituximab maintenance can add relevantly to the

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long-term CLL control in these patients groups. However, in the group of patients with MRDnegativity measured from peripheral blood, the median risk reduction for PFS was still 25% (hazard ratio (HR) 0.75) so there is a relevant option that such a population may turn out to have benefitted from maintenance with longer follow up. Indeed, in the absence of curative potential, those patients with a very good initial control may benefit most from the addition of maintenance strategies, since they may be selected for a good response to the rituximabcontaining induction and thus may have equally good response to the maintenance (this is discussed in more depth further below). Furthermore, one may assume that the necessary trough levels to control a very minimal tumor mass may in fact be lower than those for a larger remaining tumor mass (due to a possible “sink” effect). Importantly, our trial reports randomized toxicity data for an intensively treated cohort. We found a clear increase in severe adverse events driven by infections, with no significant difference in other categories of serious adverse events for the maintenance group. However, while severe infection toxicities of Grade 4 and 5 were not more common in the maintenance arm, infections of grade 3 and of any grade were more common in the rituximab maintenance arm. Importantly, even the observation arm reported 50% of infections of all grades and 11% G3-5 infections during the observation period, underlining the importance of late induction-related toxicities as mentioned above [57-59]. Overall infections increased from 50% to 66% in the rituximab maintenance arm and about two thirds of the difference resulted from grade 3 events. However, this increase in infections proved manageable and an analysis of an EFS definition including infection events showed a sustained benefit for the maintenance group [51].

A second randomized trial testing rituximab maintenance has been reported most recently by the French group [49]. The trial recruited patients older than 65 years without del17 in 1st line of treatment to receive an abbreviated schedule of 4 cycles of FCR followed by a randomization to either observation or relatively intensive maintenance with 500mg/m2 rituximab in two-monthly intervals. From 542 patients enrolled, 409 were randomized to receive maintenance or observation. PFS was reported to be significantly improved with a HR of 0.55 and no OS benefit was reported with a median FU of 47.7 months, similar to the other two trials. Neutropenia and grade G3 or 4 infections were reported significantly more often in the maintenance group (19% vs 10% infections when comparing maintenance with observation). Interestingly this trial, reports a significant benefit for patients also in patients achieving MRD-negativity (in bone marrow AND peripheral blood) and confirms accentuated benefits for patients with high risk cytogenetics (i.e. del11q) and unmutated IgVH.

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A third rituximab maintenance trial mainly conducted by the Polish PALG recruited patients after a cladribine cyclophosphamide rituximab induction in 1st line of treatment[52]. Patients were randomized to 2 years of 3-monthly rituximab maintenance or observation. This trial had been stopped prematurely, due to low recruitment and randomized only 66 patients, only about half of what had originally been intended. Next to the premature recruitment stop this was also due to exclusion of 32% of patients after induction, mostly due to hematopoietic toxicities remaining from the induction regimen. However, this trial also reported a significantly longer PFS with a HR of 0.41. Similar to the other trials, there was an increase in overall adverse events in the maintenance arm, but this was mostly driven by reported neutropenia and did not translate into a major difference in reported infections. Finally, a Russian trial investigating rituximab maintenance after FC or FCR induction has been presented in abstract form at a Russian meeting in 2010 [61], but no international presentation followed. The abstract stated a significant PFS benefit for rituximab maintenance applied over 2 years when compared with observation, but does not allow any insight into baseline characteristics, including the line of treatment of induction.

Similar results, to those observed with rituximab, were reported by van Oers et al in a trial exploring ofatumumab maintenance after induction in 2 nd or 3rd line in the PROLONG trial [53]. The induction regimes were less homogenous as can be expected for later lines of treatment, but importantly included 20% of patients that had received no antibody in their last induction, likely increasing the anticipated effect of the intervention. Only 19% reported a CR after induction and post-induction baseline MRD assessments showed 15% of CR (with 33% missing the assessment overall). PFS was improved with a hazard ratio of exactly 0.5 and subgroup analyses, showed beneficial hazard ratios across all subgroups. Interestingly, no difference was reported for the distinction between patients entering maintenance treatment in MRD positive and negative states. This may be due to the expectedly higher rate of progression events from MRD-negative patients in 2nd and 3rd line of treatment. In parallel to the rituximab maintenance trial overall toxicities were increased in the ofatumumab maintenance group. Specifically grade 3 neutropenia as well as an increase in grade 3-4 infections from 8% to 13% in the maintenance group was reported.

Both the AGMT Mabtenance and the PROLONG trial [51, 53] showed a delay in time to next treatment, but with current follow up no overall survival benefit was observed in any of the trials.

Overall, despite the heterogeneity of the patient samples, the trials report remarkably similar effectivity of anti-CD20 directed maintenance for the prolongation of remission in CLL

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patients that had responded to induction chemo(immuno)therapy in 1st through 3rd line for treatment. Indeed, the magnitude of a 40-50% risk reduction in these settings compares well to the results of adding CD20 antibodies to FC induction regimens in 1 st or 2nd line[3, 62]. The added effect observed “on top” of chemoimmunotherapy induction regimens suggest that the benefit of the antibody maintenance is indeed a prolonged feature and is observed even if effective antibody induction has been used. In addition, the effectivity across different remission depths and treatment lines, combined with a relatively modest and manageable increase in toxicities, suggest that CD20 maintenance may be used to improve outcomes also for groups that may not be able to tolerate intense remission induction. Importantly, the presence of positive effects observed in high risk cytogenetic and unmutated IgVH subgroups may point to an important role of antibody maintenance for these groups, even if overall benefits seem slightly smaller in the IgVH unmutated subgroup.

In contrast with the situation in non-transformed CLL, a phase 2 trial exploring ofatumumabCHOP followed by ofatumumab maintenance for Richter’s Syndrome concluded that there was no relevant benefit over R-CHOP without maintenance in indirect comparison [63, 64]. Lenalidomide Lenalidomide treatment in CLL has been explored in a number of trials that used treatment until progression either in monotherapy or in combination with limited courses of rituximab [65-70], but no randomized datasets of induction treatment powered for outcome measures and comparing lenalidomide with other approaches are currently available. Still, available phase 2 data point to relevant tolerability problems with prolonged lenalidomide treatment with up to 50% discontinuation due to toxicity [67, 69, 70]. On the other hand some patients have been reported to have very durable remissions with prolonged lenalidomide treatment [69]. Thus, two major obstacles for further development seem to be the lack of a well-defined optimal dose for maintenance and the lack of biomarkers for identifying responders or toxic events early on. Three randomized lenalidomide trials reported interim results at the ASH meeting 2016, two of which are now fully published. For a lenalidomide maintenance trial, presented by the German CLL study group (CLL M1) [71, 72], a very specific design had been chosen in order to focus on pre-selecting patients at highest risk for early relapse. This was achieved by using a post-induction definition of high relapse risk, as had previously been defined in an FCR treated cohort (i.e. patients with response but limited reduction of measured MRD (>102) or molecular risk factors as del17p or p53 mutation or unmutated IgVH) [73]. Thus the trial recruited patients from this high risk group after at least 4 cycles of FCR induction in 1 st line of treatment, by screening 468 patients to randomize 89 patients in a 2:1 ratio to either

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lenalidomide maintenance (n=60) or placebo (n=29). Lenalidomide was introduced in a ramp up dosing design from 5 mg to the target dose of 15 mg daily. Lenalidomide was given until progression or intolerance. Due to the inclusion after FCR induction the patients were relatively young and fit at the start of maintenance. About a third of patients had finished with high MRD levels and the rest showed intermediate MRD levels. The formal interim analysis was performed after 20% of the calculated PFS events and with 89 of the 200 planned patients. The PFS analysis showed a large and significant benefit for the lenalidomide treated arm with a hazard ratio of 0.168 after a median follow up of 17.9 months. Conversions to MRD negativity were only observed in the lenalidomide arm and OS was not different (with only 3 deaths in the trial so far). The toxicity was as expected with neutropenia rates (34% vs 6% G3/4 neutropenia) showing a large difference, and further relevant differences in skin and gastrointestinal toxicities and more reported disorders of the nervous and respiratory systems in the lenalidomide maintenance arm. Surprisingly, regarding the large difference in neutropenia rates, no overall difference in infection rates was reported, however, infections of grade 3 to 5 seemed more common in the lenalidomide maintenance arm when compared to placebo (15% vs 9%). No significant signal for an increase in thrombosis was observed. With the large difference in PFS, an independent data monitoring committee has recommended the unblinding of the trial and the continuation of lenalidomide treatment. Thus, we can expect more mature data in the future, but the relatively small numbers in the trial will remain. It is interesting to note that this trial effectively excluded lower risk patients from the maintenance. As mentioned above, it may, however, be that the group of patients starting maintenance with low or very low tumor burden may benefit most from such strategies. This may in part be due to the fact that these groups contain a selection of treatment sensitive clones. Some observations in multiple myeloma support such a concept: In a British analysis thalidomide maintenance showed a PFS benefit even in the patients that started maintenance in an MRD negative state [74]. Analogous results have been presented for rituximab maintenance in mantle cell lymphoma [75]. It may be interesting to see if lenalidomide maintenance retains its positive effect in less selected patient groups. In fact another lenalidomide maintenance trial with broader inclusion criteria (CONTINUUM NCT00774345), was also reported at ASH 2016 [76, 77]. Patients were included when achieving at least PR after a broad variety of permitted induction regimens in second line of CLL treatment. Randomization was 1:1 to lenalidomide or placebo and results on 315 patients were reported. Lenalidomide was also dosed in a ramp-up design starting from 2.5 mg to a maximum of 10 mg and dosed until progression or intolerance. A minority of about 10% had MRD negative CR after induction. The median PFS was significantly longer in the lenalidomide arm with a HR of 0.4, but there was no difference in OS at a median follow up

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of 31.5 months. Next to the still relatively short follow-up this may of course be due to effective salvage treatments and, indeed, the abstract reports that around 20% of relapsing patients had received Bruton’s tyrosine kinase (BTK) or phosphoinositide 3-kinase (PI3K) inhibitors. In line with the German report, neutropenia was the most common adverse event, but the rate of infections was not significantly different. Overall the achievable does was relatively small, with a median dose in maintenance of just below 5 mg lenalidomide/day. The study also reported that second-primary malignancy rates were not different between arms. Finally, an ASH abstract report from the ALLG/FILO group details early results from their CLL6 trial [78]. Patients with residual CLL, detected by either in clinical, radiological assessment or flow cytometry of blood and bone marrow, were included after 6 cycles of FCR. Maintenance was randomized open-label and 1:1 to 10mg lenalidomide daily vs. observation for 2 years. Data on 79 patients are reported with around 16 months of median follow up. Efficacy was only analyzed by observing MRD dynamics during therapy so far. In this analysis there was a clear trend for more MRD increase in the observation arm and more MRD decreases in the lenalidomide group. Data on PFS will have to be awaited. Overall it seems that lenalidomide may be another effective maintenance option in CLL, but the trials are only presented in abstract form so far and all seem to have suffered from relatively slow recruitment, leading to presentation of the data at well under recruitment targets. Whether this was due to complicated selection procedures, arising alternative options for the patients, fear of side effects on the part of clinicians or other reasons remains unclear.

Maintenance as part of treatment with novel kinase inhibitors. As mentioned above most of the trials using ibrutinib or idelalisib use a treat-until-progression approach, making it difficult to define induction and maintenance phases for these treatments. There are, however, two reported trials using combinations of ibrutinib or idelalisib with a bendamustin rituximab chemoimmunotherapy backbone in pretreated patients [79, 80]. In these trials the limited BR phase leaves the standard arm unmaintained, while patients in the experimental arms receive kinase inhibitor monotherapy after the end of their chemoimmunotherapy phase. The comparison is of course hampered by the fact, that the experimental arms receive the kinase inhibitors also during induction, but the results of PFS show that the curves barely separate during the induction phase, while quickly separating after the end of induction, suggesting that a “maintenance effect” of the kinase inhibitors may be important for the observed differences. It is however currently unclear whether the initial combination phase really provides a meaningful benefit over a kinase inhibitor monotherapy arm. Indeed, indirect comparisons of trials suggest that this might not be the case [81]. Thus, with regards to kinase inhibitors it may remain difficult to discern

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induction versus maintenance effects and no comparison with other options of remission maintenance might become available.

Considerations of a basic tumor biology of maintenance The duration of treatment of any kind is an important parameter from both, a tumor biologic and a clinical standpoint. The current and still valid clinical paradigm of short-term induction treatment in frontline CLL is thought to lead to a number of different potential evolutionary outcomes [82-84]: In a first step, an initial clone with intrinsic heterogeneity is reduced to achieve a remission state. This can be achieved by reducing all subclones proportionally (Figure 2A, Variant (V)1) or can already lead to a first process of clonal selection, with a proportionally lower rate of reduction or even growth of a primary resistant subclone (Figure 2A, V2), such that the clonal balance may be perturbed at the end of treatment. A second selection process starts after the end of treatment (the observation period) where clones enter a competition to re-colonize the microenvironment (Fig 2A V1a,b). This situation is called “competitive release” in literature on tumor evolution. While a chemotherapy resistant clone may be the most abundant at the end of treatment and is likely to be very competitive to enter this second stage of competition (Fig 1, V2a), this is not necessarily the case (Figure 1, V2b). Overall, these two selection procedures (treatment and release) create the evolutionary pressures that drive the relapsing clones. Mathematical models may be able to pinpoint the outgrowth of such resistant clones over time [85], but need to be validated in prospective observations. Technically, important help to define such clonal dynamics in the clinics may come from observations using circulating tumor DNA to track clonal evolution even in states of relative remission or low blood counts and in the absence of multiple biopsies [86]. In contrast to a limited induction treatment approach, any prolonged treatment will face the problem of the continued selection pressures on the clonal architecture and if there are available mechanisms for resistance development to the pressure, there will likely be a cumulative outgrowth of such clones over time. Such a scenario can be observed in longterm treatment of relapsed/refractory CLL with kinase inhibitors such, as ibrutinib (Figure 2B). Indeed, recent observations point to a high rate of patients where potentially pre-existing clones with specific BTK or PLCg2 mutations are selected in continued treatment with ibrutinib [87]. The analyses also show that such mutation can be detected many months before clinical progression occurs. Maintenance strategies after induction chemotherapies (e.g. limited numbers of cycles of chemoimmunotherapy) will suffer from a similar selection pressure during induction, but

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either collapse the selection pressure to a single pressure (if one continues with a component of the induction) or changes to a different (ideally non cross-reactive) selection pressure. As mentioned earlier, competitive release of the remaining sub-clones after the end of an induction treatment may lead to an option for specific subclones to effectively colonize the microenvironment and use this input for clonal expansion and dominance (Figure 2, V2b). This may be an important moment to help shape the clonal architecture and may contribute to the dominance of specific resistant phenotypes. Indeed, putting a brake on early clonal re-growth after the end of induction treatment using a maintenance approach may be especially effective if the situation at the end of treatment is conductive to rapid colonization of the microenvironment (Figure 2C). This may be the case due to a skewed immune system [88, 89], potential parallel reconstitution of the T cell compartment or a relevant change in the inflammatory milieu [90], all of which are partly hypothetical but conceivable. Importantly, it is currently unclear whether maintaining a selection pressure may be especially effective in small clone sizes, but theoretical considerations may point to a relationship of clone size with the option to select resistant clones [91], suggesting that deep remissions may be a preferable starting point for maintenance from an evolutionary perspective. It may thus be unwise to exclude patients with very good response qualities from maintenance strategies, as is also suggested by subgroup analyses form the French rituximab maintenance trial [49]. What may thus be the ideal maintenance option from this perspective? One aspect is the “height” of a resistance barrier posed by a specific agent, another is the danger of creating cross-resistance. The latter can be seen in chemotherapy regimens where the selection of a chemo-resistant clone in CLL (mostly by selecting for a defective DNA damage response) generally means that other chemotherapeutic options vanish at the same time (for a review see [92]). This phenomenon is observed in an exemplary fashion as cross-resistance to most chemotherapeutic options after a selection of a p53 mutated/deleted clone has occurred during chemotherapy (as an example [93]). Therefore, it may be wise to use modalities where a potential resistance does not impair our most effective armamentarium. This may also be illustrated by pointing to the initially reported very bad prognosis of patients relapsing under the highly effective kinase inhibitors [94]. While outcomes of patients after ibrutinib failure have since improved, due to the availability of additional novel drugs (such as venetoclax) [7, 8], the early reports of a median survival of only about 3 months [94] illustrated the disease characteristics once all available options had been exhausted. With regards to the resistance barrier, there is relatively little data that can be used to compare available treatment options. However, the fact that chemotherapeutic approaches can be made ineffective by deleting the function of a single gene (i.e. p53) or that resistance to ibrutinib may be sufficiently induced by mutations in only one or two collaborating genes

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[95], points to the fact that alternative approaches may be more effective in achieving the goal of prolonged disease control using prolonged treatment in the face of relatively low resistance barriers. Indeed, such mutations could be detected early during ibrutinib treatment in some cases and it was even suggested that they may be preexistent at the start of ibrutinib treatment [87, 96]. In addition, early clonal dynamics, driven by specific sets of mutations or pathway activations have been associated with earlier clinical progression on ibrutinib therapy [97], suggesting that the evolutionary capacity to overcome selection pressures imposed by ibrutinib may be greater than expected. Thus, approaching maintenance with CD20 antibodies or lenalidomide may be an interesting option, since no simple molecular mechanisms have been reported as resistance mechanism to either approach. Indeed, even after 20 years of rituximab use neither fully convincing clinical definitions nor molecular resistance pathways have been observed and in lymphoma many patients receive CD20 directed treatment after failing initial rituximab [98, 99]. In addition, many of the resistance mechanisms in discussion involve changes in effector mechanisms outside of the malignant clone that may potentially be reversible after an interval or with a change of the CD20 antibody and may thus not constitute long-term archived clonal resistance [100]. On the other hand there are few instances where the selective pressure induced by anti-CD20 treatment induced changes in phenotype of lymphoma (e.g. in marginal zone lymphoma) that were associated with inferior outcome and treatment resistance [101]. A similar point can be made regarding lenalidomide, where resistance mechanisms may be molecularly complex and thus more difficult to select for and where an additional action on the microenvironment may be important for the treatment effect [102]. Such restrictions on the levels of microenvironmental interactions of the clone may not be easily “solvable” by the clone itself with simple clonal evolution strategies and thus resistance phenotypes may either not be permanently archived and may also not necessarily damage a whole class of agents (as can be seen by the incomplete cross resistance between different Imids observed in myeloma) [103]. In both cases there is some argument that rituximab and lenalidomide may have higher resistance barriers than kinase inhibitors or chemotherapy approaches. However, it is possible that the presence of such a presumed higher resistance barrier may only be important in the relapsed/refractory setting, where the complexity of the clone has been increased by multiple previous selection pressures. And this is illustrated in the increase in CLL with complex karyotype and the detrimental effect of this marker of clonal heterogeneity on outcome even with highly effective drugs such as ibrutinib [9]. The treatment of CLL in a primary treatment situation may have different rules and these will have to be elucidated from the perspective of clonal dynamics. In addition, the registration of another effective drug (i.e. venetoclax) for the treatment of CLL [104] complicates the

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landscape for clinical decision-making. While an understanding of the biologic basis for clinical resistance observed with venetoclax treatment is lacking, the effectivity of a combination with rituximab in pretreated patients to induce deep remissions has recently been shown in abstract form [105]. Interestingly in this trial a limited maintenance phase with venetoclax after combined induction with venetoclax with rituximab has been used as a treatment strategy, introducing another option for prolonged treatment in CLL with unclear evolutionary consequences. While our understanding of evolutionary processes in CLL is currently rapidly improving [84, 87, 96, 97, 106] and suggestions for adapting treatment strategies are being put forward in CLL [82], further suggestions on how to optimally invoke our increasing knowledge on evolutionary events during cancer treatment are being developed across oncology [107]. These include options such as pulsed administration strategies, variable dosing and socalled double-bind treatment strategies and will involve complex mathematical modeling [85, 108-112]. However, it is currently unclear how and if we may be able to implement such strategies into our clinical trial designs, let alone our clinical everyday practice.

Where does this leave the clinician that will have to decide on a treatment strategy? Clinically, and in keeping with the discussion above, CD20 antibodies or lenalidomide have shown relatively ill-defined mechanisms of resistance and there is little evidence that these modalities select for more aggressive clones such as del17p CLL clones or for complex karyotypes. Both substances are relatively well-tolerated and have been used in prolonged treatment situations in other hematologic malignancies. The currently available randomized database supports an idea of effective maintenance of remissions in CLL, but cannot currently provide proof-of-efficacy on the level of overall survival, nor is it likely to be able to in the future, due to the many new salvage options arising in the field. However, this setting may only be true in highly affluent societies. There is a growing difficulty of implementing the success of novel CLL drugs, on a broader level due to relevant financial constraints even in high-income countries with high healthcare budgets [113, 114] and this is of course even more true in a large part of the rest of the world. Drug pricing and payment structures may thus lead to a lack of availability or adherence for novel drugs [115] even in affluent societies. In this context the currently observed change of rituximab pricing in a post-patent situation, combined with the option to prolong remissions and thus significantly delay the next (more expensive) treatment line with a tolerable and affordable maintenance option may thus be important for clinical decision making in large parts of the world. For the affluent societies, individualized decision-making taking into account strategic information on individual risk and treatment goals, remission depth and remaining future treatment alternatives may be one way forward, while creating a better understanding of

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clonal dynamics in continued treatment of CLL should be another way of defining the best treatment algorithms. This will be especially true in view of a large number of clinical experiences that start to be reported on all sorts of combination approaches and with limited comparability of “standard” arms. A better understanding of the long-term treatment and maintenance strategies used in these trials should be among the questions asked in order to optimally design the treatment paths for our patients. Acknowledgments: We thank Daniela Wolkersdorfer, Alexandra Keuschnig and Brigitta Elsässer, AGMT, for help with editing the manuscript. This work was supported by the SCRI-LIMCR, the Province of Salzburg, the City of Salzburg. AE’s work in CLL is supported by grants from the Austrian Science Fund (Transcan-2 FIRE-CLL I2795-B28 and DACH grant I 3282-B26). RG is supported by grants from the Province of Salzburg Authors statement: Informed consent was obtained for experimentation with human subjects. All authors made substantial contributions to the manuscript and gave their approval on the final version. The present publication has not been published in this form previously and is not under consideration for publication elsewhere.

Conflict of Interest Statement: A.E. and R.G. reports grants and personal fees from Roche and grants and personal fees from Celgene, outside the submitted work; L.P. reports grants and personal fees from Celgene. Figure Legends:

Figure 1: Schematic definition of remission by defining remission depth. The y-axis represents a measure of overall tumor mass (tumorload), while the x-axis represents time. A) After the incremental reduction of tumorload during induction treatment, we define remission states either by clinical staging or using techniques to more quantitatively measure remission depth and define so-called “MRD-negative” states. After the end of induction we observe regrowth kinetics of the disease that will cross clinical (or MRD-) thresholds to define relapse. B) in the case of a maintenance approach the optimal outcome will be that molecular and clinical relapses are delayed by the maintenance approach, leading to a longer PFS.

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Figure 2: Schematic of potential clonal dynamics as described in the main text. A) Standard and finite chemoimmunotherapy will affect a clonally heterogenous tumor (three sub-clones are outlined in the left box) to achieve an overall reduction of tumor cell counts, while either leaving the clonal architecture unchanged (V1) or selecting for a shift in clonal architecture. After the end of treatment the tumor will resume its growth and either keep its architecture derived after the end of treatment (V1a or V2a) on the one hand, or enter a different form of clonal selection (e.g. competitive release) effecting a different subclonal composition (V1b or V2b). B) Novel drugs have been observed to reduce overall cell numbers while selecting for mostly pre-existing resistant subclones. There’s subclones drive the relapse. C) in a maintenance approach all variants outlined in A, may be observed at the end of the induction. While patients in an observational follow up (ob) will have outcomes as outlined in A, maintenance (M) may partially control the clone and limit competitive release phenomena as drivers of evolution (M1) or delay growth while unable to prevent clonal changes over time (M2), depending on the mode of action and efficacy of the maintenance approach.

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30 Table 1: Induction results for common induction regimens in first line (selected examples from randomized trials) Regimen

CR rate

Rate of MRD <10-4 in PB after induction

Rate of MRD <10-4 in BM after induction

Source

FCR

40%

74%

58%

CLL10 Eichhorst et al. (2016)

BR

31%

63%

32%

CLL10 Eichhorst et al. (2016)

Clb-R

7%

3%

3%

CLL11 Goede et al. (2014)

Clb-Obi

23%

38%

19%

CLL11 Goede et al. (2014)

Ibrutinib

4%

Resonate-2 Burger et al. (2015)

BM, bone marrow; BR, bendamustine and rituximab; Clb-Obi, chlorambucil and obinutuzumab; Clb-R, chlorambucil and rituximab; CR, complete response; FCR, fludarabine, cyclophosphamide, and rituximab; MRD, minimal residual disease; PB, peripheral blood;

31 Table 2: Results of randomized maintenance trials in CLL Trial

Publication

Pat

Induction regimen

Line

Maintenance regimen

Median FU (months)

Median PFS (months)

MRD results

GCLLSG_CLL 4B

Wendtner et al. (2004)

21

F (48%) FC (52%)

1st

alemtuzumab 30 mg three times a week for 12 weeks vs. observation

14.4

nr vs. 17.8 p=0.036

Conversion to MRD negativity: 5/6 vs. 0/3

Roche ML21445

Foa et al. (2014)

66

Clb-R

1st

12 8-week doses of rituximab (375 mg/m2) vs. observation

34.9

38.2 vs. 34.7 p=0.07

Conversion to MRD negativity: 3/5 patients with CR vs. 0/6

AGMT_CLL 8A Mabtenance

Greil et al. (2016)

263

FCR (73%) BR (20%)

1st (80%) 2nd (20%)

8 12-week doses of rituximab (375 mg/m 2) vs. observation

33.4

47.0 vs. 35.5 HR 0.50

MRD PFS 31.3 vs. 12.0 months (HR 0.40)

CLL 2007 SA

Dartigeas et al. (2016)

409

FCR

1st

12 8-week doses of rituximab (500 mg/m2) vs. observation

47.7

59.3 vs. 49.0 HR 0.55

na

PALG-CLL4

Robak et al (2018)

66

RCC

1st

8 12-week doses of rituximab (375 mg/m 2) vs. observation

na

nr vs. 25.2 HR 0.418

MRD-negative rate in patients with CR or PR: 20% vs. 28.6%

PROLONG

VanOers et al. (2015)

474

FCR (43%) BR (20%)

2 (70%) 3rd (27%)

ofatumumab 1000 mg (start dose 300 mg) every 8 weeks for 2 years. vs. observation

19.1

29.4 vs. 15.2 HR 0.50

na

Chanan-Khan et al. (2017)

314

FCR (48%) BR (21%)

2nd

Escalating doses of lenalidomide (2.5 – 10 mg/day) until progression vs. placebo

31.5

33.9 vs. 9.2 HR 0.40

Conversion to MRD negativity: 2% vs. 1%

GCLLSG_CLLM1

Fink et al. (2017)

89

FCR (38%) BR (61%)

1st

lenalidomide vs. placebo

17.9

nr vs. 13.3 HR 0.168

Conversion to MRD negativity: 3 (8%) after cycle 7 and 2 (7%) after cycle 12 vs. 0

ALLG/FILO CLL6 Residuum

Gottlieb et al. (ASH 2016)

27

FCR

1st

lenalidomide 10 mg for 2 years vs. placebo

16.0

na

Increased MRD 38% vs. 62% of patients (p =0.032)

CONTINUUM

nd

BR, bendamustine and rituximab; Clb-R, chlorambucil and rituximab; CR, complete response; F, fludarabine; FC, fludarabine, cyclophosphamide; FCR, fludarabine, cyclophosphamide, and rituximab; HR, hazard ratio; MRD, minimal residual disease; nr, not reached; PFS, progression free survival; RCC, rituximab, cladribine and cyclophosphamide

32 Table 3: PFS: Subgroups of patients with or without benefit from maintenance therapy Trial

Induction treatment (line) HR

Regime HR

IgVH status HR

Cytogenetics 11q-/17pHR

Remission after induction HR

MRD status BM after induction HR

Others HR

na

na

na

GCLLSG_CLL 4B

na

Roche ML21445

na

na

unmut PFS 38.2 mut PFS 22.8 (p=0.012)

17p13 deletion/TP53 mutation: OR 3/66; NR 2/4 (p=0.0221) 11q22-q23 deletion OR: 13/56; NR 2/4 (p=0.4038)

na

na

na

trisomy12: OR 14/55; NR 0/6 (p=0.9618)

AGMT_CLL 8A Mabtenance

1st 0.51 2nd 0.51

FCR 0.68 BR 0.43

mut 0.62 unmut 0.69

Unf 0.53 Fav 0.63

CR/CRi 0.51 PR 0.56

pos 0.53 neg 0.75

Pos 0.60 Neg 0.88

CD38+ 0.53 CD38- 0.53

na

na

mut 0.96 mnmut 0.46

del11q yes 0.42 del11q no 0.65

CR/CRi 0.82 PR 0.49

pos 0.57 neg 0.43

na

del11q 0.37 del17p 0.28

CR 0.64 PR 0.47

Pos 0.37 Neg 0.31

β2M high 0.44 β2M low 0.50

CLL 2007 SA

The difference between both groups was statistically not associated with response to F or FC, IgVH mutational status or cytogenetic aberrations

MRD status PB after induction HR

PALG-CLL4

na

na

PROLONG

2nd 0.53 3rd 0.41

Alkylating monotherapy 0.21 Chemoimmu notherapy 0.55

mut 0.30 unmut 0.49

CONTINUUM

na

na

mut 0.40 unmut 0.27

GCLLSG_CLLM1

na

FCR/FC 0.20 BR 0.165

na

na

na

Intermediate (≥10-4 and < 10-2) 0.171 High (≥ 10-2) 0.165

na

Deleted/mutated TP53 status 0.134 Not deleted/mutated TP53 status 0.173

ALLG/FILO CLL6 Residuum

na

na

na

na

na

na

na

na

CR/CRi 0.78 PR 0.32

BM, bone marrow; BR, bendamustine and rituximab; CR, complete response; CRi, CR with incomplete bone marrow recovery; F, fludarabine; FC, fludarabine, cyclophosphamide; FCR, fludarabine, cyclophosphamide, and rituximab; HR, hazard ratio; IgVH, mutational status of the immunoglobulin heavy chain variable region genes; MRD, minimal residual disease; NR, no response; OR, overall response; PB, peripheral blood; PFS, progression free survival

33 Highlights: 

Remission induction in CLL has become very effective in recent years



No cure is available for a majority of patients



Remission maintenance improved PFS in 6 randomized trials



The position of remission maintenance in treatment algorithms is unclear



Arguments of evolutionary biology are discussed

36