Immune Recovery After Fludarabine–Cyclophosphamide

Immune recovery after ﬂudarabine–cyclophosphamide–rituximab treatment in B-chronic lymphocytic leukemia: implication for maintenance immunotherapy

L Ysebaert1,2,3,6, E Gross1,2,6,EKu¨hlein4, A Blanc1,2, J Corre5, JJ Fournie´1,2, G Laurent1,2,3 and A Quillet-Mary1,2

1Inserm U563, Toulouse F-31300, France; 2Universite´ Toulouse III Paul-Sabatier, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, France; 3CHU Toulouse, Hopital Purpan, Service d’He´matologie, Toulouse F-31300, France; 4CHU Toulouse, Hopital Rangueil, Laboratoire d’Immunologie, Toulouse F-31300, France and 5CHU Toulouse, Hopital Purpan, Laboratoire d’He´matologie, Toulouse F-31300, France

Thirty B-cell chronic lymphocytic leukemia patients were during the post-treatment period.1 It has also been proposed that treated with fludarabine–cyclophosphamide–rituximab (FCR) immunosuppression could also contribute to the emergence of and immune cell counts (natural killer (NK) cells, CD4, CD8, Tcd second malignancies, including leukemia and Richter’s syn- and monocytes) were monitored from the end of treatment drome, which has been observed in the setting of both B-CLL (EOT) up to 36 months (M36). Moreover, nonleukemic peri- 3–5 pheral blood lymphocyte cytotoxicity (PBL/CTC) as well as and Waldenstro¨m’s disease. rituximab (RTX)-dependent PBL/CTC was also measured at the Whether FCR therapy could affect some specific immune cell initiation of therapy, EOT and M12. These parameters were population and natural effector cell function is unknown. This correlated with post-FCR monitoring of the minimal residual question is still of interest based on two considerations. First, it disease (MRD) level in blood using a four-color flow cytometry is conceivable that host immune function may contribute to technique. FCR induced a profound and sustained depletion of all T-cell populations, Tcd being the most affected, whereas NK the control of the disease; for example, B-CLL relapse reflects cells were relatively preserved. Both basal and interleukin-2- escape to innate immunity through proper deficit in natural stimulated nonleukemic PBL/CTC against MEC-2, a CLL cell killer (NK) or Tgd cell function, which can be further worsened line, increased during the post-FCR period. There was no by treatment.6–8 Second, in the perspective of maintenance correlation between immune recovery parameters and MRD therapy with monoclonal antibodies,9 including rituximab progression profile, except that patients with high post-FCR (RTX), it is important to know whether antibody-dependent CD4 þ counts experienced rapid MRD progression. MRD at M12 predicts clinical relapse. The limited data show RTX-mediated cellular cytotoxicity (ADCC) capacity of natural effector cells LBL/CTC activity against autologous B-cell cells in individuals is preserved during the post-FCR period. For this reason, we with o1% residual disease at M12, opening avenues for have measured at different times immune cell counts (NK, immunomodulation post-FCR with anti-CD20 antibodies. To CD4, CD8, Tgd), in vitro nonleukemic peripheral blood conclude, our study suggests that MRD increase at M12 lymphocyte (PBL) cytotoxicity with or without interleukin precedes disease evolution post-FCR, and should be assessed (IL)-2, reflecting NK and lymphokine activated killer (LAK) as a surrogate marker for proactive management of CLL relapse. Leukemia (2010) 24, 1310–1316; doi:10.1038/leu.2010.89; function, respectively, as well as RTX-dependent PBL ADCC published online 13 May 2010 against autologous leukemic cells. Finally, these values were Keywords: FCR; MRD; immune recovery; NK; antibody-dependent correlated with post-FCR monitoring of the minimal residual cellular cytotoxicity; RTX disease (MRD) level in blood used in this study as a parameter of disease control.

Introduction Patients and methods

Recent therapeutic successes in B-cell chronic lymphocytic Patients and treatment leukemia (B-CLL) management were achieved by combinations From December 2005 to December 2008, 30 patients with of purine analogs, alkylating agents and monoclonal anti- B-CLL referred to our University Hospital who received FCR as bodies.1 In this regard, the fludarabine, cyclophosphamide, their frontline treatment were prospectively evaluated on rituximab (FCR) schedule is now generally considered as one of informed consent (according to the local ethics board recom- the best first-line regimens, yielding a 95% overall response rate mendations). Treatment initiation was based on the National and 52% complete response (CR). Moreover, FCR induces a Cancer Institute-International Workshop on Chronic Lympho- molecular response rate as high as 66%.2 However, FCR therapy cytic Leukemia (NCI-IWCLL) criteria, and associated fludarabine results in a profound immunosuppression, mainly due to 2 2 (40 mg/m at days 1–3, per os), cyclophosphamide (250 mg/m neutropenia during the treatment, but also related to severe 2 at days 1–3, per os) and RTX (375 mg/m at day 1 for course 1, and sustained B- and T-cell depletion over the 2-year post- 2 and then 500 mg/m at day 1 for courses 2–6, intravenously). treatment period. FCR-mediated immunosuppression is reflected Response was assessed 3 months after the last FCR course, and by a significant rate of opportunistic infections, which occur patients were classified according to the NCI-IWCLL criteria, except for bone marrow biopsy (patients with clinical and Correspondence: Dr L Ysebaert, CHU Toulouse, Service d’He´mato- biological evidence of CR are therefore considered as CR or logie, Hopital Purpan, Toulouse F-31300, France. nodular PR patients). Bone marrow cytological and phenotypi- E-mail: [email protected] 6These authors contributed equally to this work. cal studies were only provided to PR patients, to stratify them as Received 16 September 2009; revised 11 March 2010; accepted 30 CRi (incomplete bone marrow recovery) or PRd (disease March 2010; published online 13 May 2010 related), as described elsewhere.1,10 Immune cell recovery and MRD follow-up post-FCR L Ysebaert et al 1311 Immunological studies months after the completion of FCR treatment (to classify At different times following treatment completion (end of patients into MRD group A or B). Events relevant for the end treatment, EOT), fresh whole blood was used for immune cell point event-free survival were clinical progression, disease counts and immunophenotype profile. PBLs were stained with recurrence or death from any cause, transformation of any kind two combinations of monoclonal antibodies: CD3-FITC/CD8- (one Richter’s syndrome, one secondary acute myeloid leuke- PE/CD45-PerCP/CD4-APC in trucount tubes and TCRVg9d2- mia due to treatment). Data were analyzed as of September FITC/CD56-PE/CD3-PerCP-Cy5.5/CD16PE-Cy7/CD45-APC-H7, 2009. purchased from BD Biosciences (Becton-Dickinson France SAS, Le Pont de Claix, France), gated on CD45/SS, and acquired on a FACSCANTO II BD. The lymphocyte absolute counts were obtained directly with the CANTO software on the Results trucount tube. Patients and clinical follow-up Patient demographics are described in Table 1. FCR yielded MRD flow cytometry CR þ nodPR rate as high as 80%, with 10% CRi and 10% PRd MRD monitoring after FCR was made according to published rate (no stable disease or progressive patient). Response rate was international recommendations.11 MRD levels are given correlated with stage C but not with other clinical or biological parameters. At 3 months after the last FCR course (EOT), four- according to this consensus, as the percentage of positive cells 4 among total leukocytes with the two four-color combinations color flow cytometry peripheral blood MRD level o10 was proposed: CD43-FITCint/CD79b-PElo/CD5-PerCP-Cy5.5/CD19- obtained in 16 of 30 (53.3%) patients. During follow-up (ranging APC and CD81-FITClo/CD22-PElo/CD5-PerCP-Cy5.5/CD19-APC. from 12 to 46 months with a median of 30 months), 8 disease Monoclonal antibodies were purchased from BD Biosciences.11,12 progressions occurred (disease-free survival (DFS) for the entire All MRD data were obtained from fresh blood samples using a cohort was 67% at 26 months). In all cases, increase in MRD 4 À2 FACSCalibur BD. Acquisition was stopped when all leukocytes 10 was detected at 3–6 months before clinical relapse. On in samples had been processed (a median of 115 000 events molecular progression, three patients died (one from Richter’s were analyzed). The sensitivity of this assay is one CLL cell among syndrome, two from severe sepsis), one needed re-treatment and 104 leukocytes. These combinations allowed a distinct determi- four remained treatment-free (owing to Binet stage A disease, nation of B-CLL and normal B cells. with discrete hyperlymphocytosis and palpable lymph nodes). Collectively, these results suggest that in the first year post-FCR, an MRD increase 41% of total leukocytes predicts clinical Cell- and antibody-mediated cellular cytotoxicity relapse. The natural cytotoxicity and ADCC function of effector cells from CLL patients were tested using the classical chromium Table 1 Characteristics of 30 patients treated with FCR as a release assay. Briefly, PBL from CLL patients were stimulated or frontline regimen not with recombinant glycosylated IL-2 (10 ng/ml, a kind gift from Sanofi-Aventis, Toulouse, France) during 3 days in RPMI Characeristics n Percentage of patients 10% fetal calf serum at 37 1C, 5% CO2 and used as effector cells. Target cells (MEC-2 or B cells from autologous patients, Binet stage and frozen before treatment) were incubated in RPMI 1% fetal A 2 6.7 calf serum for 1 h at 37 1C with 51Cr (Sodium Chromate, Perkin B 19 63.3 6 C930 Elmer, Courtaboeuf, France) (100 mCi for 10 cells). Cells were Median age (years) 30 57 (36–69) then washed and incubated or not with RTX (10 mg/ml) for Gender (M/F) 30 70/30 15 min at room temperature. Cells were washed again and Median leukocytosis per ml 30 84500 plated at 104 cells per well in round-bottom 96-well plates. Increasing amounts of effector cells were added to triplicate FISH wells as an NK/target ratio ranging from 0.1:1 to 2:1 depending Del13q alone/normal 17 63 Del11q 8 30 on the patients. This ratio was calculated from the NK Tri12 2 7 percentage obtained by flow cytometric analysis. Control wells contained only target cells (pre-coated or not with RTX) to IgVH measure spontaneous release or target cells with 0.1% Triton MUT/UNMUT 20 40/60 X-100 to measure maximal release. After centrifugation, plates were incubated for 4 h at 37 1C with 5% CO2. Then 50 ml were CD38 420% 26 50/50 collected from each well and counted in a gamma counter. The percentage of specific lysis was calculated as follows: ((sample Response release–spontaneous release)/(maximal release–spontaneous ORR 30 100 release)) Â 100. CR+nod PR 24 80 CRi 3 10 PRd 3 10 MRD 10À4 after FCR 30 53.3 Statistical methods o Student’s t-test was used for comparison of independent Abbreviations: CR, complete response; CRi, incomplete bone marrow samples, and Mann–Whitney U-test for comparison of nonpara- recovery; Del13q/11q, deletion 13q/11q; F, female; FCR, fludarabine– cyclophosphamide–rituximab; FISH, fluorescent in situ hybridization; metric variables. All statistical tests were two-sided and used IgVH, immunoglobulin variable heavy chain gene status (MUT, a P-value of 0.05 for statistical significance. Survival data were mutated; or UNMUT, unmutated); M, male; MRD, minimal residual carried out using Statistica software (Statsoft, Tulsa, OK, USA) disease; nod, nodular; ORR, overall response rate; PR, partial according to the Kaplan–Meier method, data being measured 12 response; PRd, disease related; Tri12, trisomy 12.

Leukemia Immune cell recovery and MRD follow-up post-FCR L Ysebaert et al 1312 MRD group A (n=81) MRD group B (n=12) 100% 100%

10% 10%

1% 1%

0.1% 0.1% MRD level 0.01% 0.01% (% of leucocytes) <0.01% <0.01% EOT M6 M12 M18 M24 EOT M6 M12 M18 M24

MRD FU MRD MRD p= MRD group A group A Group B Binet stage A 50% 50% ns B 58% 42% C 44.5% 55.5% Log-rank p<0.01 FISH : no del11q 60% 40% ns del11q 50% 50%

IgVH MUT 54.5% 45.5% ns UNMUT 50% 50% Percent surviving MRD group B CD38 >20% 46% 54% ns <20% 58% 42% MRD <10-4 EOT 75% 25% 0.014 months

Figure 1 Minimal residual disease (MRD) kinetics after completion of FCR therapy. (a) Groups A and B are defined at 1 year of follow-up as having MRD levels below (a) or above (b) 1% of total leukocytes (set up as a clinically relevant relapse value, represented by a black thick line), each curve depicting the MRD monitoring from one patient. The value o0.01% symbolizes the threshold of MRD detectability with our flow technique, and MRD-negative patients are therefore plotted at this value for graphic representation purposes. (b) Disease-free survival (DFS) post- FCR according to MRD group. After 1 year of follow-up, MRD groups A and B have statistically different DFS by Kaplan–Meier analysis (log-rank Po0.01). (c) Correlation between MRD control and disease characteristics. MRD kinetics between EOT and M12 is only correlated with the quality of molecular response after FCR (MRD EOT o0.01%), not with baseline CLL risk factors. EOT: end of treatment; FCR: fludarabine, cyclophosphamide, rituximab; M6–24: months 6–24 post-FCR; MRD FU: minimal residual disease follow-up; P ¼ NS: not significant.

Flow cytometry follow-up NK cells were relatively preserved, although they remained MRD was assessed every 6 months for at least 1 year after FCR below the normal range (Figure 2). From EOT to M12, NK cells completion. At 1 year, 30 of 30 patients were informative. From accounted for up to 32% of PBL, endowed with potential ADCC, EOT, different MRD profiles were observed, but we chose because they express CD16 (Figure 2b). Normal B cells to classify patients according to the MRD outcome after 1 year (Figure 2c, calculated from MRD dot plot analyses) rapidly into two groups (Figure 1a). Group A patients had MRD levels increased, to reach almost normal ranges (2–3% of leukocytes, below 1% of total leukocytes (n ¼ 18 of 30), and group B instead of 3–6%) in the first year post-immunochemotherapy. patients had progressive MRD levels 41% of total leukocytes The only patient with decreased B cell numbers is the patient (n ¼ 12 of 30) at 12-month (M12) evaluation. Interestingly, who required second-line treatment at 15 months after FCR. two patients in group A showed progressive decrease in MRD Altogether, these findings showed that Tgd and CD4 þ T cells levels (without receiving any treatment). From M12, group B were much more sensitive to FCR than NK cells. had significantly shorter DFS (18 months vs not reached in group A, log-rank Po0.01) (Figure 1b). Group A and group B showed no significant differences for Binet stage, del11q, IgVH NK function recovery mutational status and CD38 expression (data not shown). As NK cells are believed to be important immune effectors However, undetectable MRD at EOT evaluation was predictive for both disease control and defenses against pathogens, we of a favorable molecular outcome over the first year after treat- investigated whether FCR could have influenced intrinsic NK ment (P ¼ 0.014) (Figure 1c). These data indicate that patients function. For this reason, we measured at different times the with MRD levels remaining o1% of leukocytes did not natural PBL lysis capacity against MEC-2, a CLL-derived cell experience clinical relapse, suggesting the occurrence of some line, at the low effector/target ratio of 0.4:1. As shown in immunomodulating events. Figure 3a, we found that resting PBL from healthy donors showed a modest activity against MEC-2 (4.7±3.5%), the latter being dramatically boosted when PBLs were stimulated by IL-2 Immune subset recovery after FCR (LAK activity) (24.8±14%). As shown in Figure 3a, when tested NK, monocytes, T CD4/CD8 and Tgd lymphocyte counts at the overt phase of the disease, resting B-CLL PBL showed reconstitution are presented in Figure 2a. FCR induced a sharp similar cytotoxicity against MEC-2, compared with healthy and prolonged decrease in values for all lymphoid subpopula- donors (1.4±1.1%). However, when IL-2 was used, LAK activity tions, compared with the pre-FCR period. The median time was much lower, compared with normal healthy donors to reach CD4 þ 4200 cells per ml was 6 months (but up to (6.5±4.3%, P ¼ 0.004), suggesting that LAK function was 24 months to reach 400 cells per ml), and the median CD8 þ indeed depressed in B-CLL samples (Figure 3a). Moreover, as levels reached the normal range after 12 months. T Vg9d2 depicted in Figure 3b, LAK activity progressively increased lymphocytes showed profound and sustained depletion. and was totally restored at M12 compared with EOT in eight

Leukemia Immune cell recovery and MRD follow-up post-FCR L Ysebaert et al 1313 3 Absolute count/mm3 Absolute count/mm

NK CD4

n= 22 25 17 8 8 8 n= 24 24 23 10 10 8 EOT M6 M12 M18 M24 M36 EOT M6 M12 M18 M24 M36 Mono CD8

n= 24 25 21 9 9 8 n= 27 26 19 13 9 7 EOT M6 M12 M18 M24 M36 EOT M6 M12 M18 M24 M36 NK/CD16 EOT T 350 R2= 0,9564 300

250

200

150

100

Absolute cell number 50 n= 21 24 19 11 10 7

0 EOT M6 M12 M18 M24 M36 0 50 100 150 200 250 300 350 Absolute cell number NK/CD16 M12 10 800 R2= 0,9158 700 600 1 500 400 0,1

300 Normal B cells (% of leucocytes) 200

Absolute cell number 0,01 100 EOT M6 M12 M18 M24 0 0 100 200 300 400 500 600 Absolute cell number

Figure 2 Time kinetics of absolute numbers of different cellular subsets in the blood of CLL patients following FCR treatment. (a) Each box shows the median value (À) and the 25–75% percentiles, and bars represent the range of values. The gray zone within each category represents our laboratory’s normal values for each cellular subset. (b) Correlation between CD16-expressing cells and CD3ÀCD56 þ NK cells (results shown are EOT and M12, but are also true at M18 and M24). (c) Normal B-cell recovery (four-color ﬂow cytometry) is rapid and almost reaches the normal range within 6–12 months). Same representation is used as for MRD kinetics data, and the results shown are percentage of B lymphocytes among total leukocytes. n: the number of patients analyzed at each time point during follow-up; EOT: end of treatment; M ¼ months. paired samples (19.9±15.2% vs 4.6±2.4% at EOT, P ¼ 0.02), Correlation between MRD and immune recovery as the results were comparable to what was seen with healthy On the basis of the above results, we investigated whether donors. This result suggests that, whereas the NK function was immune recovery could inﬂuence disease control attested by altered in B-CLL at the overt phase of the disease, FCR restored the MRD level remaining below 1%. For this reason, we have LAK activity (using IL-2) within 1 year after therapy completion. compared group A and group B patients for CD4, CD8, NK, Tgd,

Leukemia Immune cell recovery and MRD follow-up post-FCR L Ysebaert et al 1314 p=0.004 p=0.02 p=0.03

50 p=0.002 -IL-2 40 + IL-2

20 p=0.02

% specific cytotoxicity 0

Normal donors CLL before FCR CD4+ ber absolute numb

p=0.02 50 -IL-2 p=0.01 + IL-2 MRD group A group B 40 n= 14 12

þ 30 Figure 4 Low median CD4 T-cell count at EOT is predictive of good disease control during the ﬁrst year post-FCR (MRD remaining below 1% of total leukocytes), according to the Mann–Whitney 20 p=0.002 analysis in 26 available patients (P ¼ 0.02). EOT: end of treatment. 10 % specific cytotoxicity p=0.03 0 MRD group A (M12) 40 EOT M12 MRD group B (M12)

Figure 3 Natural cytotoxic capacities of CLL patients’ PBL recovered 30 after FCR. (a) Comparison of natural cytotoxicity (ÀIL2) or LAK ( þ IL2) activity of PBL taken from 8 CLL patients (just before receiving FCR) or 10 healthy donors against the MEC-2 cell line. Effector/target ratio 20 shown is always 0.4:1, and lysis experiments were carried out as stated in the Patients and methods section. (b) LAK activity against MEC-2 is 10 restored in the ﬁrst year post-FCR treatment. PBL from eight CLL

patients were collected at EOT and M12, and lysis experiments were % specific cytotoxicity carried out. Paired t-test analyses indicate a significant recovery of LAK 0 activity at M12 post-FCR. EOT: end of treatment; FCR: fludarabine, CLL CLL RTX CLL RTX cyclophosphamide, rituximab; IL-2: interleukin-2; M12: 12 months IL-2 after FCR. Figure 5 Rituximab-mediated ADCC against autologous CLL cells. PBL from six patients (three in group A, and three in group B) were collected 1 year after completion of FCR, and assessed for natural PMN counts, as well as for NK and LAK activities against cytotoxicity (control) and ADCC with RTX with or without IL-2, against MEC-2, measured at EOT, M6 and M12. As illustrated in autologous CLL cells (frozen before FCR treatment) as targets. Results þ shown are mean cytotoxicity (±s.d.) at a calculated effector/target Figure 4, group A showed significantly lower CD4 counts at ratio of 0.4:1. EOT (median ¼ 133 vs 225 per ml, n ¼ 26, P ¼ 0.02), and a slow CD4 þ T-cell recovery during the first year post-FCR (median at M12: 233 vs 419 per ml, n ¼ 25, P ¼ 0.02). We found no correlation with the other parameters at any other time point, group, A and B). Against autologous B-CLL cells, group A including NK or LAK function (data not shown). Best CD4 count patients show statistically different RTX-ADCC (28.8±7% vs cutoffs to predict DFS were o150 per ml at EOT (DFS 30 vs 8.1±2.8% in group B, P ¼ 0.03, Figure 5). These results suggest 16 months, P ¼ 0.008), and o300 per ml at M12 (DFS from that although the LAK function was dramatically improved by M12 post-FCR: 19 vs 7 months, Po0.01). FCR therapy, NK remained ineffective against the leukemic clone, unless being used in association with RTX, which may redirect intrinsically efficient NK cells toward their targets, or PBL cellular cytotoxicity against autologous B-CLL cells trigger secondary signals resulting in boosting their cytolytic after FCR function. On the basis of the above findings, which indicated that the NK/LAK function was improved during the first year post-FCR, we speculated that PBL from CLL patients treated with FCR Discussion could exert potent RTX-mediated ADCC against autologous leukemic cells. From unpaired analyses, we first compared Although FCR has become one of the major standards for B-CLL, RTX-ADCC ±IL-2 from CLL patients before and 12 months after scarce information is available on long-term immune recon- FCR, but detected no statistical difference (mean RTX-ADCC stitution after therapy. Recently, concerns about FCR-induced þ IL-2 was 8.1±4% at baseline vs 18±12% at M12, n ¼ 6). We immunosuppression were raised about the relatively high rate of then focused on these six patients at M12 (three from each MRD opportunistic infection during the post-FCR period.1 We also

Leukemia Immune cell recovery and MRD follow-up post-FCR L Ysebaert et al 1315 addressed the kinetics of MRD levels after FCR, and its evolution of course, but future strategies aiming at modulating relevance for the prediction of clinical relapse. Correlative CD4 þ subsets (Tregs, Th17) may prove of interest in CLL. studies between both recovery data (immune cells and MRD) In a view of immunomodulation strategies early post-FCR, we point toward a putative role of the immune system in the control present data indicating that natural effectors, presumably NK of CLL post-FCR. cells, exert in the presence of RTX cellular cytotoxicity against Unsurprisingly, we have found that FCR induced a severe and autologous leukemic cells. Interestingly, such RTX-mediated prolonged depletion of all T-cell subsets. Previous studies have ADCC of patient PBL improved during the post-FCR period and largely documented the sensitivity of CD4 and CD8 T cells to reached levels comparable to that of normal healthy donors fludarabine.13,14 However, the impact of the drug on Tgd subset (data not shown). We believe that ADCC increase is due to has not so far been reported. These cells were found to be even enhanced NK function during the same period of time. This more sensitive than Tab cells. The extent to which severe Tgd finding is important to consider because ADCC is the major depletion contributes to FCR-associated opportunistic infection mechanism by which RTX exerts its anti-leukemic effect. This remains to be determined. This question is of interest because of consideration, combined with the observation that MRD control the introduction of phosphoantigen-based preparations able to during the 1-year post-FCR period seems to be critical for future expand in vivo Tgd cell population.9 Interestingly, NK cells were clinical outcome, argues for the use of RTX as maintenance or relatively preserved. This observation is in sharp contrast with consolidation therapy during the first year following FCR alemtuzumab therapy, which was found to be harmful toward completion. RTX-based maintenance therapy is currently being NK cells.15 PBLs derived from B-CLL patients have much lower tested in the setting of FCR-treated B-CLL (French collaborative cellular cytotoxic capacity, compared with those from healthy CLL2007-SA phase III trial). Whether these studies will confirm donors. As NK cells support most, if not all, PBMC natural our hypothesis is still unknown. Our study also suggests that this cytotoxicity,16 this observation suggests that, before treatment, strategy should benefit from co-administration of NK-stimulating NK basal function is profoundly altered in B-CLL. Decrease in cytokines such as IL-2,22 IL-1523 or IL-2124 or drugs susceptible NK-mediated B-CLL lysis has been largely described. However, to enhance NK function, such as Lenalidomide.25 it remains unclear whether CLL cell natural resistance or To conclude, our study shows that FCR therapy induces a decreased function of B-CLL patient NK cells has the more severe and prolonged T-cell depletion; however, NK cells important role. Our study shows that NK activity in autologous are less affected and NK function even increases 1 year after RTX-ADCC improved during the post-FCR period in some treatment, with potential autologous RTX-ADCC restoration. patients (who could benefit from RTX maintenance treatment), We also provide evidence that MRD monitoring the first year but remained very low in others, suggesting that these latter post-FCR is of clinical importance to predict relapses and patients are endowed with highly NK-resistant CLL cells. manage preemptive therapeutic strategies. Altogether, these MRD eradication during and post-FCR is predictive of better results support the use of anti-CD20-based maintenance outcome in the CLL8 trial. Extensive data on MRD monitoring therapy in B-CLL to maintain low levels of MRD, prolong PFS post-FCR are lacking. Such data collected after bone marrow and perhaps improve survival. grafting procedures indicated MRD increase as a good predictive marker for clinical relapse.17–19 In our cohort of 30 patients, 8 out of 12 patients from group B (MRD 41% at M12) Conflict of interest suffered a relapse, with increasing levels of MRD 41% being always detected 3–6 months before clinical relapse. MRD The authors declare no conflict of interest. monitoring at 12 months after FCR identified patients with a high relapse risk, who could benefit from investigational Acknowledgements treatments aiming to prevent overt relapse. The choice of MRD threshold is nonetheless questionable. Using a 0.05% 19 We are grateful to the patients who participated in the study and threshold (as proposed recently in the post-allograft setting ), their referring physicians. We also thank Pr C Rećher for a critical four patients from group A would fall into group B, none with reading of the paper. This work was supported by grants from 4 overt clinical relapse (despite 24 months of follow-up). Thus, INSERM and Association Laurette Fugain (ALF/No 07-05). the size of our cohort may limit the significance of a 1% We thank F Hoffmann-La Roche Ltd for providing rituximab used threshold: the main message is clearly that MRD monitoring in our experiments. EG is the recipient of a grant from Ministe`re should be implemented in the follow-up of FCR patients de´le´gue´ a` l’Enseignement supe´rieur et a` la Recherche. because of its relevance in predicting clinical evolution of the disease. Another interesting result suggests a negative impact of CD4 þ cell recovery on molecular disease control. This observation is in agreement with another study presented very References recently.20 The significance of these observations remains unclear. They may simply reflect a surrogate biomarker of FCR 1 Tam CS, O’Brien S, Wierda W, Kantarjian H, Wen S, Do KA et al. efficacy against lymphocytes. Characterization of these CD4 þ Long-term results of the fludarabine, cyclophosphamide, and cells was beyond the scope of this study. It seems unlikely that rituximab regimen as initial therapy of chronic lymphocytic þ leukemia. Blood 2008; 112: 975–980. these cells are CD4 Treg, which could have thus facilitated 2 Hallek M, Fingerle-Rowson G, Fink A, Busch R, Mayer J, Hensel M tumor cell escape. Indeed, Treg cells are exquisitely sensitive to et al. Immunochemotherapy with fludarabine (F), cyclophos- fludarabine and indeed efficiently depleted by this drug.14 phamide (C), and rituximab (R) (FCR) versus fludarabine and Alternatively, it is possible that these CD4 þ cells correspond to cyclophosphamide (FC) improve response rates and progression- an expansion of cytokine-producing cells, which may exert a free survival (PFS) of previously untreated patients (pts) with direct helper function toward tumor cells, as for example advanced chronic lymphocytic leulemia (CLL). Blood 2008; 112 þ þ abstract no. 325. CD4 CD154 cells, a subset that was found to facilitate 3 Milligan DW, Kochethu G, Dearden C, Matutes E, MacConkey C, 21 proliferation, survival and immuno-escape of CLL cells. CD4 Catovski D. High incidence of myelodysplasia and secondary monitoring cannot be considered as a surrogate marker for MRD leukaemia in the UK medical research council pilot of autografting

Leukemia Immune cell recovery and MRD follow-up post-FCR L Ysebaert et al 1316 in chronic lymphocytic leukaemia. Br J Haematol 2006; 133: subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, 173–175. CAMPATH-1H) treatment as first-line therapy for B-cell chronic 4 Cheson BD, Vena DA, Barrett J, Freidin B. Second malignancies as lymphocytic leukaemia. Leukemia 2004; 18: 484–490. a consequence of nucleoside analog therapy for chronic lymphoid 16 Beum PV, Lindorfer MA, Taylor RP. Within peripheral blood leukaemia. J Clin Oncol 1999; 17: 2454–2460. mononuclear cells, antibody-dependent cellular cytotoxicity of 5 Tam CS, Seymour JF, Miles Prince H, Kenealy M, Wolf M, rituximab-opsonized Daudi cells is promoted by NK cells and Januszewicz EH et al. Treatment-related myelodysplasia following inhibited by monocytes due to shaving. J Immunol 2008; 181: fludarabine combination therapy. Haematologica 2006; 91: 2916–2924. 1546–1550. 17 Moreno C, Villamor N, Colomer D, Esteve J, Gine´ E, Muntanõla A 6 Ravandi F, O’Brien S. Immune defects in patients with chronic et al. Clinical significance of minimal residual disease, as assessed lymphocytic leukemia. Cancer Immunol Immunother 2006; 55: by different techniques, after stem cell transplantation for chronic 197–209. lymphocytic leukemia. Blood 2006; 107: 4563–4569. 7 Burton JD, Weitz CH, Kay NE. Malignant chronic lymphocytic 18 Ritgen M, Bo¨ttcher S, Stilgenbauer S, Bunjes D, Schubert J, leukemia B cells elaborate soluble factors that down-regulate T cell Cohen S et al. Quantitative MRD monitoring identifies distinct and NK function. Am J Hematol 1989; 30: 61–67. GVL response patterns after allogeneic stem cell transplantation for 8 Maki G, Hayes GM, Naji A, Tyler T, Carosella ED, Rouas-Freiss N chronic lymphocytic leukemia: results from the GCLLSG CLL3X et al. NK resistance of tumor cells from multiple myeloma and trial. Leukemia 2008; 22: 1377–1386. chronic lymphocytic leukemia patients: implication of HLA-G. 19 Oelschlaegel U, Bornhaüser M, Kiani A, Platzbecker U, Ro¨llig C, Leukemia 2008; 22: 998–1006. Wermke M et al. Flow cytometric detection of minimal residual 9 Gertner-Dardenne J, Bonnafous C, Bezombes C, Capietto AH, disease one year post allogeneic stem cell transplantation predicts Scaglione V, Ingoure S et al. Bromohydrin pyrophosphate outcome in patients with B-CLL. Blood 2009; 114 abstract no. 202. enhances antibody-dependent cell-mediated cytotoxicity induced 20 Egle A, Weiss L, Gassner F, Russ G, Pleyer L, Gunsilius E et al. by therapeutic antibodies. Blood 2009; 113: 4875–4884. Minimal residual disease (MRD) and T/NK cell dynamics during 10 Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Fludarabine, Cyclophosphamide plus Rituximab (FCR) followed Do¨hner H et al. Guidelines for the diagnosis and treatment of by Fludarabine plus Rituximab (FR) and remission maintenance chronic lymphocytic leukemia: a report from the International therapy with Rituximab in previously untreated B-chronic lympho- Workshop on Chronic Lymphocytic Leukemia updating the National cytic leukemia (B-CLL): risk factor stratification in the Chairos Cancer Institute-Working Group 1996 guidelines. Blood 2008; 111: study. Blood 2008; 112 abstract no. 3175. 5446–5456. 21 Ghia P, Chiorazzi N, Stamatopoulos K. Microenvironmental 11 Rawstron AC, Villamor N, Ritgen M, Bo¨ttcher S, Ghia P, Zehnder JL influences in chronic lymphocytic leukaemia: the role of antigen et al. International standardized approach for flow cytometric stimulation. J Intern Med 2008; 264: 549–562. residual disease monitoring in chronic lymphocytic leukaemia. 22 Golay J, Manganini M, Facchinetti V, Gramigna R, Broady R, Leukemia 2007; 21: 956–964. Borleri G et al. Rituximab-mediated antibody-dependent cellular 12 Rawstron AC, de Tute R, Jack AS, Hillmen P. Flow cytometric cytotoxicity against neoplastic B cells is stimulated strongly by protein expression profiling as a systematic approach for develop- interleukin-2. Haematologica 2003; 88: 1002–1012. ing disease-specific assays: identification of a chronic lymphocytic 23 Moga E, Alvarez E, Canto´ E, Vidal S, Rodrı´guez-Sańchez JL, Sierra J leukaemia-specific assay for use in rituximab-containing regimens. et al. NK cells stimulated with IL-15 or CpG ODN enhance Leukemia 2006; 20: 2102–2110. rituximab-dependent cellular cytotoxicity against B-cell lymphoma. 13 Keating MJ, O’Brien S, Lerner S, Koller C, Beran M, Robertson LE Exp Hematol 2008; 36:69–77. et al. Long-term follow-up of patients with chronic lymphocytic 24 Gowda A, Roda J, Hussain SR, Ramanunni A, Joshi T, Schmidt S leukaemia (CLL) receiving fludarabine regimens as initial therapy. et al. IL-21 mediates apoptosis through up-regulation of the BH3 Blood 1998; 92: 1165–1171. family member BIM and enhances both direct and antibody- 14 Beyer M, Kochanek M, Darabi K, Popov A, Jensen M, Endl E et al. dependent cellular cytotoxicity in primary chronic lymphocytic Reduced frequencies and suppressive function of CD4+CD25hi leukemia cells in vitro. Blood 2008; 111: 4723–4730. regulatory T cells in patients with chronic lymphocytic leukemia 25 Wu L, Adams M, Carter T, Chen R, Muller G, Stirling D et al. after therapy with fludarabine. Blood 2005; 106: 2018–2025. Lenalidomide enhances natural killer cell and monocyte-mediated 15 Lundin J, Porwit-MacDonald A, Rossmann ED, Karlsson C, Edman antibody-dependent cellular cytotoxicity of rituximab-treated P, Rezvany MR et al. Cellular immune reconstitution after CD20+ tumor cells. Clin Cancer Res 2008; 14: 4650–4657.

Leukemia