Consolidation with alemtuzumab in patients with chronic ... - Nature

Leukemia (2004) 18, 1093–1101 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu

Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission – experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG) C-M Wendtner1,6, M Ritgen2, CD Schweighofer1,6, G Fingerle-Rowson1,6, H Campe3, G Ja¨ger3, B Eichhorst1, R Busch4, H Diem5, A Engert6, S Stilgenbauer7, H Do¨hner7, M Kneba2, B Emmerich8, M Hallek1,6 and the German CLL Study Group (GCLLSG) 1

Klinikum Grosshadern, Medical Clinic III, Ludwig-Maximilians-University, Munich, Germany; 2Medical Clinic II, University Hospital, Kiel, Germany; 3Max-von-Pettenkofer-Institute, Ludwig-Maximilians-University, Munich, Germany; 4Institute of Medical Statistics and Epidemiology, Technical University, Munich, Germany; 5Clinical Chemistry, Ludwig-MaximiliansUniversity, Munich, Germany; 6Medical Clinic I, University Hospital, Cologne, Germany; 7Medical Clinic III, University Hospital Ulm, Germany; and 8Medizinische Klinik Innenstadt, Ludwig-Maximilians-University, Munich, Germany

Patients with CLL responding to initial chemotherapy with fludarabine alone (F) or in combination with cyclophosphamide (FC) were randomized for treatment with alemtuzumab (30 mg i.v. TIW, 12 weeks) or observation. Of 21 evaluable patients, 11 were randomized to alemtuzumab before the study was stopped due to severe infections in seven of 11 patients. These infections (one life-threatening pulmonary aspergillosis IV; four CMV reactivations III requiring i.v. ganciclovir; one pulmonary tuberculosis III; one herpes zoster III) were successfully treated and not associated with cumulative dose of alemtuzumab. In the observation arm, one herpes zoster infection II and one sinusitis I were documented. At 6 months after randomization, two patients in the alemtuzumab arm converted to CR, while three patients in the observation arm progressed. After alemtuzumab treatment, five of six patients achieved a molecular remission in peripheral blood while all patients in the observation arm remained MRD-positive (P ¼ 0.048). At 21.4 months median follow-up, patients receiving alemtuzumab showed a significant longer progression-free survival (no progression vs mean 24.7 months; P ¼ 0.036). In conclusion, a consolidation therapy with alemtuzumab is able to achieve molecular remissions and longer survival in CLL, but a safe treatment regimen needs to be determined. Leukemia (2004) 18, 1093–1101. doi:10.1038/sj.leu.2403354 Published online 8 April 2004 Keywords: CLL; alemtuzumab; consolidation; survival; MRD; CMV

Introduction Fludarabine induces higher remission rates and longer progression-free survival in comparison to alkylating agents in untreated patients with CLL.1,2 Nevertheless, the majority of patients in remission relapse after a median of 20–30 months. One strategy to prolong progression-free survival (PFS) in these patients is to combine fludarabine with alkylating agents.3,4 Another option currently under investigation is high-dose chemotherapy with autologous or allogeneic hematopoietic stem cell support,5 or more recently to combine established chemotherapy regimens Correspondence: Prof Dr Michael Hallek, Klinik I fu¨r Innere Medizin, Klinikum der Universita¨t zu Ko¨ln, Joseph-Stelzmann-Strasse 9, D-50924 Ko¨ln, Germany; Fax: þ 49 221 478 5455 Presented, in part, at the 39th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, May 31–June 3, 2003, and the 45th Annual Meeting of the American Society of Hematology, San Diego, CA, December 6–9, 2003. Supported by a research grant of Schering AG, Berlin and MedacSchering Onkologie, Germany. VH sequencing was supported by a research grant from the Else Kro¨ner-Fresenius Stiftung, Germany. Received 13 November 2003; accepted 1 March 2004; Published online 8 April 2004

with immunotherapy. The anti-CD20 antibody rituximab was shown to be very effective against CLL in combination with fludarabine-based chemotherapy with high rates of complete remissions.6 The monoclonal antibody alemtuzumab, a humanized anti-CD52 antibody, has shown activity alone and in combination for patients with refractory CLL with an overall response rate of 33%, and was also proven effective in initial treatment of CLL patients, with approximately 80% of patients achieving durable responses.7–12 Encouraging results have been published for the in vivo purging of residual disease after alemtuzumab consolidation.13 Alemtuzumab lyses normal and malignant lymphocytes by antibody-dependent cellular cytotoxicity (ADCC), complement activation and direct induction of apoptosis.14–17 Besides effects against malignant lymphocytes in blood, alemtuzumab shows activity against lymphocytes derived from bone marrow and spleen, but is less active against nodal or extranodal masses.9 Since CD52 is not expressed on CD34 þ hematopoietic stem cells, therapy with alemtuzumab does not preclude stem cell collection and further high-dose concepts.18 Therefore, the aim of this study was to assess the safety profile, feasibility and efficacy of alemtuzumab as a consolidation therapy to improve the quality of remission and to eradicate minimal residual disease in patients responding to a first-line fludarabine-based chemotherapy.

Patients and methods

Study design The German CLL Study Group (GCLLSG) conducted an openlabel, randomized phase III trial (CLL4B protocol) at 13 academic centers throughout Germany and Austria. The objective of the study was to assess the efficacy of alemtuzumab as consolidation therapy for patients with advanced B-CLL, who have responded to a first line chemotherapy with fludarabine (F) or fludarabine/cyclophosphamide (FC) (Eichhorst et al, Blood 2003; 102: 72, abstract). Patients were stratified according to induction treatment and response to induction treatment and randomized for treatment with alemtuzumab or observation. The primary end point was PFS defined as the time between start of F or FC until progression of disease or death. Secondary end points were clinical responses as assessed according to NCI-WG criteria,19 molecular response rates, overall survival, drug safety and in particular the occurrence of severe infections. Toxicity was graded according to the common toxicity criteria (CTC).20 The study was approved by the local ethics committee, and all patients gave written, informed consent prior to enrollment.

Alemtuzumab consolidation in CLL C-M Wendtner et al

1094

Patients Patients between 18 and 66 years of age with B-CLL in first complete or partial remission after F or FC were eligible. Alemtuzumab had to be started no less than 30 days and no more than 90 days after the last dose of F or FC. Patients with an apparent autoimmune cytopenia, severe infections during treatment with F or FC and any medical conditions requiring long-term use of oral corticosteroids could not enter the trial.

Treatment Patients received six cycles of F (25 mg/m2 d1–5 i.v. q 28 days) or FC (F 30 mg/m2 d1–3 i.v., C 250 mg/m2 d1–3 i.v. q 28 days). Responding patients being randomized to consolidation therapy received alemtuzumab three times per week (TIW) as a 2-h infusion. The starting dose of alemtuzumab was 3 mg on day 1; if well tolerated, dose was increased to 10 mg on day 2 and to the target dose of 30 mg on day 3. The 30 mg dose was subsequently given TIW for a maximum of 12 weeks. Therapy was discontinued, if an unacceptable toxicity occurred and stopping criteria for the trial were set as grade 3 or 4 infections occurring in the alemtuzumab arm in five of the first 10 patients.

Concomitant medications Premedication with antihistamines (e.g., 2 mg i.v. clemastin), paracetamol (500 mg p.o.) and prednisone (100 mg i.v.) was given with the first dose at each escalation and thereafter only if clinically indicated. Anti-infective prophylaxis including cotrimoxazole (960 mg p.o. twice daily, TIW) and famciclovir (250 mg p.o., twice daily) was given during and up to a minimum of 2 months following the discontinuation of alemtuzumab therapy.

Staging and follow-up At enrollment, a physical examination, an abdominal ultrasound and a full laboratory analysis of blood parameters was performed. For molecular MRD analysis by RT-PCR, peripheral blood was collected from all patients. During treatment, blood counts, physical examination and toxicity assessment were performed weekly. RT-PCR for assessment of MRD was scheduled at week 6 for patients in the alemtuzumab arm and for all patients at the end of treatment. During follow-up, physical examination was performed every 3 months and bone marrow examination was recommended to be repeated every 3– 6 months. RT-PCR from peripheral blood was planned to be performed 6, 12 and 24 months after randomization.

Interphase cytogenetic analysis and sequencing of clonal VDJ rearrangements Genomic aberrations were assessed by interphase FISH with a comprehensive set of DNA probes screening for the following aberrations: þ (3q26), del(6q21), þ (8q24), del(11q22–q23), t(11;14)(q13;q32), þ 12q13, del(13q14), t(14;18)(q32;q21), t(14q32) and del(17p13).21 Identification and sequencing of the clonal VDJ rearrangement to determine the VH mutational status and the CDR3 structure for MRD assessment was performed as previously described.22 Leukemia

Detection of minimal residual disease (MRD) by real-time PCR MRD detection was assessed as described previously.23 Briefly, real-time PCR was performed using an upstream allele-specific primer (aso-primer) matching the clonotypic CDR3 region and a consensus downstream JH primer and TaqMan-probe combination. Standard curves were generated by diluting DNA from samples taken at time of primary diagnosis into polyclonal genomic DNA. To equalize for differences in amount and quality of the patients DNA, an albumin TaqMan assay was additionally performed to measure the copy number of this gene as internal reference.

PNH immunophenotyping Flow cytometric analysis was performed on a FACScan (Becton Dickinson, BD, San Jose, CA, USA). Data acquisition was accomplished using CELL Quest software (BD). For PNH analysis, the following mAbs were used: CD55 (IA10; Pharmingen) and CD59 (MEM43; Cymbus Ltd.) on all cells, CD14 (MoP9; BD) on monocytes, CD16 (3G8; Immunotech) and CD24 (ALB9; Immunotech) on granulocytes.

CMV monitoring Before initiation of alemtuzumab, antibody response status against CMV antigens was tested. Furthermore, CMV-specific Tcell subsets were determined, as has been previously described.24–26 If at least one test was positive, a CMV-specific monitoring was performed by a weekly CMV-specific quantitative PCR of peripheral blood. Detection of 4500 copies/ml CMV-DNA was considered to be the threshold for treatment discontinuation even without clinical signs of CMV infection. Rising viral load in the following week was indicative for ganciclovir (10 mg/kg/day i.v., at least for 10 days) administration, while clinical CMV-disease was treated immediately. In any case, treatment of CMV resulted in discontinuation of alemtuzumab therapy.

Statistical methods Treatment efficacy was assumed upon achievement of a 25% improvement of PFS at 2 years. Based on a sample size of 45 patients for each arm, the log-rank test was calculated to have an 80% power to detect differences between the survival curves based on 23 months accrual time and 47 months overall length of the trial. Fisher’s exact test was used to compare qualitative parameters between subgroups of patients. The time from start of therapy (F or FC) to treatment failure was estimated according to the method of Kaplan and Meier.27 The 95% confidence intervals (CI) were calculated with Greenwood’s variances.28 Differences in survival parameters were calculated using the log-rank test.29 The comparison of treatment duration, cumulative alemtuzumab dosage and tumor load with rate of severe infections, response rate and MRD status was performed using the Mann–Whitney test. For all tests, Po0.05 was considered to be statistically significant.


1095 of treatment with alemtuzumab (cumulative target dose: 1033 mg) (Table 2a). The median cumulative dose of alemtuzumab administered to the patients was 283 mg (range, 213– 1033 mg).

Results

Demographics In all, 23 patients were recruited for this study, two patients refused initiation of study treatment after randomization and were excluded from analysis. Therefore, 21 patients with a median age of 60 years (range 37–66 years) were evaluable. All patients had received six cycles of F or FC as first-line therapy. A total of 11 patients with a median age of 60 years (range 38–63 years) was randomized to receive alemtuzumab (Table 1). Comparing patients’ characteristics between both groups, there were no statistically significant differences regarding age, sex, stage of disease, serum thymidine kinase (sTK) levels, cytogenetic aberrations by FISH analysis, VH mutational status, type of pre-treatment, response status to pre-treatment and WBC count at enrollment (P40.10).

Dosing of alemtuzumab All 11 patients randomized to alemtuzumab received at least 3 weeks of treatment with alemtuzumab after a median time interval of 67 days (range 45–90 days) after the last dose of F or FC. All patients were escalated to the final dose of 30 mg alemtuzumab TIW during the first week. Three patients (nos. 2, 7 and 9) received prolonged corticosteroids (cumulative dose of prednisone: 900, 475 and 750 mg, respectively) beyond the escalation phase due to allergic reactions during dosing of alemtuzumab. Only two of 11 patients completed all 12 weeks

Safety and Toxicity First-dose reactions: Maximum grade I first-dose side effects under alemtuzumab were seen in seven patients with nine documented events, grade II reactions occurred in eight patients with 12 documented events (Table 2b). No grade III/IV first-dose toxicity was documented. All other first-dose reactions have been described before.7–9 Hematologic toxicity: Severe hematologic toxicity with six events of CTC grade IV was documented in four of 11 patients receiving alemtuzumab (Table 2a, b). Three patients (nos. 2, 3 and 6) developed grade IV neutropenia with a median duration of 4 weeks (range 2 days to 48 weeks). Only one of them (no. 6) responded to a 7-day course of G-CSF and did not acquire infections subsequently. Two patients (nos. 1 and 2) were documented for grade IV thrombocytopenia requiring transfusions of platelets. Neutropenia and/or thrombocytopenia of CTC grade III was observed in five patients. One patient (no. 2) developed a pancytopenia, which resolved slowly after several months. At 3 months after the start of alemtuzumab, this patient showed loss of phosphatidylinositolglycan-anchored glycoprotein (PIG-AP) surface molecules (CD55, CD59) on monocytes (CD55: 47%, CD59: 31%), lymphocytes (CD55: 18%, CD59:

Table 1

Patients’ characteristics for arm A (alemtuzumab) and arm B (observation)

Patient

Age/sex

Arm A 1 2 3 4 5 6 7 8 9 10 11

51/M 48/F 63/M 61/M 61/M 43/M 61/F 60/F 63/M 38/M 60/M

B/II B/I B/II B/II A/II B/II A/II B/II B/II A/II B/II

Arm B 12 13 14 15 16 17 18 19 20 21

64/M 48/M 50/F 61/M 37/F 46/M 50/F 66/M 58/M 61/M

B/II C/IV A/I B/II B/II B/II B/II C/IV C/III B/II

Stage WBC (G/l) (Binet/Rai)

sTK (U/l)

Karyotype (FISH)

IgVH Pretreatment Response to pretreatment mutational status (residual BM infiltration in %)

4.6 5.7 2.3 4.4 10.3 3.4 4.3 2.5 5.5 4.9 4.2

6.8 8.8 11.1 10.0 46.7 7.8 25.2 9.2 67.0 34.3 55.3

11q, 13q +12q, 13q 6q, 11q, 13q Normal 11q 13q +12q, 13q Normal Normal 3q, 6q, t(14q32) 13q

+ + n.d. +

FC F FC F F FC FC F F FC FC

CR (11) PR (20) PR (20) PR (15) PR (n.d.) PR (n.d.) PR (12) PR (n.d.) PR (15) PR (17) PR (11)

CR (0) PR (n.d.) PR (n.d.) PR (0) CR (5) PR (34) PR (0) PR (n.d.) PR (15) CR (0) PR (n.d.)

3.2 4.6 5.6 4.9 4.8 2.0 3.3 3.2 7.8 7.9

18.5 17.9 6.4 19.0 2.5 10.7 34.1 7.1 20.1 n.d.

13q 11q, 13q 13q 13q, 14q Normal Normal 13q 6q, 11q, 14q +8q, 13q Normal

+ + n.d. n.d. +

FC FC F FC F F FC F FC F

CR (24) nPR (15) nPR (70) PR (n.d.) nPR (20) PR (30) PR (10) PR (40) CR (2) PR (35)

CR (n.d.) nPR (n.d.) PD (n.d.) PR (n.d.) nPR (n.d.) PR (n.d.) PR (n.d.) PD (90) CR (n.d.) PD (n.d.)

Response status 6 months after randomization (residual BM infiltration in %)

M ¼ male, F ¼ female; sTK ¼ serum thymidine kinase (normal: o7 U/l); IgVH ¼ variable region of the heavy chain of the immunoglobuline gene; WBC ¼ white blood cell count before start of CLL4B trial; F ¼ fludarabine, FC ¼ fludarabine/cyclophosphamide; CR ¼ complete remission, PR ¼ partial remission, nPR ¼ nodular partial remission, PD ¼ progressive disease; n.d. ¼ not done; + ¼ mutated IgVH; ¼ unmutated IgVH. Leukemia


1096 Table 2a Patient

Treatment duration and maximum toxicity (CTC grade I–IV) under/after alemtuzumab treatment Treatment duration (weeks)

Cumulative dose of alemtuzumab (mg)

1

3

223

2

4

293

3 4

11 4

943 236

5 6

12 6

1033 416

7

4

283

8 9

12 4

1033 213

10 11

3 3

223 223

Hematologic toxicity (at week)

Infection (at week)

Neutropenia III (3) Thrombocytopenia IV (3) Neutropenia IV (5) Thrombocytopenia IV (6) Anemia IV (12) Neutropenia IV (12) Neutropenia III (6) Thrombocytopenia II (6) None Neutropenia IV (8) Thrombocytopenia III (5) Anemia III (9) Neutropenia III (6) Thrombocytopenia III (4) Anemia II (3) Neutropenia III (12) Neutropenia II (4) thrombocytopenia I (2) Neutropenia II (4) Neutropenia II (6) Thrombocytopenia I (1) Anemia I (4)

CMV reactivation III (4) Aspergillosis IV (9) HSV/HHV-6 III (10) Herpes zoster III (17) TB III (7) Sinusitis I (7) None CMV pneumonia III (4) Gastroenteritis II (13) CMV reactivation II (8) Bronchitis II (4) CMV reactivation II (4) CMV reactivation III (6) CMV pneumonia III (6)

wk ¼ week(s) after initiation of alemtuzumab treatment; CMV ¼ cytomegalovirus; HSV ¼ herpes simplex virus; HHV-6 ¼ human herpes virus type 6; TB ¼ tuberculosis.

Table 2b

Maximum toxicity associated to alemtuzumab treatment No. of patients with CTC grade toxicity

Toxicity Neutropenia Thrombocytopenia Anemia Infection Nausea Vomiting Dyspnoe Hypotension Fever Chills Rash Diarrhoe Myalgias

Grade 1

Grade 2

Grade 3

Grade 4

0 2 1 1 0 0 1 0 1 2 3 1 1

3 1 1 4 1 0 0 0 6 1 1 1 2

4 2 1 7 0 0 0 0 0 F 0 0 0

3 2 1 1 F 0 0 0 0 F 0 0 F

28%), granulocytes (CD55: 45%, CD59: 33%) and to a lesser extent even on erythrocytes (CD55: 99%, CD59: 99%) corresponding to a phenotype seen in patients with paroxysmal nocturnal hemoglobinuria (PNH).30 In the control group, one patient (no. 19) developed thrombocytopenia II 12 months after randomization due to disease progression and was subsequently enrolled on a relapse protocol.

Infectious complications: Alemtuzumab consolidation was associated with an increased incidence in infectious complications. After a median of 4 weeks, alemtuzumab treatment had to be interrupted due to severe infections in seven of 11 patients and forced to stop the trial. Infections observed were one lifethreatening CTC grade IV pulmonary aspergillosis and seven CTC grade III infections including four CMV reactivations, one HSV/HHV-6 infection, 1 herpes zoster infection and one reactivation of pulmonary tuberculosis (Table 2a). In detail, we observed a grade IV pulmonary aspergillosis combined with Leukemia

a pulmonary HSV/HHV-6 infection at week 9 after initiation of alemtuzumab in a patient (no. 2), whose treatment was interrupted after 4 weeks due to severe hematologic toxicity. Infection was life-threatening and pulmonary disease finally responded to antibiotic therapy including a 6-month course of oral voriconazol. In a patient (no. 4) suffering from pneumonia, acid-resistant rods were cultured in broncho-alveolar lavage fluid 7 weeks after treatment start consistent with a pulmonary tuberculosis. After initiation of treatment with isoniazid (INH), rifampicin, pyrazinamide and ethambutol, pulmonary disease was controlled. Another patient (no. 3) presented with severe herpes zoster infection 17 weeks after start of alemtuzumab requiring systemic treatment with acyclovir. Four patients showed severe CMV reactivation detected by CMV-specific PCR,31,32 and required i.v. ganciclovir treatment because of rising viral load combined with fever (nos. 1 and 10) or because of CMV pneumonia (nos. 7 and 11) (Table 2a). CMV pneumonia was diagnosed in two patients who had increased CMV titer by CMV-specific PCR, positive CMV antigen (pp65), radiologic signs of pneumonia and clinically suffered from fever, dyspnea and coughing. Although a bronchoalveolar lavage was not performed, from a clinical perspective a CMV pneumonia was assumed. Ganciclovir therapy resolved CMV symptoms and CMV-DNA declined to baseline levels by week 8 in all treated patients. Importantly, all patients with CMV reactivation were tested positive by serology before alemtuzumab treatment and were documented to have measurable CMV-specific T-cell frequencies before study initiation (Table 3). T-cell subsets, although assessment was not mandatory on study protocol, were analyzed in some individual patients. While all patients assessed had undetectable T-cell counts during alemtuzumab treatment, some differences were apparent after consolidation treatment was discontinued. Patient 5 had a CD4 and CD8 count both below 60 cells/ml 2 months after alemtuzumab therapy, while he did not suffer from severe infections. At 6 months after therapy, CD4 and CD8 counts increased over 200 cells/ml each. In contrast, patient 7 showed a CD4/8 counts of 116 and 110 cells/ml, respectively, 2 months


1097 Table 3 Monitoring of CMV reactivation before, during and after alemtuzumab treatment detected by PCR (CMV-specific DNA) and flow cytometry (CMV-specific CD4/CD8+ T cells) Before alemtuzumab Patient

1 2 3 4 5 6 7 8 9 10 11

During alemtuzumab

Serology

CMV-specific T-cell frequency in % CD4/CD8

CMV-specific DNA (Geq)

+ + n.d. + + + + +

2.00/n.d. 0.12/1.08 0/0 0/0 n.d. 0/0 0.13/0 n.d. 0.85/0.95 n.d. 0.15/0.04

0 0 0 0 n.d. 0 0 n.d. 0 0 0

Maximum CMV-specific DNA (Geq)

3100 (week 4) 90 (week 6) 0 n.d. 0 n.d. 800 (week 4) 1700 (week 8) 450 (week 5) 10250 (week 6) 1700 (week 6)

After alemtuzumab Resolution of Treatment with CMV-specific ganciclovir DNA to baseline levels week week F F F F week n.d. week week week

7 8

7 8 8 8

Yes No F F F F Yes No No Yes Yes

n.d. ¼ not done; wk ¼ week after start of alemtuzumab; Geq ¼ genome equivalents/ml.

after discontinuation of alemtuzumab and acquired a severe CMV pneumonia, as described. After complete resolution of the infection 4 months after start of alemtuzumab, CD4/8 counts were documented to be 284 and 258 cells/ml, respectively. From these individual observations, it can only be concluded that alemtuzumab can significantly decrease T-cell counts for several months, but no direct correlation can be drawn from the absolute decrease in CD4/CD8 counts and the risk for severe infections. In the control group who did not receive alemtuzumab, only two episodes of infections were recorded: one patient (no. 15) experienced a herpes zoster infection grade II 10 months after randomization. Another patient (no. 21) was documented with a sinusitis grade I at month 6 after randomization, while symptoms resolved without antibiotic treatment. Although based on small sample sizes, patients with severe infections did not differ from those with only mild (grade I/II) or no infectious complications with respect to cumulative dose of alemtuzumab (P ¼ 0.31) or response status after pre-treatment with F or FC (P ¼ 1.0). Besides infections, no other severe nonhematologic toxicity occurred during alemtuzumab treatment. During observation time, no toxic death was reported. In summary, four patients (nos. 1, 2, 3 and 6) were prematurely withdrawn from the treatment plan due to severe myelosuppression (one thrombocytopenia IV, three neutropenia IV), which did not resolve after an interval of 14 days. Alemtuzumab was stopped in three patients due to CMV reactivation grade III (nos. 7, 10, 11), in one patient (no. 4) because of TB reactivation and in another one (no. 9) because of a bacterial bronchitis slowly responding to oral antibiotics.

Efficacy Clinical response and progression-free survival: At month 6 after randomization, three patients were in CR and eight in PR in the alemtuzumab consolidation arm, while the observation group comprised two patients with CR, two patients with nPR, three patients with PR and another three patients with progressive disease (Table 1). All eight patients with PR after alemtuzumab treatment had residual splenomegaly, one patient (no. 6) showed residual lymphadenopathy in addition. Although patient number was small, there was a trend for a difference in

Figure 1 Progression-free survival (PFS) in CLL patients receiving alemtuzumab consolidation (n ¼ 11; dashed line) and no further consolidation treatment (observation) (n ¼ 10; solid line) after initial fludarabine-based chemotherapy; P ¼ 0.036.

response status between both arms, which was not statistically significant (11/11 vs 7/10, P ¼ 0.059). Importantly, two of 11 patients (nos. 5 and 10) improved their clinical response status after alemtuzumab treatment converting from PR to CR. Including bone marrow examination, another patient (no. 1), who entered the trial with residual lymphocytes in his bone marrow, showed clearance of lymphocytes by morphological studies, while a true molecular response by four-color-flow cytometry or PCR of the CDRIII region was not assessed in our study. Two patients (nos. 4 and 7) with residual splenomegaly after alemtuzumab consolidation showed clearance of their bone marrow by alemtuzumab. For all patients in the alemtuzumab arm, response was not significantly associated with duration of treatment (P ¼ 0.49), cumulative dose of alemtuzumab (P ¼ 0.78) or response to pretreatment (P ¼ 0.11). While in the alemtuzumab arm no patient progressed, follow-up of patients in the control arm revealed a progressive disease in four patients. At a median follow-up of 21.4 months from start of chemotherapy with F or FC, all patients were alive; therefore no difference in overall survival was seen between the two study arms. Progression-free survival (PFS) showed a significant difference in favor of patients who received alemtuzumab (P ¼ 0.036) (Figure 1). The mean PFS time for the observation Leukemia


1098 group was 24.7 months (95% CI, 19–30.3 months), while the PFS estimates could not be calculated for the consolidation group since all observations were censored, that is, no progression of disease occurred to date. The difference between both groups was statistically not associated with age, sex, disease stage, response to F or FC, VH mutational status or cytogenetic aberrations (P40.10). When calculating PFS from initiation of alemtuzumab consolidation with a limited median follow-up time of 14.4 months, PFS was also significantly longer in the alemtuzumab treatment arm (no progression vs mean 17.8 months, 95% CI 12.5–23 months; P ¼ 0.036).

Eradication of minimal residual disease (MRD): In eight patients, a quantitative allele-specific IgH RT-PCR from peripheral blood samples was evaluable, in a further patient a quantification was not possible (Table 4). Tumor burden could be reduced by pre-treatment with F or FC in all three patients in the observation arm, but RT-PCR remained positive during follow-up. In contrast, five of six patients in the alemtuzumab arm with a median treatment time of five weeks showed MRD negativity in peripheral blood at a median of 6 weeks after start of alemtuzumab. These five patients were in clinical PR due to residual splenomegaly or lymphadenopathy. One patient turned MRD negative 18 weeks after start of alemtuzumab, further

Table 4 Detection of MRD by RT-PCR of CDRIII rearrangement in peripheral blood of patients treated with alemtuzumab (arm A) and in control group (arm B) Patient

Treatment duration (wks)

Time (wks after randomization/start of alemtuzumab)

MRD

Quantitative MRD level*)

Arm A 1

3

Before FC 4 weeks after FC week 6/0 week 24/18 week 43/37

+ + + +

5.0 101 5.4 104 5.7 104 o2.6 105 6.0 106

3

11

Before FC 6 weeks after FC week 15/11

+ +

1.0 100 1.9 104 o5.0 105

6

6

Before FC 9 weeks after FC week 8/5 week 11/8

+ +

n.d. n.d. n.d. n.d.

8

12

Before F 5 weeks after F week 2/0 week 8/6 week 14/12 week 29/27

+ + +

1.4 100 7.7 103 1.8 102 o1.58 105 o4.8 105 o9.7 105

9

4

Before F 12 weeks after F week 9/5 week 10/6 week 12/8 week 14/10 week 16/12

+ +

1.0 101 4.9 102 o5.3 105 o1.6 104 o2.5 105 o2.4 105 o1.5 105

11

3

Before FC 6 weeks after FC week 7/2 week 8/3 week 12/7

+ +

1.6 100 2.5 103 o5.0 104 o4.0 105 o4.0 105

Arm B 12

F

Before FC week 16/week 52/-

+ + +

1.3 2.2 103 3.1 103

17

F

Before F week 12/week 27/-

+ + +

4.5 101 5.6 102 1.0 101

18

F

Before FC 7 weeks after FC week 12/week 24/-

+ + + +

3.0 101 1.0 103 3.4 104 3.9 104

MRD ¼ minimal residual disease; wk(s) ¼ week(s); F ¼ fludarabine; FC ¼ fludarabine/cyclophosphamide *denotes MRD-level in MRD-positive patients (+) and sensitivity level in MRD-negative patients (). Leukemia


1099 follow-up revealed a molecular relapse at week 37, although at a very high sensitivity level (6 106). At 12-month-follow-up this patient is still in CR with morphologically normal bone marrow. Two patients (nos. 6 and 9) showed clearance of CLL cells in their peripheral blood as assessed by PCR, despite the fact that their bone marrow showed residual disease by morphology at the same time point. Based on small patient numbers, there was no significant association between MRD status in peripheral blood and duration of treatment (P ¼ 0.50) or the cumulative dose of alemtuzumab given (P ¼ 0.83). The differences regarding eradication of MRD in peripheral blood between alemtuzumab treatment arm and control group were significant (P ¼ 0.048). MRD status was not associated with other factors, especially not with the type of pre-treatment (F vs FC) (P ¼ 1.0).

Discussion This randomized phase III trial shows that a consolidation with alemtuzumab in a standard dose of 30 mg i.v. TIW in CLL patients responding to fludarabine-based chemotherapy is associated with an increased incidence of severe infections and myelotoxicity. Therefore, this multicenter trial was prematurely closed. The explanations for this unexpected result are manifold: the number of fludarabine cycles might have been too high, and the time for regeneration after chemotherapy too short. The time interval between the last dose of fludarabine and start of alemtuzumab was quite short with a median elapse time of only 67 days. In another trial using alemtuzumab for consolidation, the median time from fludarabine discontinuation to start of alemtuzumab consolidation was much longer, that is, 5 months (range 2–11 months).33 It has to be taken into account that bone marrow and T-cell function may be impaired for several months after therapy with purine analogues.34 Therefore, less extensive pre-treatment in combination with long time intervals between last chemotherapy and start of alemtuzumab consolidation might be preferable. Although we could not prove a statistical association between the incidence of infections and cumulative dose of alemtuzumab in our trial, it has to be noted that lower cumulative doses of alemtuzumab for consolidation were used in other trials, which did not show high incidences of infections. Dyer et al33 described six patients receiving a median of 6 weeks of alemtuzumab, with no major infections being reported.13 In an Italian study consolidating CLL patients with alemtuzumab, patients received alemtuzumab three times a week for 6 weeks in escalating doses up to 10 mg. In an US trial, a schedule of three times weekly 10–30 mg for 4 weeks was used.35 Therefore, it seems that lower doses of alemtuzumab in a consolidation setting may be equally efficacious without producing major infections. Another argument for high infection rates in our series is that the tumor load before alemtuzumab might have impacted negatively on our results. Previously, it was shown that a higher tumor load before alemtuzumab treatment correlated not only with an inferior response rate, but also with a higher incidence of infections (Albitar et al, Blood 2002; 100: 804, abstract). The majority of our patients entered the trial with a PR status; in the alemtuzumab arm, 10 of 11 patients had palpable residual lymphadenopathy and/or organomegaly. In the previously mentioned trial by O’Brien et al only 21 of 41 patients were in PR, while the other patients received alemtuzumab treatment with an nPR or CR status.35 Although 15 patients (37%) developed infectious complications, this rate was still lower than in our trial. The route of application for alemtuzumab

might also impact on side effects, since longer time periods and a higher amount of drug is required after s.c. administration until similar trough levels are reached compared to infusion of alemtuzumab (Hale et al, Blood 2002; 100: 207, abstract). Montillo et al33 described nine patients who were consolidated with s.c. alemtuzumab at 10 mg TIW over 6 weeks. An immunophenotypic CR was achieved in most patients including also molecular CRs. No severe infectious episodes were recorded, although three patients developed CMV reactivation of grade II being treated with oral ganciclovir. Furthermore, patients’ inclusion criteria for treatment with monoclonal antibodies should be critically checked for concomitant risk factors: one patient suffered from TB reactivation after a past history of tuberculosis, which was not reported when the patient entered the trial. Finally, an extended premedication with corticosteroids beyond the dose escalation phase could have supported the development of severe infections at least in one patient in our series: a patient suffering from aspergillosis and an HSV/HHV-6 infection received a cumulative dose of 900 mg of prednisone. In other trials in which prolonged use of corticosteroids was discouraged and only applied on an as-needed basis for infusion-related reactions, more severe infusion-associated adverse effects were recorded, but the rate of severe infections was low.9 Also in the trial by Montillo et al33 where no severe infections were documented, steroids were not routinely used for premedication. Comparing safety data from the presented consolidation trial with use of alemtuzumab even in combination with fludarabine – although in a different group of patients, that is, patients with relapsed or refractory B-CLL – reveals that alemtuzumab can be safely combined with other drugs, but patient population and schedule have to be well defined. In this trial, the combination of fludarabine and alemtuzumab was well tolerated with only one severe infection and no clinical apparent CMV reactivations (Elter et al, Blood 2002; 100: 803). In these patients with an even significant higher tumor load compared to patients enrolled in our consolidation trial, the dosage of alemtuzumab was quite lower (maximum cumulative dose of 360 mg) and the drug was not given continuously at a weekly base, but in monthly intervals. The major infectious complication of alemtuzumab in our trial was the reactivation of CMV detected by quantitative PCR. Importantly, CMV-specific T cells detectable before initiation of alemtuzumab did not preclude development of CMV reactivation. In the CALGB Trial 19901, based on 36 patients receiving alemtuzumab (30 mg TIW over 6 weeks), CMV infection was the most frequent cause of nonhematologic toxicity, being even fatal in one case (Rai et al, Blood 2002; 100: 205, abstract). Both studies underline the necessity of vigorous surveillance for CMV infection using weekly quantitative PCR-based testing. Most patients being treated with alemtuzumab developed mild to severe neutropenia before they suffered from infectious complications. Two patients with severe neutropenia grade IV suffered from herpes viral infections; in another patient with grade IV neutropenia, it can be speculated that use of G-CSF might have prevented development of infectious complications. Interestingly, one of the patients with severe neutropenia in our study experienced a long-lasting pancytopenia and her blood cells were found to express a phenotype resembling PNH. Similar immunophenotypic changes were described for patients with rheumatoid arthritis and refractory B-NHL including B-CLL receiving alemtuzumab.30,36,37 PIG-AP-deficient cells were not only observed in T cells and monocytes,30 but also in granulocytes and erythrocytes which showed diminished expression of CD52. It is hypothesized that alemtuzumab, Leukemia


1100 which targets the PIG-AP CD52, leads to the expansion of a PIG-anchor-deficient cell clone with the phenotypic characteristics of PNH cells. In future trials, it has to be examined whether patients with a PNH immunophenotype are at increased risk for severe infectious complications. In this patient, a hemophagocytic syndrome based on the observed pancytopenia was formerly ruled out by bone marrow biopsy, which did not show typical signs of a marked histiocyte hyperplasia with hemophagocytosis.38 Nevertheless, EBV-related hemophagocytosis can be observed after alemtuzumab treatment of patients with peripheral T-cell lymphomas, as has been reported recently.39 A residual lymphadenopathy after alemtuzumab treatment was observed in most patients of our study. Only two of 11 patients converted from PR to CR after alemtuzumab therapy, resulting in a low response rate of only 18%. Interestingly, two of the patients in PR with residual splenomegaly after alemtuzumab consolidation showed a clearance of their bone marrow. This is in line with several reports describing a high efficacy of alemtuzumab for bone marrow and peripheral blood, while disease eradication is incomplete in nodal masses: Based on a large trial from the MD Anderson Cancer Center, only two of 13 patients with lymph node disease responded, while most responses occurred in patients who had only residual bone marrow disease.35 This emphasizes the importance of targeted combination regimens to reduce disease involvement in all sites.8 Patients who received F or FC with alemtuzumab had a significantly higher rate of molecular remissions in peripheral blood than patients who received only F or FC. MRD negativity was attained at a high sensitivity level by use of allele-specific primers, in contrast to use of less-sensitive consensus primer PCR, and compares favourably with MRD levels after high-dose chemotherapy. Nevertheless, we recognize that assessment of MRD in peripheral blood is only a surrogate marker and that clearance of bone marrow by antibody treatment is crucial. In future trials, it has to be prospectively assessed whether MRD analysis in peripheral blood which is more convenient and costeffective mirrors changes in bone marrow samples, as has been shown for MRD assessment in other leukemia entities.40–42 Although based on a few patients due to incomplete accrual, a significant trend towards longer PFS for alemtuzumab-treated patients was seen at a median follow-up of almost 2 years. We are fully aware that these results have to be taken cautiously and that this trend needs to be confirmed after a longer follow-up, especially in light of residual splenomegaly and/or lymphadenopathy in most patients. Although there are several limitations to these data, this is the first observation in a randomized setting of the potential benefit of an alemtuzumab consolidation approach. The dosing and scheduling of alemtuzumab in consolidation therapy of CLL are not well defined. Only few reports exist on pharmacokinetics and pharmacodynamics.43,44 Toxicity observed in our trial should deter from the use of alemtuzumab consolidation treatment of patients with residual disease outside a clinical trial. A phase I/II trial of the GCLLSG evaluating escalating doses of alemtuzumab in different application routes combined with close detection of MRD in peripheral blood and bone marrow, CMV status and plasma levels of alemtuzumab will determine how this antibody can be used safely in consolidation therapy of CLL. Besides alemtuzumab, other monoclonal antibodies like rituximab, epratuzumab or apolizumab have to be studied for their safety profile and efficacy in controlling MRD in CLL patients.45 Leukemia

Acknowledgements We are grateful to the following colleagues taking care of patients being enrolled on study protocol: Dr Abenhardt, Munich; PD Dr Benz, Tu¨bingen; Professor Dr Du¨hrsen, Essen; Dr Fiedler, Chemnitz; Dr Freier, Hildesheim; Dr Hopfinger, Wien; Dr Kamp, Wendlingen; Dr Lambertz, Garmisch-Partenkirchen; Dr Lindemann, Hagen; Dr Middecke, Lemgo; PD Dr Mittermu¨ller, Munich; Dr Pasold, Potsdam; Dr Srock, Berlin; Dr Stauch, Kronach; Dr Ziegler, Erlangen. We appreciate especially the support of D. Egger and Dr Grassmann, Medac-Schering Onkologie GmbH, and Dr Kuhlmann, Schering AG. We thank Dr Hillmen and Dr Hale for inspiring discussions. This study was supported by a grant from Schering AG and ILEX Oncology.

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