An open-label, pilot study of fludarabine, cyclophosphamide, and alemtuzumab (FCC) in relapsed/refractory patients with B-cell chronic lymphocytic leukemia

Although combination regimens have improved outcomes over monotherapy in chronic lymphocytic leukemia (CLL), patients eventually relapse. Combined fludarabine, cyclophosphamide, and monoclonal anti-CD52 antibody alemtuzumab (FCC) provided synergistic cytotoxicity with effective clearing of minimal residual disease. This phase 2 study determined FCC efficacy and safety in relapsed/refractory CD52-positive B-CLL following ≥ 1 line of treatment. From January 2005 through June 2008, up to six courses of oral fludarabine 40 mg/m 2 /d, oral cyclophosphamide 250 mg/m 2 /d, and subcutaneous alemtuzumab (Mab-Campath ® ) 10 mg (increased to 20 mg after first 10-patient cohort) were administered days 1 to 3 every 28 days. The primary objective was overall response rate (ORR); secondary objectives included response duration, time to disease progression, and safety and tolerability. ORR was 67% in 43 patients; 30% achieved complete response (CR). ORR significantly improved with one versus therapies ( P =.018), and without versus with previous monoclonal antibody treatment ( P =.003). Median progression-free survival was 24.4 months, not reached in patients achieving CR. Median overall survival was 33.6 months. Myelosuppression was the most common adverse event, with a low percentage of cytomegalovirus reactivations and manageable infections. However, a close vigilance of opportunistic infections is warranted. FCC provides effective immunotherapy in relapsed/refractory CLL, including in patients with poor-risk prognostic factors.


Introduction
B-cell chronic lymphocytic leukemia (B-CLL) constitutes a third of adult leukemic malignancies, with an age-adjusted incidence rate of 4.2 per 100,000 men and women per year in the United States. 1,2 Approximately 15,000 new cases of chronic lymphocytic leukemia (CLL) are diagnosed each year 1,2 in the United States, with similar rates in Europe. Patients usually respond well to initial therapy; however, progressively shorter times to relapse or progression are typical until patients eventually become refractory to treatment.
Fludarabine is an effective treatment for CLL, leading to high overall response rates (ORRs) of 60% to 80% for patients in first-line treatment and 45% to 60% in previously treated patients. [3][4][5][6] In vitro, fludarabine combined with cyclophosphamide demonstrates additive or synergistic cytotoxic effects in leukemic cells. 7,8 This cell-killing activity, led to their development as a combination therapy, 9 demonstrating an improvement of patient outcomes and remission times.
Continued investigation of single-agent fludarabine versus fludarabine plus cyclophosphamide (FC) showed significantly improved response rates and progressionfree survival (PFS) 10-12 in first-and second-line settings for FC combination . Despite these improvements compared with historical therapy, minimal residual disease (MRD) is detectable even in patients achieving a complete response (CR), leading to eventual relapse.
The monoclonal antibody alemtuzumab is one of several agents demonstrating evidence of the ability to eradicate MRD and affect overall survival (OS) in CLL. 13,14 Alemtuzumab targets cells positive for CD52, an antigen present in high levels on a For personal use only. on August 10, 2017. by guest www.bloodjournal.org From majority of normal and malignant T-and B-cell lymphocytes, but not hematopoietic stem cells. [15][16][17] Single-agent alemtuzumab showed durable ORRs and CR rates in first-line (83%-87% ORR, 19%-24% CR) or relapsed or refractory (33%-34% ORR, 2%-4% CR) CLL, including in patients refractory to prior fludarabine treatment. [18][19][20][21] Alemtuzumab plus fludarabine demonstrated significant clinical activity and achievement of MRD negativity in patients refractory to either monotherapy. 22,23 The greatest challenge in CLL is to provide a treatment regimen maintaining durable hematologic and molecular remission while overcoming potential drug resistance. This study's goal was to examine therapeutic efficacy and safety effects of combined fludarabine, cyclophosphamide, and alemtuzumab (FCC) in patients with relapsed or refractory CLL.

Materials and methods
This was a single-arm, open-label phase 2 study of the combination oral fludarabine, oral cyclophosphamide, and subcutaneous (SC) alemtuzumab for patients with refractory or relapsed B-CLL following ≥ 1 line of chemotherapy including alkylating agents, purine analogs, alone or in combination, immunochemotherapy including rituximab or single-agent alemtuzumab. All patients provided informed written consent in accordance with the Declaration of Helsinki and the institutional guidelines of each participating site.

Patients
Male or female subjects ≥ 18 years of age with confirmed CD52-positive B-CLL (defined as peripheral lymphocyte count >5×10 9 /L and clonal CD5, CD19, CD23, and weak Patients were excluded if they had no previous treatment with chemotherapy or immunotherapy, or had received prior investigational agents, stem cell transplant or alemtuzumab combined with chemotherapy. Also excluded were patients with fewer than 3 weeks since last treatment, HIV positivity or active viral hepatitis C or B or other active infection, or autoimmune anemia and/or thrombocytopenia poorly responsive to corticosteroid therapy.

Study objectives
The primary objective of the study was to determine ORR (CR or PR) following combination treatment with oral fludarabine, oral cyclophosphamide, and SC alemtuzumab. Secondary objectives included duration of response (DR) in responders, time to disease progression (TTP), and safety and tolerability.

Study design and treatment
Up to six courses of combination treatment were repeated every 28 days (Table 1), including oral fludarabine 40 mg/m 2 /d, oral cyclophosphamide 250 mg/m 2 /d, and SC For personal use only. on August 10, 2017. by guest www.bloodjournal.org From alemtuzumab (MabCampath ® ) 10 mg on days 1 to 3. According to the described schedule's safety profile, after the first cohort of 10 treated patients the alemtuzumab dose was increased from 10 to 20 mg. Alemtuzumab therapy began with dose escalation (3, 10, 20 mg) beginning 2 days before chemotherapy day 1.
Premedication included oral paracetamol 1 g and intravenous (IV) chlorphenamine 10 mg given 30 to 60 minutes before alemtuzumab. Tumor lysis syndrome prophylaxis with allopurinol 300 mg/d was recommended for ≥ 28 days of therapy. Anti-infective prophylaxis included acyclovir 400 mg twice daily and trimethoprim sulfamethoxazole 1000 mg every other day from treatment initiation until 6 months after treatment end.
Patients with antigen-positive cytomegalovirus (CMV) received oral valganciclovir 400 mg for at least 3 weeks or 2 weeks after they became antigen negative.
The fludarabine dose may be 50% reduced for patients with creatinine clearance of 30 to 60 mL/min. In the event of hematologic toxicity (ie, neutrophils <1.0x10 9 /L, platelets <75x10 9 /L), treatment was delayed ≤ 2 weeks and doses reduced 25% for subsequent cycles; the dose was reduced another 25% (to 50% of original dose) if further grade 3/4 hematologic toxicity occurred. Granulocyte colony-stimulating factor was allowed per physician discretion. Alemtuzumab was not dose-reduced.

Response and survival assessments
Disease response was evaluated 2 months after treatment discontinuation. Efficacy was assessed by ORR, comprising CR and PR, as defined according to National Cancer Institute Working Group (NCI-WG) response criteria 24 . Response assessments included clinical examination, CT scan of lymph node regions involved at baseline, and peripheral blood evaluation. Bone marrow biopsy confirmed the CR.
For personal use only. on August 10, 2017. by guest www.bloodjournal.org From Patients with PD per clinical parameters (eg, lymphocytosis, lymphadenopathy, splenomegaly) could discontinue treatment after 2 cycles. Patients with clinical SD after 3 cycles could continue treatment at the investigator's discretion. In case of clinical response achievement after at least four cycles administered, at physician discretion, treatment could be discontinued and patients underwent disease reassessment.
MRD was assessed in patients achieving CR. Bone marrow was analyzed at the time of response evaluation; analyses via quadruple antibody detection followed the method described by Rawstron et al. 25

Safety
Patients were monitored before, during, and following each cycle and throughout the 4-

Statistical analysis
Absence of MRD was defined as CR(MRD-), with no detection of neoplastic B cells by multichannel flow cytometry on bone marrow cells. DR was defined for all responding patients (CR or PR) as time from date of first response assessment to date of documented progression, death from any cause, or relapse. Differences in response rate between groups were analyzed by Fisher's exact test; association of continuous or discrete variables with the end points was measured with Wald's test after logistic regression. TTP was measured from treatment initiation in responding patients to disease progression. OS was measured from treatment initiation until death. Time to retreatment in responding patients was measured from end of FCC to start of a further treatment line. Time-to-event data (TTP, DR) and survival were analyzed using the Kaplan-Meier method. Confidence intervals for median time were calculated with nonparametric methods. Statistical significance was assumed for P<.05.

Baseline demographics and clinical characteristics
From January 2005 through June 2008, 43 patients were enrolled in this phase 2 study at 10 centers in Italy. Their median age was 60 years, and 60% were male (

Response
In all 43 patients, the ORR was 67%, with 30% of patients achieving CR (Table 3).
Fourteen percent of patients maintained SD, whereas 18.5% progressed. Pretreatment variables associated with response achievement are listed in Table 4. The ORR was significantly higher in patients receiving only one prior line of therapy (100%) compared with those receiving two (70%) or more (38%) prior treatments (P=.018). No difference in ORR was observed between patients with fludarabine-refractory disease versus patients with fludarabine-sensitive disease. In contrast, patients who received previous monoclonal antibody treatment showed a significantly inferior ORR compared with For personal use only. on August 10, 2017. by guest www.bloodjournal.org From those without prior monoclonal antibody therapy (38% vs 85%, respectively; P=.003).
Longer time from last treatment to FCC initiation indicated a significantly higher probability of attaining a response (cutoff 21 months; P=.018).
No clinical or biological variables had prognostic significance for response achievement.
Moreover, no considered variables affected the attainment of CR. MRD-negative CR was observed in six patients. Notably all but one patient received two or more previous lines of therapy; all had received previous fludarabine, demonstrating treatmentsensitive disease in all cases. In 29 responding patients, 8 observed deaths were related to disease progression in 5 patients, Richter's syndrome in 2, and late pulmonary tuberculosis in 1. Among nonresponding patients, 11 deaths were recorded and were due to PD in 10 patients and pneumonia in 1 patient.

Survival and time to retreatment
Grade 1-2 AEs related to SC alemtuzumab administration included skin reactions in 43 courses (28%), fever in 39 courses (25%), and hypotension in 1 course. As previously reported, one case of grade 3/4 hypotension was observed. Over a third of skin reactions (37%) and fever (44%) were experienced during the first therapy cycle.

Discussion
Superiority in number of responses and PFS following combined fludarabine and cyclophosphamide, compared with single-agent fludarabine, has been confirmed by numerous phase 3 trials in CLL. 10-12 Alemtuzumab induces a response in relapsed or refractory disease, although patients with bulky lymphadenopathy usually do not respond to therapy. 14 However, alemtuzumab plus fludarabine showed increased response rates compared with either single agent 22 and represents a promising chemoimmunotherapeutic approach with an acceptable toxicity profile. 23 The study's major limitation was that biologic risk factors (IgVH, FISH, and ZAP70) were not determined. Poor prognostic factors were strongly represented in our series, including 68% unmutated IgVH and 46% del17p or del11q. Importantly, after FCC treatment, response achievement was independent of mutational status or cytogenetics; ORR was 79% in patients with unmutated IgVH, and 67% in patients with del17p or del11q. These data are similar to the results from the Badoux et al phase 2 trial of using the same combination without cyclophosphamide and a higher alemtuzumab dose. In addition, among our patients who achieved a CR, 83% showed a persistent response after a median follow-up of 23.3 months. The increasing importance conferred to MRD eradication in patient outcomes has been confirmed in the six patients who For personal use only. on August 10, 2017. by guest www.bloodjournal.org From As previously reported, SC alemtuzumab demonstrated at least equivalent efficacy compared with IV alemtuzumab with reduced AEs. 19 In our experience, the SC regimen was well tolerated. Skin reactions at the injection site were mild. Myelosuppression is commonly the most frequent toxicity in these studies. In the present study, grade 3/4 neutropenia was detected in 46% of courses; this is comparable with other studies of previously treated patients with chemo-immunotherapeutic regimens. 27,28 A major concern is the low percentage of patients who completed the expected six courses of treatment. However, the reason for halting therapy prior to the planned six cycles in eight cases was the achievement of response in fewer treatment cycles.
Furthermore, in our series more than one-half of the patients (16 of 29) showing an objective response had been treated with less than six courses. This may lead to the speculation that when planning treatment with this combination, a lower number of courses should be considered. Based on these study results, it is our opinion that the use of the FCC combination is recommended in selected relapsed or refractory patients such as those showing unmutated IgVH status, and high risk genetic abnormalities.

Acknowledgments
We would like to thank the following for research support of this study: Schering AG Berlin, Germany; Bayer Healthcare AG, Leverkusen, Germany; and Genzyme Corporate, Cambridge, USA. The authors would like to thank Julie Kern, PhD, for editorial support, Chiara Colombo, PhD, for data management, and Michele Nichelatti, PhD, for statistical analysis.   Statistically significant difference between patients treated with 1 prior line of therapy vs those treated with > 1 prior line of therapy (P < .05). ‡ Statistically significant difference between patients treated with monoclonal antibodies vs no monoclonal antibodies (P < .05).
For personal use only. on August 10, 2017. by guest www.bloodjournal.org From Table 5  For personal use only. on August 10, 2017. by guest www.bloodjournal.org From