VOLUME 23 NUMBER 18 JUNE 20 2005

JOURNAL OF CLINICAL ONCOLOGY ORIGINAL REPORT

Early Results of a Chemoimmunotherapy Regimen of

Fludarabine, Cyclophosphamide, and Rituximab As
Initial Therapy for Chronic Lymphocytic Leukemia
Michael J. Keating, Susan O’Brien, Maher Albitar, Susan Lerner, William Plunkett, Francis
Giles, Michael Andreeff, Jorge Cortes, Stefan Faderl, Deborah Thomas, Charles Koller,
William Wierda, Michelle A. Detry, Alice Lynn, and Hagop Kantarjian
From the Departments of Leukemia,
A B S T R A C T
Hematopathology, Experimental
Therapeutics, Blood and Marrow
Transplantation, and the Biostatistics Purpose
and Applied Mathematics, The Univer- Fludarabine and cyclophosphamide (FC), which are active in treatment of chronic lympho-
sity of Texas M.D. Anderson Cancer cytic leukemia (CLL), are synergistic with the monoclonal antibody rituximab in vitro in
Center, Houston, TX. lymphoma cell lines. A chemoimmunotherapy program consisting of fludarabine, cyclophos-
Submitted December 9, 2003; phamide, and rituximab (FCR) was developed with the goal of increasing the complete
accepted November 11, 2004. remission (CR) rate in previously untreated CLL patients to $ 50%.
Supported in part by a grant from Patients and Methods
Genentech Inc.
We conducted a single-arm study of FCR as initial therapy in 224 patients with progressive
Authors’ disclosures of potential con- or advanced CLL. Flow cytometry was used to measure residual disease. Results and
flicts of interest are found at the end of safety were compared with a previous regimen using FC.
this article.
Results
Address reprint requests to Michael
Keating, MB, BS, Department of Leuke-
The median age was 58 years; 75 patients (33%) had Rai stage III to IV disease. The CR
mia, The University of Texas M.D. rate was 70% (95% CI, 63% to 76%), the nodular partial remission rate was 10%, and the
Anderson Cancer Center, 1515 Holcombe partial remission rate was 15%, for an overall response rate of 95% (95% CI, 92% to 98%).
Blvd, Unit 428, Houston, TX 77030; e- Two thirds of patients evaluated with flow cytometry had less than 1% CD5- and CD19-
mail: mkeating@mdanderson.org. coexpressing cells in bone marrow after therapy. Grade 3 to 4 neutropenia occurred during
© 2005 by American Society of Clinical 52% of courses; major and minor infections were seen in 2.6% and 10% of courses,
Oncology respectively. One third of the 224 patients had $ one episode of infection, and 10% had a
0732-183X/05/2318-4079/$20.00 fever of unknown origin.
DOI: 10.1200/JCO.2005.12.051 Conclusion
FCR produced a high CR rate in previously untreated CLL. Most patients had no detectable
disease on flow cytometry at the end of therapy. Time to treatment failure analysis showed
that 69% of patients were projected to be failure free at 4 years (95% CI, 57% to 81%).

J Clin Oncol 23:4079-4088. © 2005 by American Society of Clinical Oncology

INTRODUCTION
of in vitro evidence of synergism between
4
fludarabine and cyclophosphamide (FC),
The introduction of purine analogs, such as a combination regimen was developed and
fludarabine, into the treatment of chronic was associated with increased CR and OR
lymphocytic leukemia (CLL) has improved rates compared with the rates seen with
5-7
the complete remission (CR) and overall re- treatment with fludarabine alone.
sponse (OR) rates beyond those seen with Rituximab is a chimeric monoclonal
alkylating agents such as chlorambucil and antibody that targets the CD20 antigen;
cyclophosphamide. Fludarabine was associ- cytotoxicity may occur through complement-
ated with a CR rate of 20% to 30% as initial mediated lysis, antibody-dependent cellu-lar
1-3
single-agent therapy for CLL. On the basis cytotoxicity, and direct induction of

4079

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 Keating et al

8 history, physical examination, CBC with differential, platelet

apoptosis. In a pivotal trial using rituximab in the treat-ment
of relapsed low-grade lymphoma, disappointing re-sults were count, liver and renal function tests, bone marrow aspiration and
noted in patients with small lymphocytic lymphoma (SLL), biopsy, and bone marrow sample for immunophenotyping. All
9 patients had a monotypic expansion of CD5-, CD19-, and
the lymphomatous counterpart of CLL. This finding was CD23-coexpressing lymphoid cells in the peripheral blood ( 10
attributed to the lower expression of CD20 on the surface of 9
10 /L), which were morphologically consistent with CLL, more
SLL cells and the shorter half-life of the antibody in patients than 30% lymphocytes in the bone marrow, and adequate renal
9
with SLL compared with patients with follicular lymphoma. (creatinine 2 mg/dL) and hepatic (total bilirubin 2 mg/dL)
The shorter half-life was postulated to be a result of an function. Fluorescent in situ hybridization studies and analysis
antigen sink. Subsequent studies showed that a soluble form of the mutation status of the immunoglobulin (Ig) gene or
of CD20 is present in the plasma of patients with CLL and ZAP70 expression were not performed.
that immune complex formation with the antigen sink may Flow cytometry testing was performed in the MDACC labora-
26
10 tory using established techniques. The serum level of beta2-
explain the shorter half-life of the antibody. Rituximab had
microglobulin (b2M) was obtained in 220 patients. One hundred
limited activity in previously treated patients with CLL, with fifty-five patients (69%) were men. The median age was 58 years
a partial response rate of 10% to 15% at conventional (range, 24 to 86 years). Thirty patients (13%) were older than 70
11,12
doses. Subsequent studies have shown that more years. Seventy-five patients (33%) had advanced Rai stage disease
intensive dose regimens can in- (stage III to IV), and the rest had evidence of progressive stage 0 to
crease the OR rate in previously treated CLL patients to II disease. Nine patients with Rai stage 0 were treated because
12,13 of repeated infections, B symptoms, or a lymphocyte doubling
40% to 75%, and higher response rates are noted time of less than 6 months. The Zubrod performance status was
when rituximab is used as initial therapy at the conven- 0 in 77 patients (34%), 1 in 138 patients (62%), and 2 in nine
14,15
tional dose and schedule. patients (4%). The median time from diagnosis to treatment was
Studies in lymphoma cell lines have shown that ritux- 26 months (range, 0 to 157 months).
imab enhances the cytotoxicity of both fludarabine and
16,17 Therapy
cyclophosphamide. In some cell lines, fludarabine ex- On day 1 of the first cycle of FCR, patients received 375
posure results in downregulation of the complement- 2
mg/m of rituximab with 25 mg of intravenous (IV) diphenhydra-
18,19
defense proteins CD55 and CD59. Clinical trials in mine and 650 mg of oral acetaminophen as premedication. No
low-grade and intermediate-grade lymphoma strongly sug- corticosteroids were administered as premedication. The infusion
gest higher response rates and even longer survival when was interrupted if patients showed signs of grade 3 or 4 toxicity, and
25 to 50 mg of meperidine was administered IV for chills. If patients
rituximab is added to chemotherapy regimens. These ob-
had an elevation of temperature to more than 39°C, 100 mg of
servations provided the rationale for the combination of hydrocortisone was administered IV. After symptoms sub-sided, the
fludarabine, cyclophosphamide, and rituximab (FCR) as infusion was reinstituted at a slower rate and was com-pleted in all
therapy for CLL. Subsequently, later studies confirmed patients on the first day of therapy. On days 2, 3, and 4 of the first
2
the activity of chemotherapy combined with rituximab in cycle of therapy, the first nine patients received 30 mg/m of
20-22 2
lymphoma. fludarabine IV over 30 minutes and 300 mg/m of
The goal of this study was to improve the CR rate using cyclophosphamide IV over 1 hour. Because of significant tumor
lysis noted in three of the first nine patients, all subsequent patients
the National Cancer Institute Working Group (NCIWG) 2
criteria for response to $ 50% for patients receiving initial received fludarabine 25 mg/m /d and cyclophosphamide 250 mg/
2
23 m /d for 3 days; in addition, allopurinol was administered to all
therapy for CLL. There is a clear correlation between the patients in the study at a dose of 300 mg daily for 7 days on the first
response to treatment of CLL and the time to progression cycle. In cycles 2 to 6, the rituximab dose was increased to 500
2,24 23 2
(TTP) and survival. The NCIWG criteria for CR do not mg/m on day 1. Fludarabine and cyclophosphamide were admin-
incorporate an evaluation of residual CD5- and CD19- istered on days 1, 2, and 3. Courses were repeated every 4 weeks,
coexpressing cells by flow cytometry of the bone marrow depending on recovery of blood counts, with courses delayed until
9
at the time remission is documented. In many cases of CR the platelet count was greater than 80 10 /L and the absolute
9
by NCIWG criteria, cells that coexpress CD5 and CD19 neutrophil count was more than 1 10 /L. For patients beginning
25 therapy with thrombocytopenia, courses were delayed only if the
are still detectable. We report that the FCR combination
platelet count had not returned to the baseline level by 4 weeks.
as initial therapy for CLL achieves a high frequency of Doses of fludarabine and cyclophosphamide were reduced if blood
CRs with no detectable disease by flow cytometry. counts had not recovered to the levels described earlier 5 weeks
after the last course of therapy or if major infections oc-curred. Both
fludarabine and cyclophosphamide were reduced by one dose level
PATIENTS AND METHODS 2
(20 and 200 mg/m , respectively) or two dose levels (15 and 150
2
mg/m , respectively). The rituximab dose was not reduced. One
Between July 1999 and April 2001, 224 patients with CLL requiring hundred nine patients received oral trimethoprim-sulfamethoxazole
23
therapy as indicated by the NCIWG guidelines were treated after (TMP-SMX) twice weekly as Pneumocystis cari-nii pneumonia
informed consent was obtained according to The University of prophylaxis, and 154 patients received 500 mg daily of valacyclovir
Texas M.D. Anderson Cancer Center (MDACC) institutional for herpes simplex and herpes zoster infection prophylaxis. The use
guidelines. All patients had a pretreatment evaluation including of prophylactic antimicrobials, therapeutic

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 Rituximab Chemoimmunotherapy for CLL

antibiotics, and growth factor support were at the discretion of the new lymphadenopathy, splenomegaly, or Richter’s syndrome; to
treating MDACC or community physician. Sixty-three percent of a 50% increase in the size of nodes or the spleen in patients who
patients received their therapy under the care of their community achieved a PR; to an increase in the peripheral lymphocyte count
physician after receiving the first course at MDACC. Laboratory 9
to more than 10 10 /L on two occasions; or to an increase in
data and toxicity and treatment delivery information were re-ported bone marrow lymphocytes to more than 50%. This definition of
to MDACC by fax and entered into the study database. 2,5,24
time to relapse is similar to other MDACC studies. The
Response Criteria time to treatment failure was measured from the time of the
Response criteria were those previously defined by the initiation of therapy until patients failed to respond to FCR and
23
NCIWG. CR required disappearance of all palpable disease and were re-moved from the study, died, or suffered progressive
9 disease or Richter’s syndrome. Response was evaluated with
normalization of blood counts (neutrophils 1.5 10 /L, plate-lets 100
9 bone marrow examination on completion of the therapy and
10 /L, and hemoglobin 11 g/dL), less than 30% lymphocytes on again 6 months later. Some patients who had residual disease in
bone marrow aspirate, and no evidence of disease on bone marrow the bone marrow or cytopenia on completion of therapy had
biopsy. A nodular partial remission (NPR) re-quired the same normal values when re-evaluated 6 months later. The final
criteria as CR except that one or more lymphoid nodules or response, using NCIWG criteria, was identified at the time of
aggregates were present on bone marrow biopsy. The criteria for the better response. A general linear mixed model was used to
partial remission (PR) included at least 50% reduction in measurable create three models for the re-peated measurements, IgG, IgA,
disease and one or more of the following features: neutrophils $ 1.5 and IgM, as a function of time since baseline measurement.
9
10 /L or a 50% improvement over baseline, platelets more than 100 Because of skewed distributions, each outcome variable was log
9
10 /L or a 50% improvement over baseline, and hemoglobin more transformed before analysis. Ig measurements were taken before
than 11.0 g/dL or a 50% improve-ment over baseline without treatment, after 6 months of treatment, and 12 months after
transfusions. Computed tomography scans were not required to treatment began. However, patients did not necessarily have all
evaluate response. Remission (partial or complete) occurred only if the measurements. Partial data was included in the model.
it persisted for more than 2 months. Bone marrow evaluation was Significance was assessed at the 5% level. Separate models were
usually performed at the end of therapy, although it was not required built for each of the three Ig measures studied.
for determination of PR. After completion of therapy, patients were
re-evaluated at 3-month intervals with history, physical
examination, and blood counts. If possible, bone marrow RESULTS
examination was performed 6 and 12 months after treatment.

Flow Cytometry One hundred fifty-six patients (70%; 95% CI, 63% to 76%)
Bone marrow samples were evaluated by multicolor immu- achieved a CR, 23 (10%) achieved a NPR, and 34 (15%)
nostaining and flow cytometry analysis before treatment and achieved a PR (Table 1). Eleven patients did not respond to
26
after the third and sixth cycles of therapy. Flow cytometry CR FCR; six of these patients had an inadequate antitumor
was defined as CD5- and CD19-coexpressing cells of less than response with persistent cytopenia, one developed Richter’s
1%, with normalization of the kappa:lambda ratio ( 3:1 in transformation after the first course of treatment, two died of
patients with monotypic kappa and 1:3 in patients with
pneumonia after one or two courses of treatment, and two
monotypic lambda). Flow cytometry relapse was defined as a
return to $ 5% CD5- and CD19-coexpressing cells, together with were lost to follow-up after one and two courses and were
an abnormal kappa: lambda ratio. considered as having experienced treatment failure. The
responses in 34 patients who had PRs were classified as such
Statistical Section
for various reasons. Five patients had residual lymph-
The CR rate in previously untreated patients receiving flu-
2
darabine as a single agent was 29%, and the CR rate with FC in
adenopathy. Four patients had a clinical CR but did not have
5
a similar patient population was 35% ; the major infection rates
a confirming marrow. One of these four patients later had a
were 19% and 18% of patients, respectively. The goal of this Richter’s transformation after five courses of therapy.
study was to achieve a CR rate of more than 50%, with less than Twenty-five patients were classified as PRs because of per-
40% of patients experiencing major infection. The design by sistent anemia (five patients) or thrombocytopenia (20 pa-
27
Bryant and Day was used to test this outcome. If there was a tients). These 25 patients often had no measurable residual
CR rate of more than 22% or there were less than 38 major disease in nodes (16 of 18 assessable patients), spleen (10 of
infections in the first 57 patients, the study was to continue until
11 assessable patients), liver (five of five assessable
the 95% CI did not include a CR rate of 50%. This was
accomplished, and after internal discussion, additional patients patients), and peripheral blood (25 of 25 assessable patients).
were recruited onto the study to broaden experience with this Thirty-four patients who achieved a PR had a follow-up
effective regi-men. Type I and II errors were less than 10%. marrow; 16 of these patients had a marrow CR, eight had
Because no systematic evaluation of remissions confirmed by persistent nodules, two had 50% reduction in marrow
flow cytom-etry had been performed in earlier trials to allow
2,5,26 infiltrate but more than 30% marrow lymphocytes, and four
compari-son, this analysis was descriptive.
failed to respond. No marrow evaluation was performed in
Survival was measured from the time of the initiation of
therapy in all patients. The last follow-up data available for this four patients. Applying the NCIWG criteria for response to
analysis was from July 31, 2003. Time to relapse was measured var-ious sites of disease, CR was seen in 214 (96%) of 224
from the time of final response evaluation to the development of assessable patients in the blood, 177 (91%) of 195 patients

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 Keating et al

were advanced Rai and Binet stages, older age, higher

Table 1. NCI Working Group Response to FCR by
Pretreatment Characteristics WBC counts, lower platelet counts, higher serum b2M
No. of
level, bone marrow biopsy cellularity more than 50%, and
Characteristic Patients CR (%) OR (%) splenomeg-aly $ 5 cm below the left costal margin.
Total 224 70 95 Patients were usually evaluated for response after three
Rai stage courses of therapy and at the end of their therapy (Table 2).
0 to II 149 76 96 Fifty-eight of the 60 patients in CR after three courses were
III to IV 75 64 94 still in CR at the end of therapy. One patient was found to
Binet stage
have residual nodules, and one patient developed thrombo-
A 61 82 98
B 109 70 95 cytopenia and was classified as a PR. The corresponding
C 54 56 91 values for obtaining CR at the end of therapy for patients
Age with NPR, PR, and stable disease after three courses of
55 years 81 80 96 therapy were 58 (87%) of 67 patients, 39 (59%) of 66
55-69 years 113 68 96
patients, and three (43%) of seven patients, respectively.
$ 70 years 30 47 87
WBC
9
Survival and Time to Treatment Failure
100 10 /L 130 75 98
9 The overall survival and time to treatment failure are
100-199 10 /L 77 66 91
9
$ 200 10 /L 17 41 88
shown in Figure 1. One hundred fifty-four (99%) of the 156
Spleen patients who achieved CR are alive (Fig 2). One patient died
0-4 cm below LCM 166 74 97 of infection after developing delayed pancytopenia, and one
$ 5 cm below LCM 58 57 90 patient died in CR from gall bladder cancer. Nine patients
Beta2-microglobulin have relapsed (Fig 3), and all were still alive as of 2003. One
3 mg/L 68 88 98
3.1-4 mg/L 59 78 98
patient relapsed with a Richter’s transformation. Eight of 23
4 mg/L 93 53 92 patients who had a NPR have relapsed, and two have died.
Marrow cellularity, biopsy Nine of 34 patients who achieved a PR have had disease
50% 53 79 96 progression (one with Richter’s syndrome, seven with re-
50%-80% 98 65 93
current CLL, and one with worsening cytopenia), and six
$ 90% 32 63 97
have died. Three additional patients died of infection while
Abbreviations: NCI, National Cancer Institute; FCR, fludarabine, cyclo-
phosphamide, and rituximab; CR, complete remission; OR, overall re-
still in PR. Five of the nine patients whose CLL did not
sponse; LCM, left costal margin. respond to therapy have died; one of these patients died after
P .05. a Richter’s transformation, two died from pneumonia while
on therapy, and two died from infection after being taken off
the study because of resistance. Two patients ex-perienced
early deaths from infection (4 and 7 weeks). Two additional
in the lymph nodes, 108 (92%) of 117 patients in the patients who were classified as having experi-enced
spleen, and 48 (91%) of 53 patients in the liver. One treatment failure because of lack of follow-up infor-mation
hundred seventy-four patients (78%) had a CR in the were still alive at 5 and 14 months.
marrow, and 30 patients (13%) had an NPR.
Fifty-eight patients (26%) did not complete the pre- Response As Measured by Flow Cytometry
scribed six courses of therapy. The major cause of prema- Two hundred seven patients had flow cytometric stud-
ture discontinuation of therapy was persistent cytopenia, ies on bone marrow aspirates to evaluate residual disease
which was noted in 28 patients. Neutropenia led to discon-
tinuation of therapy in 21 patients. Other reasons for dis-
Table 2. Comparison of End of Therapy Response With Response at the
continuation were patient preference (n 9), infection (n 6), End of Three Courses
Richter’s syndrome (n 3), resistant disease (n 3), cardiac Response at End of Therapy
symptoms (n 3), myelodysplastic syn-drome (n 1), gall (No. of patients)
bladder cancer (n 1), rash (n 1), elevated creatinine (n 1), and No
Course 3 Total No. Further
early death (n 2). Early discontinuation of therapy was Response of Patients CR NPR PR Fail Therapy
significantly associated (P .05) with advanced Rai or Binet
CR 63 58 1 1 — 3
stage, age more than 65 years, hemoglobin less than 11 g/dL,
NPR 68 58 8 1 — 1
and serum creatinine more than 1.4 mg/dL and b2M levels PR 72 39 11 16 — 6
more than 4 mg/dL. NR 13 3 — 3 1 6
Responses to FCR by patient pretreatment characteris- Abbreviations: CR, complete remission; NPR, nodular partial remission;
tics are listed in Table 1. Characteristics significantly asso- PR, partial remission; NR, no response.
ciated with a lower probability of achieving CR (P .05)

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 Rituximab Chemoimmunotherapy for CLL

Fig 3. Time to Relapse by National Cancer Institute Working Group 96

response criteria. CR, complete remission; CRN, nodular partial remis-sion;
Fig 1. Overall survival and time to treatment failure. PR, partial remission.

after six courses of therapy (or after their last course of probability of relapse and NCIWG responses, as well as
therapy, if only three to five courses were administered). One the CD5 and CD19 flow cytometry response (Fig 4 and
hundred thirty-eight patients (67%) had less than 1% CD5- Table 4). Too few deaths have occurred to analyze risk of
and CD19-coexpressing cells (flow cytometry CR), 41 dying according to flow cytometry response.
patients (20%) had 1% to 5% coexpressing cells, and 28
Toxicity
patients (14%) had more than 5% coexpressing cells. The
The first dose of rituximab was associated with infu-
proportion of patients in CR with CD5- and CD19-
sional symptoms, which were usually responsive to meper-
coexpressing cells less than 1% was 78% (120 of 153 pa-
idine or hydrocortisone; grade 3 to 4 toxicities were rare
tients), which was significantly higher than the five (24%) of
(Table 5). Adverse reactions to rituximab in courses 2
21 patients in NPR (P .001) and 13 (50%) of 26 patients in
through 6 were noted in only three patients. In courses 2
PR (P .001). Flow cytometry CR was significantly less
through 6, rituximab was usually infused over 2 to 4
common in patients with marked splenomegaly, b2M levels
more than 3 mg/L, and marrow cellularity $ 90% (Table 3).
The relapse rate was analyzed according to the
Table 3. Probability of Achieving Flow Cytometry CR by
NCIWG criteria and the percentage of CD5- and CD19- Pretreatment Characteristics
coexpressing cells in the marrow aspiration at the end of Flow CR
FCR treatment. There was strong correlation between
Total No. No. of
Characteristic of Patients Patients %

Rai stage
0 to II 139 97 70
III to IV 68 41 60
Age
55 years 80 49 61
55-69 years 104 75 72
$ 70 years 23 14 61
Spleen
0-4 cm below LCM 152 108 76
$ 5 cm below LCM 55 30 55
Beta2-microglobulin
3 mg/L 65 52 80
3.1-4 mg/L 57 34 60
4 mg/L 82 50 61
Marrow cellularity, biopsy
90% 138 103 75
$ 90% 30 10 33

NOTE. Flow cytometry CR CD5- and CD19-coexpressing cells 1%.

Fig 2. Survival time by National Cancer Institute Working Group 96 Abbreviations: CR, complete remission; LCM, left costal margin.
response criteria. CR, complete remission; CRN, nodular partial remission; P .05.
PR, partial remission.

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 Keating et al

Table 5. Toxicities Associated With the First Infusion of Rituximab

Grade 1 to 2 Grade 3 to 4
No. of No. of
Toxicity Patients % Patients %

Fever and chills 94 42 2 1

Hypotension 22 10 2 1
Dyspnea 28 13 — —
Nausea 25 11 — —
Vomiting 10 5 — —
Back pain 7 3 — —
Urticaria 7 3 — —
Headache 5 2 — —

Fig 4. Time to Relapse by CD5 and CD19 flow cytometry response.

cellulitis, urinary tract infections, upper respiratory infec-
tions, sinusitis, or bronchitis, were reported in 10% of the
hours. Toxicities associated with FC were similar to the courses. The organisms identified in the documented
5 infec-tions were P carinii (n 3), Aspergillus (n 2),
toxicities seen in previous studies, with nausea occurring
during 23% of the courses and vomiting occurring during torulopsis glabrata (n 1), Pseudomonas (n 2), Escherichia
7%. Nausea and vomiting tended to occur late and often coli (n 1), Enterococcus (n 2), and cytomegalovirus (n 1).
began on day 3 of the chemotherapy or after it had been Eleven patients experienced a reactivation of herpes
discontinued. Prophylactic ondansetron was administered simplex (n 8) or herpes zoster (n 3).
to all patients. One patient complained of hair loss, and TMP-SMX (one 800 mg/160 mg tablet twice a day) was
one patient developed hemorrhagic cystitis. administered to 109 patients on a Saturday and Sunday or
every Monday, Wednesday, and Friday regimen for P cari-
Cytopenia and Infection
nii pneumonia prophylaxis. There was no difference in the
The adverse effects observed with FCR were myelosup-
incidence of grade 3 to 4 neutropenia or major or minor
pression and infections. Grades 3 and 4 neutropenia oc-
infections in patients administered TMP-SMX compared
curred in 24% and 28% of 927 assessable courses,
with patients who did not receive prophylaxis. Prophylactic
respectively. Grades 3 and 4 thrombocytopenia occurred in
valacyclovir (500 mg) was administered to 154 patients;
4% and less than 1% of courses, respectively. Despite the
none of these patients developed herpes zoster reactivation
significant incidence of neutropenia, only 2.6% of the
courses were associated with major infections, including
pneumonia (20 episodes) or septicemia (11 episodes; Table
6). Minor infections, such as fever of unknown origin, Table 6. Incidence of Infections With FCR Therapy by NCIWG Response,
Lowest Neutrophil Count per Course, and Course Number
No. of Total No. of No. of Major Risk per
Infection Courses Infections Infections Course†
Table 4. Relapse by NCIWG and Flow Cytometry Responses at the End Response
of FCR to Determine Residual Disease (CR NPR PR) CR 889 105 18 0.12
Relapse Died NPR 126 11 — 0.09
No. of No. of No. of PR 165 26 11 0.16
Response Criteria Patients Patients % Patients % NR 24 8 2 0.33
Nadir neutrophils‡
NCIWG 9
500 10 /L 257 23 5 0.09
CR 156 9 5.8 2 1.3 9
500-999 10 /L 224 36 5 0.16
NPR 23 8 34.8 2 8.7 9
$ 1,000 10 /L 442 46 9 0.10
PR 34 9 26.5 9 26.5
Course number
CD5 CD19 (BM)
Courses 1 to 3 627 86 17 0.14
1% 138 5 3.6† 4 2.8†
Courses 4 to 6 527 76 14 0.14
1%-4.9% 41 8 19.5 — —
$ 5% 21 9 43 3 14.3 Abbreviations: FCR, fludarabine, cyclophosphamide, and rituximab;
NCIWG, National Cancer Institute Working Group; CR, complete re-
Abbreviations: NCIWG, National Cancer Institute Working Group; FCR, mission; NPR, nodular partial remission; PR, partial remission; NR, no
fludarabine, cyclophosphamide, and rituximab; CR, complete remission; response.
NRR, nodular partial remission; PR, partial remission; BM, bone marrow. Assessable data.
CR v NPR PR, P .001. †Total infections number of courses.
† 1% v 1%-4.9% v $ 5%, P .001. ‡Not available for all infections.

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 Rituximab Chemoimmunotherapy for CLL

compared with three of 70 patients who did not receive patients for IgM. There was considerable oscillation in Ig
prophylaxis. The incidence of fever and infection was no levels during follow-up. There was a significant decrease
different in the first three courses of therapy compared in the mean and median distributions of IgG at the start of
with courses 4 to 6. There was no statistically significant therapy and at 6 months after the end of treatment. Of the
9
association between the degree of neutropenia ( 1 10 /L) 224 patients, 105 (47%) had all three IgG measurements,
and the incidence of major, minor, or herpetic infection 70 (31%) had two measurements, 45 (20%) had one
(Table 6). measure-ment, and four (2%) had no IgG data. The
The Coomb’s (direct antibody) test was performed general linear mixed model examined time in months as
within 3 months of enrollment on the study in 101 patients the only co-variate. Because of skewed distributions, the
and was positive in 13 patients. Autoimmune hemolysis IgG mea-surements were log transformed. The model
developed in three of these 13 patients, and one patient had revealed that there was a significant association (P .001)
red cell aplasia. Autoimmune hemolytic anemia (AIHA)
be-tween time and log IgG level, with a coefficient of
developed in seven (8%) of the 88 patients who were
0.014 (SE 0.003). Therefore the log IgG levels decreased
Coomb’s negative. In 123 patients without a Coomb’s test
0.014 units per month over the 12 months of follow-up.
before therapy, there were six episodes (5%) of AIHA and
There was no significant difference in the mean values for
one episode of red cell aplasia. A clear episode of immune
IgA and IgM, although values 6 months after the end of
thrombocytopenia was noticed in one patient.
treatment were somewhat lower than at the start of ther-
Dose reductions were required in 35 patients. Nine of
apy. The mean and median levels of Ig and the SEs and
these patients had to discontinue therapy despite dose re-
standard deviations are listed in Table 7.
ductions. The incidence of dose reduction was 13% (21 of
156 patients) in patients who had a CR and 25% (14 of 57 Comparison of the Current FCR Study and
patients) in patients who had a NPR or a PR. The inci-
Historical Experience With FC
dence was significantly higher in patients older than 60
Thirty-four patients who had received FC as their ini-
years and in patients with pretreatment Rai stage IV 5
disease (P .01). tial therapy for CLL were previously reported. These pa-
2
Hypogammaglobulinemia was noted for IgG in 75 pa- tients received higher doses of fludarabine (30 mg/m
tients, IgA in 46 patients, and IgM in 50 patients. Sequential 2
three times daily) and cyclophosphamide (300 mg/m
follow-up of Ig levels were not mandated in this study. There three times daily) than the doses administered with FCR.
was no significant difference in the levels of IgG, IgA, or Only 14 (41%) of 34 patients treated with FC were able to
IgM between the starting level and the levels at the end of complete all six prescribed courses. Twelve patients
therapy (Table 7). Although some patients improved their (35%) discontinued FC because of persistent cytopenia.
IgG, IgA, and IgM levels after therapy, an equal number of Four patients had AIHA, and two of these patients had
patients decreased their level. No significant trend was noted AIHA before starting FC. The clinical characteristics,
with the Ig levels according to response. response data, and comparison of survival, time to treat-
However, patients who had low IgG, IgA, or IgM levels ment failure, and TTP of responders are listed in Table 8.
at the start of therapy; had achieved a CR; and had Ig levels The CR rate, proportion of patients with CD5 and CD19
measured at the end of therapy or at later times were found to cells in their bone marrow less than 1%, time to treat-
be in the normal range in seven (18%) of 40 patients for IgG, ment failure, TTP, and survival were significantly better
five (9%) of 54 patients for IgA, and 14 (36%) of 39 with FCR than with FC.

Table 7. Levels of Serum IgG, IgA, and IgM at Various Time Points for FCR Patients
IgG IgA IgM
6 Months 6 Months 6 Months
Start End of Rx After Rx Start End of Rx After Rx Start End of Rx After Rx

Patients, No. 198 159 143 197 158 141 197 158 141
Levels, mg
Mean 855 747 688 96 94 89 66 47 60
SE 38 27 28 6 5 6 10 4 7
Median 749 690 636 76 75 67 35 32 35
Standard deviation 541 338 338 81 69 73 143 48 84

Abbreviations: Ig, immunoglobulin; FCR, fludarabine, cyclophosphamide, and rituximab; Rx, therapy.
P .05.

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 Keating et al

20,21
actions of these three agents. Preclinical data suggested
Table 8. Comparison of Patient Characteristics and Outcome
Between FC and FCR that rituximab sensitized cells to both fludarabine and
16-18
Characteristic and Outcome FC (n 34) FCR (n 224)
cyclophosphamide and that fludarabine may enhance
one of rituximab’s modes of action by downregulating
Age, years 19
Median 53 57
complement-resistance proteins.
Range 33-92 24-86 The improvement in the CR rate with FC over fludara-
Sex, No. 2,5
bine alone was modest (35% v 29%, respectively), sug-
Male 25 155
gesting that the addition of rituximab was crucial to the
Female 9 69
Rai stage, No.
success of this FCR regimen. A combination of fludarabine
0-II 17 149 and rituximab has been investigated in the treatment of CLL
III-IV 17 75 in a Cancer and Leukemia Group B (CALGB) study that
9
WBC, 10 /L showed a higher CR rate when the two agents were admin-
Median 93.9 84.5 28
istered simultaneously as opposed to sequentially. The
Range 6.6-304 1.8-619.5
b2M, mg/dL
CALGB study evaluated six 5-day courses of fludarabine
Median 3.9 3.8 either administered alone (51 patients) or combined with 375
Range 1.6-11.1 1.8-16.4 2
mg/m of rituximab administered on day 1 of each course
CR, % 35 70 and on day 5 of the first course (53 patients). Two months
NPR, % 29 10
OR, % 88 95
later, all stable or responding patients on both arms of the
CD5 CD19, end of Rx, % study received 4 weekly consolidation doses of ritux-imab.
Median 12.8 0.45 Several patients showed improved response with the
Range 0-94.3 0-97.7 consolidation phase. The OR and CR rates for the concur-
Major infections per course 0.03 0.03 rent arms were 90% and 47%, respectively. The OR and CR
Minor infections per course 0.12 0.10
rates for the patients treated with sequential therapy were
Toxicity, %
Neutropenia
77% and 28%, respectively. The CR rate after the six cycles
Grade 3 20 24 of concurrent fludarabine and rituximab was 33% (approx-
Grade 4 18 28 imately half the rate seen after six cycles of FCR). The early
Thrombocytopenia progression-free and overall survival results for the CALGB
Grade 3 4 4 study are similar to those reported here to date. A
Grade 4 0 0.3 comparison of the results of this study with the fludarabine-
Median survival, months 73 NR
alone study population of the immediately preceding
TTF, months 40 NR
CALGB study showed a higher response rate and improved
TTP, months 47 NR
progression-free survival for the group receiving
Abbreviations: FC, fludarabine and cyclophosphamide; FCR, fludarabine, 29
cyclophosphamide, and rituximab; b2M, beta2-microglobulin; CR, complete rituximab.
remission; NPR, nodular partial remission; OR, overall response; Rx, therapy; The criteria for response in CLL have been formalized
TTF, time to treatment failure; NR, not reached; TTP, time to progression. 23
P .05. by the NCIWG. Application of these criteria has helped to
compare responses in previous clinical studies. A clear rela-
tionship between response and both survival and TTP has
2,24
been established. Additional criteria for response have
DISCUSSION been suggested. A decrease in the percentage of CD5- and
CD19-coexpressing B cells has been associated with a pro-
1-3 25
The use of fludarabine for initial treatment of CLL has longed TTP and is supported by this study. A recent study
increased the CR and OR rates compared with the rates seen suggested that four-color flow cytometry to detect CD5/19/
with alkylating agents. Studies comparing fludarabine with 20/79b-expressing cells was more sensitive than polymerase
cyclophosphamide, doxorubicin, and prednisone and chain reaction in detecting residual disease and predicting
1,3 30
chlorambucil confirmed the single-institution studies that relapse. FCR therapy resulted in a marked decrease in the
2
showed high response rates. Combinations of FC have been percentage of CD5- and CD19-coexpressing cells in the bone
5-7 marrow to less than 1% in two thirds of responding patients.
associated with promising CR and OR rates. Ritux-imab,
as a single agent, was more active in CLL when used in The median value for patients treated with FC alone was
5
higher dose-intensity schedules than those recommended for 12.8%. Thus, the addition of rituximab to FC is associated
follicular lymphoma and in previously untreated pa-tients with significantly greater reductions in residual CLL cells in
12-15
with CLL. the marrow than the reductions seen with FC alone. In a
The CR rate of 70% with FCR (using NCIWG crite-ria) study using single-agent fludarabine, only nine (39%) of 23
is the highest rate reported for initial therapy for CLL, which patients with NCIWG CR were in remission by flow
supports the concept of additive or synergistic inter- cytometric criteria (CD5- and CD19-coexpressing

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 Rituximab Chemoimmunotherapy for CLL

31
cells 10%) compared with 120 (78%) of 153 patients was the most common cause for early discontinuation of
with CD5- and CD19-coexpressing cells less than 1% therapy. Stem-cell harvesting was not attempted in the
with FCR. The follow-up time was not adequate to assess study, so no conclusions can be drawn as to the impact of
the effect of flow cytometry remissions as a surrogate end the regimen on this procedure.
point of long-term prognosis. The combination of two chemotherapy drugs, FC, with
The FCR regimen was well tolerated. Grade 3 to 4 the monoclonal antibody rituximab was associated with a
symptoms of fever, chills, and hypotension with high CR rate and evidence of a high degree of clearing of
rituximab occurred in nine patients (6%), and 6% of first malignant cells in the bone marrow. A similar high CR rate
infusions were associated with dyspnea, but the infusions has been reported for the fludarabine, cyclophosphamide,
33
were com-pleted in all patients on the first day. The and mitoxantrone regimen. The effect of this increase in
frequency of nausea and vomiting with FCR was similar high-quality CR (morphologic, molecular, and by flow cy-
to that seen with FC alone and was usually easily tometric analysis) raises the possibility that the use of such
controlled. Only three pa-tients chose to discontinue regimens may translate into an improved prognosis.
therapy after three courses be-cause of toxicity. Comparative studies are needed to confirm the effect
Despite the addition of a B-lymphocyte– depleting agent of the addition of rituximab to FC. Long-term follow-up
to FC, no substantial difference between the infection of the effect of flow cytometric–measured remissions will
profiles of FCR and FC was noted. Neutropenia was more help to establish the role of these measurements as a
common with FCR than with FC, although the mechanisms surrogate for improved time to relapse or survival.
for this observation remain uncertain. A higher incidence of
■■■
neutropenia was noted when rituximab was administered
with fludarabine (76%) compared with the same dose of Authors’ Disclosures of Potential
fludarabine administered alone (39%) in the CALGB study. Conflicts of Interest
Thrombocytopenia was rare. AIHA and red cell aplasia The following authors or their immediate family
occurred in 18 patients (8%), which is comparable with other members have indicated a financial interest. No conflict
32
fludarabine-based regimens. Tumor lysis was not observed exists for drugs or devices used in a study if they are not
in the FCR patients who received allopurinol, which should being evaluated as part of the investigation. Acted as a
be used prophylactically. consultant within the last 2 years: Michael Keating,
Several patients developed prolonged myelosuppres- Berlex, Genentech. Received more than $2,000 a year
sion ( 6 weeks), which was more common in older pa- from a company for either of the last 2 years: Michael
tients with Rai stage III and IV disease. Myelosuppression Keating, Berlex, Genentech.

support in patients with previously untreated phocytic leukemia. J Clin Oncol 19:2165-2170,
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