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Blood, 1 April 2002, Vol. 99, No. 7, pp. 2625-2626
CORRESPONDENCE
To the editor:
Enhanced clearance of leukemic lymphocytes in B-cell chronic
lymphocytic leukemia with etodolac
Chronic lymphocytic leukemia (CLL) is a neoplastic disease
characterized by the accumulation of small mature-appearing
lymphocytes in the blood, bone marrow, and lymphoid
tissues.1 Current therapy has not been shown to prolong
survival.2 Etodolac is a racemic mixture of R-(-) and
S-(+)-1,8-diethyl-1,3,4,9,-tetrahydropyrano-(3,4-b)indole-1-acetic
acid.3 Etodolac selectively inhibits
cyclooxygenase-2,4 and it is approved for use in various
parts of the world for treatment of degenerative joint disease and
rheumatoid arthritis and for use as an analgesic.5 We report the chance observation that racemic etodolac lowers the
lymphocyte count in a patient with B-CLL and show that challenges with
13 other nonsteroidal anti-inflammatory drugs (NSAIDs) produced no
significant effect. We also present studies to show that at standard
anti-inflammatory doses, racemic etodolac achieves this action by
enhancing the selective clearance rather than by the direct killing of
leukemic lymphocytes. The patient's condition was diagnosed as B-CLL in 1994 at age 57. Her
leukemic lymphocyte phenotype was CD5+, CD19+,
CD20+, CD23+, CD25+,
FMC7+, and  dim+. She was clinically staged
at Rai/Binet 0/A. On October 23, 1997, her white blood cell count (WBC)
was 34 300 and her lymphocyte count was 27 440. From October
25, 1997, to October 27, 1997, she took etodolac 300 mg PO
twice a day for neck pain. On October 28, 1997, because of the
development of petechiae etodolac was discontinued and a complete blood
count was performed. Her platelet count was normal but her WBC had
dropped to 13 400 with a lymphocyte count of 6 700. To determine
whether this was a reproducible effect, etodolac at the same dose of
300 mg was administered twice a day from November 5, 1997, to November
11, 1997; January 21, 1998, to January 25, 1998; February 26, 1998, to
March 2, 1998; and April 8, 1998, to April 13, 1998, all with
similar effects (Figure 1A). No
clinically significant changes in the hemoglobin level, neutrophil
count, or platelet count were seen with etodolac (platelet data not
shown). Also seen in Figure 1A and B are the results of the
administration of 13 other NSAIDs. None produced any significant decrease in the lymphocyte count. The effect of etodolac appears not to
be due to its anti-inflammatory property since none of the other NSAIDs
elicited this response. Furthermore, the effect does not appear to be
related to cyclooxygenase specificity since celecoxib, rofecoxib, and
meloxicam, selective cylooxygenase-2 inhibitors, did not significantly
affect the lymphocyte count. A 4-month course of etodolac was then
administered between July 20, 1998, and November 23, 1998 (Figure 1C).
Taken together with the previous data, the reduction of lymphocyte
count in response to etodolac administration was prompt, with a nadir
occurring after 2 days of administration. The duration of the effect
after drug withdrawal was short, with a return of lymphocyte count to baseline within 2 weeks of drug cessation independent of the duration of therapy. The effect was also reproducible and did not appear to be
lessened with repeated or prolonged drug challenges.
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| Figure 1.
Cell counts as a function of
NSAID administration.
(A) Effects of E, etodolac, 300 mg twice a day for 3 to 6 days,
compared with 9 other NSAIDs: N, naproxen, 250 mg twice a day for 4 days; D, diclofenac, 50 mg 3 times a day for 4 days; S, sulindac, 200 mg twice a day for 4 days; R, nabumetone 500 mg twice a day for 6 days;
O, oxaprozin, 600 mg twice a day for 4 days; P, piroxicam, 20 mg every
day for 3 days; I, indomethacin, 25 mg 3 times a day for 3 days; T,
tolmetin, 400 mg twice a day for 3 days; and M, ibuprofen, 400 mg 3 times a day for 3 days. (B) Effects of C, celecoxib, 100 mg twice a day
for 4 days; V, rofecoxib, 25 mg every day for 4 days; F, flurbiprofen,
100 mg 3 times a day for 3 days; and X, meloxicam, 7.5 mg every day for
3 days. (C) Effect of a 4-month course of etodolac. A 400 mg sustained
release preparation was administered twice a day from July 20, 1998 until July 28, 1998, followed by 300 mg twice a day of the standard
release preparation until September 8, 1998, followed by reinstitution
of the 400 mg sustained release preparation twice a day. There was a
rapid decline in the lymphocyte count in the first several days to a
nadir of 12 510 followed by rebound and gradual increase to 33 968 on
November 23, 1998, the last day of etodolac administration. After
discontinuation of etodolac, there was a rapid rise of the lymphocyte
count to 43 132 on November 30, 1998, and to 50 199 on December 16, 1998. To determine if a full response to etodolac was still possible
after a prolonged course, etodolac was administered at 400 mg sustained
release twice per day for 2 days from December 19, 1998, to December
21, 1998. The lymphocyte count on December 21, 1998, dropped to 24 548
with a rapid return to 59 148 by December 28, 1998.
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To determine whether cell killing could be responsible for the
drop in circulating lymphocytes, mononuclear cells from the patient
were cultured for 72 hours in presence of increasing concentrations of
naproxen or etodolac up to 240 µM and analyzed at 24, 48, and 72 hours for the binding of monoclonal antibody to internucleosomal DNA by
enzyme-linked immunosorbent assay (ELISA) as a measure of apoptosis
using a standard kit assay (Cell Death Detection ELISA Plus,
Boerhinger-Mannheim, Indianapolis, IN). No significant enhancement of
apoptosis was achieved by etodolac compared with naproxen (data not
shown). The same results were obtained in a separate experiment when
assayed by analysis of the binding of FITC annexin V to and the uptake
of propidium iodide by the patient's mononuclear cells by flow
cytometry (data not shown). The fact that an increase in apoptosis or
necrosis could not be detected for etodolac compared with naproxen in
vitro did not exclude the possibility that a metabolite of etodolac or
a serum factor might be necessary to achieve the effect in vivo. To
examine this possibility, the percentages of viable, apoptotic, and
late apoptotic/necrotic cells were measured by analysis of the binding
of FITC annexin V to and the uptake of propidium iodide by isolated
mononuclear cells after administration of etodolac to the patient (data
not shown). As the lymphocyte count dropped after administration of etodolac, the percentage of apoptotic cells remained the same. These
data suggested that etodolac at standard anti-inflammatory and
analgesic concentrations did not achieve reduction of leukemic lymphocyte count by direct killing but by increasing the clearance of
leukemic cells into other compartments. To directly determine whether etodolac was enhancing the
clearance of the patient's leukemic lymphocytes, a WBC scan was
performed in the standard fashion with and without the administration
of etodolac. As shown in Figure 2, an
increase in the radiolabel appeared in the spleen with the
administration of etodolac. No difference was detected in the amount of
radiolabel that appeared in the liver.
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| Figure 2.
Effect of etodolac on the
clearance of Indium 111 labeled patient leukocytes.
Two experiments are superimposed. One experiment without drug
begun on March 21, 2000 and one with etodolac extended release
administered 400 mg twice a day beginning at 7:30 AM after
blood collection for radiolabeling on November 8, 1999 and continued
for 48 hours. Patient leukocytes were Indium 111 labeled according to
supplier's instructions (Mediphysics, Amersham Healthcare, Arlington
Heights, IL). Radiolabeled material left in the syringe after injection
into the patient was submitted to the clinical laboratory at Scottsdale
Healthcare Shea, Scottsdale, AZ for manual WBC differential counting.
For the study of November 8, 1999 the radiolabeled cells were 100%
lymphocytes and for the study of March 21, 2000, 67% were lymphocytes,
29% neutrophils, 3% eosinophils, and 1% bands. Indium-labeled
patient leukocytes were injected at 11 AM in both
experiments. Total body WBC scans were performed in the Department of
Radiology at Scottsdale Healthcare Shea using an ADAC Vertex Plus gamma
camera (Milpitos, CA) under the supervision of Ronald Korn. The same
camera was used for both studies. Scans were taken at 15 minutes, 2 hours, 24 hours, and 48 hours after injections and the percent of
counts in the liver and spleen after background subtraction were
determined at each time point. As the lymphocyte count dropped in
response to etodolac administration, the percent of radiolabel in the
spleen was increased in comparison to the percent of radiolabel in the
spleen found without drug. No significant change in the percent of
radiolabel in the liver was found with etodolac
administration.
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These data show that at standard anti-inflammatory doses, etodolac
reproducibly lowers the lymphocyte count in a patient with B-CLL.
Furthermore, data is presented to show that etodolac achieves this
action by affecting changes in leukemic cell compartmentalization. Further work should clarify the prevalence of this effect in B-CLL, the
molecular mechanisms involved, and whether or not racemic etodolac, one
of its chiral isomers, or similar compounds could be useful in B-CLL or
other B-cell neoplasms.
Francis A. Nardella and Jane A. LeFevre
Correspondence: Francis A. Nardella, Scottsdale Rheumatology,
10210 North 92nd Street, Suite 202, Scottsdale, AZ 85258; e-mail:
fnardella{at}cox.net
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