Chemosensitivity of Lymphocytes From Patients

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Chemosensitivity of Lymphocytes From Patients With B-Cell Chronic
Lymphocytic Leukemia to Chlorambucil, Fludarabine,
and Camptothecin Analogs
By Robert Silber, Barbara Degar, Dan Costin, Elizabeth W. Newcornb, Mathew Mani, Carl R. Rosenberg,
Laura Morse, John C. Drygas, Zoe N. Canellakis, and Milan Potrnesil
Chemosensitivity of B lymphocytes, obtained from 65 patients with B-cell chronic lymphocytic leukemia (B-CLL), Rai
stages 0 through IV, was determined using theMTT assay.
The results were expressed by the drug concentration required for 50% inhibition of cell viability
The cytotoxicwith that of
i t y ofchlorambucil
(CLB) wascompared
fludarabine and
the
DNA topoisomerase I inhibitors,
camptothecin, 9-aminocamptothecin, 10.1 l-methylenedioxy-20(S)-camptothecin (10.11-MDC) and 9-amino-l0,llmethylenedioxy-20(S)-campthothecin(9-A-lO.Il-MDC). and
topotecan. Considerable heterogeneity in sensitivity t o CLB
was observed, with a median ICw of 40.5 pmol/L in untreated patients. B-CLL cells from patients treated
with CLB
had a significantly higher median ICw of 86.0 pmol/L ( P <
.01). Untreated as well as CLB-treated patients were divided
into two subsets. For the purpose of thisstudy, B-CLL lymphocytes with an ICsoCLB of less than 61.0 p m o l / t were
designated as "sensitive," and those with an ICso CLB of
261.0 pmol/L weredesignated as "resistant." After baseline
assays, 15 untreated patientsreceived CLB; after treatment,
the ICsoincreased in B-CLL lymphocytes from 13 of 15 patients. The response t o CLB treatment, determined by its
by the Eastern
effect on theabsolute lymphocyte count and
Cooperative Oncology Group clinical criteria, was signifi-
cantly better in patients whose lymphocyteshad anICw CLB
of less than 61.0 pmol/L before therapy ( P < .01).B-CLL
lymphocytes also had a variable degree of sensitivityin vitro
t o each of the other drugs. There was significant cross-resistance between CLB and fludarabine ( P < 0.01 1. Whereas only
29% of CLB-resistant B-lymphocyte specimens obtained
from individual patients were sensitive t o fludarabine in
vitro, 52% and 67% of CLB-resistant lymphocyte samples
were sensitive t o 10.11-MDC and 9-A-10,11-MDC, respectively. We have previously reported thatp53 gene mutations
were associated with aggressive B-CLL and a poor prognosis. B lymphocytes from seven patients with these mutations were resistant t o CLB, and five of six were resistant
t o fludarabine. Lymphocytes from four ofseven were resistant to10,11-MDC, and three of four were resistant t o 9-A10,Il-MDC. This study implies that theMTT assay may be
useful in identifying subsets of CLL patients resistant t o conventional chemotherapy. However, definitive conclusions
can not be drawn in view of the small number of patients
studied prospectively. In addition, these resultssuggest the
potential of camptothecin-based therapy for patients unresponsive t o standard treatment.
0 1994 by The Americsn Societyof Hematology.
C
HRONIC LYMPHOCYTIC leukemia (CLL) is an inMTT assay correlated well with the dye exclusion method.*
These earlier studies evaluated the efficacy of CLB, as comcurable clonal disease with a variable response to therapy.' Until recently, standard treatment had been chlorambucil pared with other drugs, and its synergism with interferon.'.'
Using the MTT assay, in this report, we present longitudinal
(CLB) with or without corticosteroids. Over the last decade,
three nucleoside analogs, pentostatin, fludarabine, and 2'chloro- studies with patients whose lymphocytes showedvarying
degrees of sensitivity to CLB. Our results show a concordeoxyadenosine, have become available for treatment
of CLL.
dance between the in vitro response to CLB and the effect
Among these, fludarabine has been most widely used?-'
Anin vitro assay predicting the clinical response to a
of therapy on the patient's clinical and absolute lymphocyte
particular agent would be of value in the selection of drugs
count (ALC) response.
In the present study, we also progide data that compare
with which to treat CLL. To this end, several studies have
the cytotoxicity of fludarabine, camptothecin (CPT), and sevdetermined chemosensitivity using a dye exclusion method
eral of its analogs with that of CLB in CLL B lymphocytes.
to measure cell ~iability.~.'
More recently, a viability assay
The CPTs are inhibitors of DNA topoisomerase I." Among
that depends on the enzymatic reduction of 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( M P ) to
these drugs, 9-aminocamptothecin (9-AC) and 10,ll -methylenedioxy-20(S)-camptothecin(10,l l-MDC)showed an unMTT formazan has beentested in CLL. Results with the
precedented effectiveness against various resistant human
cancers carried as xenografts by immunodeficient mice.""3
From the Departments of Medicine, Radiology, and Pathology,
In phase I1 studies, the analog 7-ethyl-l0-[4-(l-piperidyl)-I New York University School of Medicine, New York, NY.
piperidyll-carbonyloxycamptothecin showed substantial acSubmitted March 18, 1994; accepted July 13, 1994.
tivity
against a variety of carcinomas, as well as against nonSupported by US Public Health Service Grants No. CA 50529,
Hodgkin's lymphoma and acute
Several CPTs
CA 54484, CA 56/29, and T32 HL 07151,by the Marcia Slater
are undergoing preclinical screening, and some are already
Society for Research in Leukemia, and bythe Harry and Gussie
Wallerstein Foundation. D.C. was a Fellow of the American Cancer
in clinical trials.13"*We now show that 10,ll-MDC and 9Society.
amino- 10, I l-methylenedioxy-20(S)-campthothecin(9-AAddress reprint requests to Robert Silber, MD, Duke University
10,ll-MDC) are effective in killing B-cell CLL (B-CLL) B
Medical Center, Box 3250, Durham, NC 27710.
lymphocytes in vitro.'"'"
The publication costsof this article were defrayed in part by page
We have previously documented that the presence of mucharge payment. This article must therefore be hereby marked
tations
in the p53 gene had anadverse effect on the prognosis
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
of CLL2'; the present study reports the correlation between
indicate this fact.
these mutations and chemosensitivity to fludarabine, 10,ll0 1994 by The American Society of Hematology.
MDC, and 9-A- IO, 1 1"DC.
0006-4971/94/88410-0024$3.00/0
3440
Blood, Vol 84, No 10 (November 15). 1994: pp 3440-3446
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DRUG RESISTANCE
IN
3441
B-CLL
I
Our results indicate that the "IT assay may be a valid
predictor of the clinical response to CLB. In addition, the
CPTs were found to be more cytotoxic than fludarabine to
CLB-resistant B-CLL lymphocytes in vitro.
1
0
300
0
0
n
MATERIALS AND METHODS
Patient population. A total of 65 patients withB-CLL seen at
the New York University Medical Center (New York, NY) were
studied. The diagnosis of B-CLL required the demonstration of at
least 5 X 103/pL monoclonal B lymphocytes positive for CD5,
CD19, and CD20. The disease was staged according to Rai et al."
The patient distribution was as follows: 18 patients, stage 0; 24,
stage I; 17, stage II; 5, stage 111; and 1 stage IV. The patients
were divided into untreated and treated groups. The untreated group
consisted of 43 patients who had never received treatment and 3
patients who hadbeen treated more than 1 year before this study. The
treated group consisted of 34 patients, 15 of whom were originally
included in the untreated patient group. In addition to earlier CLB
treatment with or without prednisone, 9 patients had received cyclophosphamide; 5 , fludarabine; 2, etoposide or pentostatin; and single
patients received either vincristine, mitoxantrone, or CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone). Responses were
evaluated according to the criteria of the EST 2480 Eastern Cooperative Oncology Group (ECOG) protocol.'.23
Lymphocyte isolation. Heparinized blood was obtained from
consenting patients. Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gradients (PH Pharmacia, Uppsala, Sweden);
T lymphocytes were removed by sheep erythrocyte rosette formation, and monocytes were depleted by adherence to Falcon plastic
dishes (Fisher, Springfield, NJ). Enrichment of B lymphocytes was
assessed byflow cytometry with anti-CD5, anti-CD19, and antiCD20 antisera. Purity of greater than 95% B-CLL B lymphocytes
with fewer than 2% T lymphocytes, as determined by flow cytometry
with anti-CD3, was obtained by this procedure.
Tissue culture. A T-cell acute lymphoblastic leukemia line
RPMI 8402 and its CPT-K5 subline, which is resistant to CPT- 11I s
were obtained from Dr T. Andoh (Aichi Cancer Center Research
Institute, Nagoya, Japan) and grown as a cell suspension in RPMI
1640-10% heat-inactivated fetal calf serum at 37°C in 95% air/5%
COz. The culture medium contained 100 U/mL penicillin G-sodium,
100 pg/mL streptomycin sulfate, 0.25 pg/mL amphotericin, 2 gm/
L sodium bicarbonate, and 1% L-glutamine (GIBCO, Grand Island,
NY).
Drugs. CLB was purchased from Sigma (St Louis, MO), and
fludarabine was provided by the Division of Cancer Treatment (National Cancer Institute, Bethesda, MD) and purchased from Berlex
Laboratories (Alameda, CA); topotecan was supplied by SmithKline
Beecham (King of Prussia, PA). CPT and the analogs 9-AC, 10,llMDC, and 9-A-10,ll-MDC were obtained from Dr Monroe C. Wall
(Research Triangle Institute, Research Triangle Park, NC). Stock
solutions of CLB in dimethyl sulfoxide (DMSO; Sigma) and fludarabine in RPMI 1640 (GIBCO) were prepared fresh for each experiment; stock solutions of the CPTs in DMSO (1.0 to 6.0 mom)
were stored at -20°C. When needed, stock solutions were diluted
with RPMI 1640. The final maximum concentration of DMSO in
all studies was less than 0.035 mom. Drug concentrations for the
camptothecins were established spectrophotometrically. The following millimolar extinction coefficients were used: 10,ll-MDC AJS3,
37.3; 9-A-10,ll-MDC A382. 24.0.
M ~ a ~ s a B
y lymphocytes
. ~ ~ ~ ~(2.5 X 105/well)or tissue culture
cells (2.0 X 104/well)were cultured in round-bottom microtiter plates
in RPMI 1640-10% fetal calf serum in the presence of drugs or
controls containing the equivalent amounts of DMSO in a final
volume of 100 pL. At 72 hours, 25 pL of MTT (Sigma; 2 mg/mL
0
T
3
U
250
0
OD
0
0
0
0
0
0
B0
0
0
cn
I
I
"-S0-0
0
000
00
0
6
0 8A
B
Fig1.Comparison
of CL6 chemosensitivity of B lymphocytes
from 46 untreated (A) and 34 treated (B) patients. The median IC,
for the entire group was 61.0 pmol/L i--4
B;lymphocytes with an
IC, less than 61.0 pmollL were designated as sensitive, and those
with an IC, 2 61.0 pmollL were designated as resistant.
phosphate-buffered saline) was added for an additional 3 hours. The
precipitated MTT formazan was dissolved in 100 pL of isopropanol:
1 N HCl (24:l). After 30 minutes of incubation at 37"C, the A570
was measured on a MultiWell Plate Reader (Dynatech MC 600;
Dynatech Laboratories, Inc, Chantilly, M).Viability was expressed
as the ratio of A570 drug-treatedcontrol sample. Cells from all patients were studied on multiple occasions, ranging from 4 to 25
times.
Statistical a n a l y s i ~ . ~Comparison
~.~~
between patient samples
was assessed by Fisher's exact test for nominal variables and by a
nonparametric test of serial randomness. P values in all analyses
were two-tailed. The SAS system was used for all statistical calculations.
RESULTS
Correlation of MTT assay results with response to CLB
treatment. The drug concentration required for 50% inhibition of cell viability (ICs0) of CLB in B lymphocytes from
untreated patients ranged from 7 to 275 pmoVL. The median
ICs0 in this group (Fig 1A) was 40.5 p m o n (n = 46),
whereas cells from patients who were treated with CLB or
with CLB and prednisone (Fig 1B) within 12 months before
the "IT assay had ahigher median ICs0value of 86.0 pmol/
L (n = 34; P < .01). Inboth the treated and untreated
groups, the preparation used in the MTT assay contained
more than 90%CD5+ B lymphocytes. No correlation was
found between ICs0 and Rai stage.
The median ICs0 CLBfor the total (treated and untreated)
patient group was 61.0 pmol/L (n = 80;see Fig 1 [----I). A
nonparametric test showed no evidence against randomness
either in the less than 61 .O pmol/L or the 2 6 1.O p m o L
subset. However, this test of serial randomness was significantly positive for the pooled data (P < .005), suggesting
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SILBER ET AL
3442
the presence of two runs (clumps), one at the low and the
other at the high range of ICsovalues. Based on this analysis,
B lymphocytes with ICso CLB less than 61.0 pmol/L were
designated as “sensitive,” and those with ICs0CLB 261.0
pmoVL were designated as “resistant” within the context
of this study.
Fifteen patients were entered into a prospective study to
correlate the pretreatment ICs0 CLB with the decrease in
ALCand clinical response. The baseline pretherapy and
maximal posttherapy ICs0values for 15 previously untreated
patients were determined (Fig 2). The median posttherapy
ICso was 5 times the median baseline pretreatment value,
with an increase in ICs0 of up to 20-fold after therapy. The
increase was observed as early as 2 weeks and as late as 3
months after therapy was instituted. With a maximal followup time of 2 years, the ICso remained elevated in 9 of 13
surviving patients. Among the 46 untreated patients on
whom serial assays were performed over a 2-year period,
we noted 10 instances of fluctuations in ICs0 (data not
shown). The effect of therapy on the clinical response and
ALC is shown in Table 1. Eleven patients whose lymphocytes had an ICso less than 61.0 p m o m during baseline
studies all had a partial clinical remission and showed a
greater than 70% decrease in ALC after therapy. In contrast,
of the 4 patients whose lymphocytes hadan ICs0 2 61.0
pmoVL, l had no response, 1 had a partial remission, and 2
had disease progression. The difference in partial remissions
between the two groups is also significant by Fisher’s exact
test (P < .009). The difference inALC decrease between
these two groups was significant (P < .001) as determined
by a randomized test.
The pattern of response to treatment is illustrated in sequential studies with B lymphocytes in 6 of the patients over
a span of 16 to 30 months Fig 3).Lymphocytes from patients
no. 2, 3, 5 , and 7 had ICso CLB values determined immediately pretherapy, which placed them into the sensitive group.
/
300
3
-J
0
3
200
3
0
-I
I
us 100
0
0
Pretreatment
Post-treatment
Fig 2. Effect of treatment on CLB sensitivity. The IC, was determined in 15 patienta before treatment with .ither CLB or CLB and
prednisone. The poattreatment value shown was the maximal observed after therapy.
During treatment, there was a rapid increase in IC50concomitant with a decrease in ALC. The change was noted as early
as within 1 week (patient no. 2) and as late as 6 months
(patient no. 5). Beyond the observation ofan increase in
ICso, the small number of patients hampers further correlation between therapy andICso oscillations. Lymphocytes
from patients no. 13 and 15 had pretreatment IC5” CLB
values that placed them in the resistant group. With therapy
these also showed an increase in ICsoand a decrease in ALC,
but the ALC remained greater than 60 X 103/pL.
Comparison of CLB, jludarabine, and the camptothecins.
The chemosensitivity of lymphocytes from treated and untreated patients to fludarabine, lO,ll-MDC, and 9-A-10,llMDC is shown (Fig 4). The selected limits of sensitivity for
each drug (Fig 4 [---I) were defined as the mean IC50plus
2 SD. As with CLB, lymphocytes with an ICsovalue above
these limits were defined as resistant. The ICs0 values for
each drug are shown for cells sensitive to CLB (ICso< 61 .O
pmoVL) and CLB resistant (ICs0 2 61.0 pmoVL). The ICso
for fludarabine ranged from 6 to greater than 100 pmoVL;
the ICso for CLB-sensitive was lower than that of the CLBresistant group (Fig 4A; P
.01). As for CLB, a nonparametric test of serial randomness applied to pooled data was
significantly positive (P < .01) suggesting the presence of
two runs with a clustering of lower and higher IC50s.
For 10,ll-MDC (ICs0, 0.10 to greater than 1.0 pmol/L)
and 9-A-10,l l-MDC(ICso,0.03 to greater than 1.O pmol/L),
there were no significant differences (P > .05) between the
subsets of CLB-sensitive and CLB-resistant B lymphocytes
(Fig 4B and C), and there was no evidence against randomness of ICso distribution. Among the CLB-sensitive
cells, there is a cluster with low ICso values. Clusters were
also observed with fludarabine in cells from 23 of 33 patients, as well as with 10,ll-MDC in 17 of 24 patients, and
with 9-A-10,ll-MDC in 22 of 23 patients. CLB-resistant
lymphocytes from 13 of 23 patients were sensitive to 10,llMDC, and 10 of15 were sensitive to 9-A-10,ll-MDC. In
contrast only 7 of 24 CLB-resistant lymphocyte preparations
were sensitive to fludarabine.
When compared with 10,l l-MDC and 9-A10, 11-MDC,
equitoxic concentrations were generally higher for CPT (N
= 5), 9-AC (N = 19), and topotecan (N = 1 l), with median
ICs0 values of approximately 1.0 pmol/L, 1.0 pmoVL, and
1.6 pmol/L, respectively. There was no difference in median
ICso values for B-CLL lymphocytes between treated and
untreated patients.
CPT-sensitive and CPT-resistant tissue-culture lines were
used as controls for drug sensitivity. The parent RPM1 8402
line was sensitive to 10,ll-MDC and 9-A-10,ll-MDC (Fig
4), whereas the CPT-K5 subline showed resistance to these
drugs.
Relationship of drug resistance to p53 mutations. In an
earlier study, we reported the relationship between CLB resistance and p53 gene mutations in B-CLL lymphocytes.*’
Chemosensitivity of B lymphocytes to fludarabine and the
CPTs was compared with that of CLB and correlated with
the presence of p53 mutations. Lymphocytes with these mutations from all 7 patients were resistant to CLB, and 5 of
6 were resistant to fludarabine (Table 2). Lymphocytes from
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DRUG RESISTANCE IN B-CLL
3443
Table 1. Effect of Therapy on Response
Chlorambucil
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Rai
Stage
Ill
II
II
II
I
II
II
II
II
I
I
II
I
111
II
Therapy
497
63
44
76
82
133
265
115
82
41
55
41
262
25
190
93
87
77
80
93
92
84
73
74
90
82
85
0
0
49
8
11
13
17
19
23
28
34
38
41
54
66
103
112
124
12
15
20
2
6
3
5
2
>9
7
1
2
2
1
2
PlJ
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
CLB
CLB
CLB + PRED
CLB
CLB
CLB
CLB + PRED
CLB + PRED
CLB
CLB
CLB + PRED
CLB
CLB f PRED
CLB
CLB f PRED
ICm Avg
Pretreatment
I C , Ratio:
Pretreatment
Maximal
% Decrease
in ALC
ALC lxlO'/
ECOG
Clinical
Response
PR
PR
DP
DP
NR
PosVPre
Abbreviations: PRED, prednisone; PR, partial response; NR, no response; DP, disease progression; ECOG, Eastern Cooperative Oncology
Group.
4 of 7 patients were resistant to 10,11-MDC, and 3 of 4
were resistant to 9-A-10,ll-MDC.
Chemosensitivity of B lymphocytes without p53 mutations from 39 patients was also analyzed in terms of CLB
resistance.Amongthe
19 patients with CLB-sensitive
lymphocytes, 15 of 17 tested were also sensitive to fludarabine, 14 of 17 were sensitive to 10,11-MDC, and 15
of 15 were sensitive to 9-A-10,ll-MDC. Among the 20
patients with CLB-resistant lymphocytes, 6 of 18 tested
weresensitive to fludarabine, 9 of 16 were sensitive
' YL.4
OO
Fig 3. Effect of therapyon
IC, CLB and ALC in 6 representative patients. Patients no.
2,3,5,
and 7 from Table 1 had B lymphocytes with pretreatment IC,
CLB
values
in the sensitive
range, and patients no. 13 and
15hadpretreatment
IC, CLB
values in the resistant range. Pa2, 3,
and
5 were
tients
no.
treated with CLB; patients no.7,
13,and15
( 1 - 1 ) weretreated
with CLB andpredniaone.Patient no. 13 wasalsotreated
with fludarabine ( 1 = I).
mr
S
l0
1s
20
I
P
H
W
'
6
TIME (months)
10
1s
TIME (months)
10
1
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3444
SILBER ET AL
0
0
0
0
0
0
o.8
8
0
0
0
I
0
0
0
0
"""
51
0.2
9
11
<61
0
S 1
261
e61
261
IC,, CHLORAMBUCIL (PM)
Fig 4. Chemosensitivity of B lymphocytes from untreated patients.I& values for fludarabine, (A); 10.11-MDC, (B); and S-A-10.1l-MDC. (C).
in the text {---I. Tissueculture linesused were RPhV 8402
Sensitive and resistant lymphocyte demareationlimits are designated as described
(A)and CPT-K!5 ( + 1. Results obtained with these lines validatethe MlT assay for the camptothecins.
to 10,11-MDC, and 9 of 1 1 were sensitive to 9-A-10,
1 I-MDC.
DISCUSSION
The MTT assay has been used to predict the response to
therapyz9and to investigate drug resistance in relapsed acute
le~kemia.2~.
O
' The assay has been less widely applied to the
lymphoid malignancies, including CLL.*' In this report, we
establish a positive correlation between in vitro chemosensitivity to CLB and a favorable response to treatment in BCLL. We have also investigated the in vitro cross-resistance
of CLB to fludarabine as well as to CPT and its analogs, a
new class of anticancer agents. Three findings withCLB
emerge from this study. The first is the marked heterogeneity
in the in vitro sensitivity to CLB and the other drugs investi-
Table 2. Relationship of Chemosensitivity
to Lymphocyte p53 Mutations
Fludarabine
p53 Mutations
Present
CLB-resistant
(n = 71
Not found
CLB-resistant
(n = 20)
CLB-sensitive
In = 19)
10.11-MDC
9-A-10.11-MDC
Resistant Sensitive Resistant Sensitive Resistant Sensitive
5
1
4
3
3
1
12
6
7
9
2
9
2
15
3
14
0
15
p53 Mutations were detected as previously described." Chemosensitivity of B
lymphocytes was assayed as describd under Materials and Methods. Data in
table refer to number of patients who were studied.
gated. Cells from the majority of untreated patients are sensitive to CLB, whereas cells from about one third of the cases
are consistently more resistant to this drug. This may reflect
a manifestation of de novo resistance, a commonand serious
problem in chemotherapy. The second is the positive correlation between the in vitro chemosensitivity of CLL B lymphocytes and the patients' response to CLB treatment. The wellrecognized variable therapeutic efficacy of CLB may be the
in vivo counterpart of the heterogeneity in cytotoxicity observed in vitro. Further studies with a larger number of patients will be required to determine if the MTT assay has a
predictive value in the treatment of B-CLL.
A third finding documents the effect of therapy on the
CLB. When cells from treated patients were compared
with cells from patients who had not received CLB, the
medianIC5O CLB of the former group was significantly
higher than the median ICsoof the latter. The possibility was
considered that the higher IC50 in patients needing treatment
merely reflected a more advanced disease stage rather than
a consequence of CLB therapy. Contrary to this concept is
the lack of correlation between Rai stage and IC50.Further
evidence against this interpretation is the relatively rapid
increase in ICm CLB observed after therapy that was not
noted in untreated patients.
The CLB resistance observed in vitro after treatment occurred in the leukemic CD5+ B lymphocytes. The increase
in ICso was not caused by the presence of T cells or monocytes, because T-cell depletion was complete and monocyte
contamination was below 1%. Although some of the 15 patients studied prospectively received prednisone as well as
CLB, the effect of therapy on IC50 wasthe same in patients
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DRUGRESISTANCE IN B-CLL
receiving CLB alone as in those who were also treated with
prednisone. The increase in IC50 most likely stems from the
killing of sensitive lymphocytes by CLB, with selection of
a resistant subpopulation. The increase observed in IC5,, in
some cases appears reversible, despite continued therapy.
The fluctuations in ICs0 observed in about one fifth of the
untreated patients indicate that factors other than CLB therapy can cause changes in drug resistance that are reflected
in the ICs0. We think it is not an artifact of the assay. The
nature of these factors remains unclear.
In the second part of the study, the in vitro effects of
fludarabine and the CPTs were evaluated. The results with
fludarabine showed considerable cross-resistance with CLB.
A clinical response to fludarabine in CLB-resistant patients
is amply documented. In contrast, our findings of crossresistance in vitro is consistent with the clinical observation
that fludarabine does not prolong the survival of patients
resistant to CLB (and predni~one).~’
Our in vitro results
differ from those of others who, using another chemosensitivity assay, find no concordance in cross-resistance between
CLB and fludarabine.6
We have previously reported the association between p53
gene mutations and CLB resistance in CLL lymphocytes.”
The present work shows that resistance to fludarabine and
the CPTs also occurs commonly in lymphocytes with a mutated p53 gene.
Our study also reports the effects of CPT and several of
its analogs on CLL lymphocytes. Among these agents, all
topoisomerase I inhibitors, two analogs, 10,ll-MDC and 9A-lO,ll-MDC, showed the greatest activity against CLL
lymphocytes. The first of these agents introduces a greater
number of breaks into CLL lymphocyte DNA than do CPT
or 9-AC.32In addition, when CLL lymphocytes are incubated
with IO,ll-MDC, the amount of cell-associated drug is
higher and its retention in the lactone form is greater than
that observed with CPT or 9-AC.19.33
These differences may
not be solely responsible for the greater cytotoxicity of
10,ll-MDC and 9-A-10,ll-MDC. Although it is tempting
to suggest that the greater cytotoxicity is caused by the increased number of breaks induced by these compounds in
CLL lymphocytes, the underlying mechanisms for cell killing by these agents remains not completely understood.
The extreme S-phase cytotoxicity of CPT has been extensively documented. In the “collision” model, the interaction
between the advancing DNA replication fork and the topoisomerase I-CPT-cleavable complex results in a doublestrand DNA break.34This event, which triggers S-phasespecific cell killing and G2-phase cell cycle arrest, may be
responsible for the highly selective killing of the S-phase
cells; however, it cannot solely account for the impressive
cytotoxicity to quiescent B-CLL lymphocytes, which are
largely arrested at the Go to G, phase of the cell cycle.
Additional processes may be involved.34
CLB an
is
alkylating agent. Fludarabine, a DNA
elongation terminator, is a DNA ligase inhibit~r.~’
The CPT
analogs, 10,ll-MDC and 9-A-10,11 MDC, are DNA
topoisomerase I inhibitors. The reason for the occurrence of
cross-resistance to these agents that have different mechanisms of action remains unknown. A common explanation
3445
may reside in the cell’s ability to repair DNA damage caused
by chemotherapeutic agents. The increased expression in BCLL lymphocyte^^^ of the ERCC-1 gene, which is involved
in radiation damage repair, supports such an interpretation.
Another mechanism may involve the induction of heat-shock
proteins recently described in CLL lymphocyte^.^' These
proteins that are induced by several cytostatic drugs may be
associated with drug re~istance.~’.
39
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From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
1994 84: 3440-3446
Chemosensitivity of lymphocytes from patients with B-cell chronic
lymphocytic leukemia to chlorambucil, fludarabine, and camptothecin
analogs
R Silber, B Degar, D Costin, EW Newcomb, M Mani, CR Rosenberg, L Morse, JC Drygas, ZN
Canellakis and M Potmesil
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