British Journal of Haematology, 1999, 106, 474±476
Response to cladribine in previously treated patientswith chronic lymphocytic leukaemia identi®ed byex vivo assessment of drug sensitivity by DiSC assay
ANDREW G. BOSANQUET,1 J. ADRIAN COPPLESTONE,2 STEPHEN A. N. JOHNSON,3 ALASTAIR G. SMITH,4 SARA J. POVEY,5JENNIFER A. ORCHARD6 AND DAVID G. OSCIER6 1Royal United Hospital, Bath, 2Derriford Hospital, Plymouth,
3Taunton and Somerset Hospital, 4Royal South Hants Hospital, Southampton, 5Janssen-Cilag Ltd, Buckinghamshire,and 6Royal Bournemouth Hospital
Received 19 October 1998; accepted for publication 14 May 1999
Summary. The ability to identify non-responders to cytotoxic
response 1´5 years, median survival 3´37 years) and two
chemotherapy has signi®cant clinical and economic bene®ts.
had a >70% fall in lymphocytes: six identi®ed as ex vivo
Differential staining cytotoxicity (DiSC) assays were per-
resistant failed to respond. The DiSC assay can accurately
formed in 34 previously treated patients with chronic
identify a subgroup of patients resistant to cladribine.
lymphocytic leukaemia prior to treatment with cladribine.
Of the 28 identi®ed as ex vivo sensitive, 26 achieved a
Keywords: cladribine, differential staining cytotoxicity assay,
complete (CR) or partial response (PR) (median length of
phase II trial.
The purine analogue cladribine (2-chloro-deoxyadenosine)
advanced disease. Patients were excluded if they had a life
can achieve high levels of overall responses and substantial
expectancy of <6 months.
numbers of complete responses in chronic lymphocytic
DiSC assay. DiSC assays were performed before cladribine
leukaemia (CLL). However, the ability to identify non-
treatment using published methods (Bosanquet & Bell,
responders to treatment would have signi®cant clinical and
1996). Mononuclear cells were isolated from blood samples
economic bene®ts. A well-validated ex vivo method, the
and incubated with cladribine at 1´024, 0´256, 0´064,
differential staining cytotoxicity (DiSC) assay, has been
0´016 and 0´004 mg/ml and the LC90 determined (minimum
previously evaluated as a tool for identifying response to
concentration of cladribine required to kill 90% of lympho-
treatment with purine analogues (Bosanquet, 1991; Bosan-
cytes). The results were not made known to the clinicians
quet & Bell, 1996; Bosanquet et al, 1999).
managing the patients.
This study aimed to investigate the prognostic value of the
Cladribine treatment. Cladribine (Leustat, Janssen-Cilag
DiSC assay in 34 patients with CLL of B-cell origin who were
Ltd) was administered as an intravenous (i.v.) 2 h infusion
either unresponsive to, or had relapsed following, one or
at a dose of 0´12 mg/kg each day for 5 consecutive days.
more treatments including chlorambucil, anthracycline/
Treatment was repeated at 28 d intervals. Depending on the
anthracenedione or ¯udarabine.
onset and degree of response, patients received between four
and six cycles. Those who showed no response to one cycle
MATERIALS AND METHODS
received no further treatment. Patients received full suppor-
tive care. Cycles could be postponed in the event of infection
Patients. 34 CLL patients entered the study: 23 men and
or thrombocytopenia (de®ned as a drop in platelets of >50%
11 women (mean age 67, range 45±83). They were either
of pre-treatment values or <60 ´ 109/l on the day before the
unresponsive to standard therapy or required a change of
next cycle). Responses were assessed using the National
therapy because of shortened remission duration or
Cancer Institute (NCI) criteria to determine study outcomes
(Cheson et al, 1996). Following cessation of treatment,
patients were monitored for duration of response (calculated
Correspondence: Dr A. G. Bosanquet, Bath Cancer Research Unit,
from 4 weeks after last cladribine treatment) and survival
Royal United Hospital, Bath BA1 3NG. e-mail: [email protected]
(calculated from treatment start).
q 1999 Blackwell Science Ltd
DiSC Assay for Cladribine Resistance in CLL 475
Table I. Response rates strati®ed according to Binet stage
and W.H.O. performance status at presentation.
Data were available from all 34 patients. Patients could be
separated into two distinct groups: 6/34 patients were ex vivo
CR PR response (%)
90 >1 mg/ml) and 28/34 patients were ex vivo
sensitive (LC90 < 0´3 mg/ml). Assay results correlated with
clinical response (Fig 1): 26 of the sensitive' group achieved
a complete response (CR) or partial response (PR), and two
had >70% reduction in lymphocytes but withdrew after one
cycle because of adverse events (fatal myocardial infarction
and haemolytic anaemia). All six patients in the resistant
W.H.O. performance status
group failed to respond: two showed a minor and short-lived
reduction in their lymphocyte count. The proportion of
patients for whom the DiSC assay correctly identi®ed clinical
outcome was therefore 32/34 (94%; 95% CI 86±100%).
>3 years after treatment; one PR patient died of pneumonia
while still in PR.
Survival of DiSC-assay-sensitive versus DiSC-assay-resis-
tant patients is presented in Fig 2. DiSC-assay-sensitive
patients survived a median of 3´37 years (CI 2´2±4´6 years).
11 patients were still alive at analysis at 3´6±4´8 years
(mean 4´2 years). Three of the assay-resistant patients died
very soon after cladribine treatment commenced, a similar
trend to that seen following ¯udarabine treatment of ex vivo
¯udarabine-resistant patients (Bosanquet et al, 1999). Of the
other three patients, one responded to splenectomy and two
survived with supportive care but poor quality of life for >3
years. Due to the long survival of these three patients, no
signi®cant difference in survival was seen between the DiSC-
assay-sensitive and DiSC-assay-resistant patients (P 0´12).
Fig 1. Comparison of cladribine sensitivity by DiSC assay (LC90, mg/
ml) with subsequent patient response to cladribine. The two ex vivo
sensitive patients who achieved a > 70% reduction in lymphocyte
count are indicated by the arrow. >2, LC90 was not observed at the
maximum concentration of cladribine tested (1´024 mg/ml).
Response and survival
7/34 patients (21%; CI 7±34%) achieved a CR, 19/34 (56%;
CI 39±73%) achieved a PR. Two patients showed a 71% and
88% reduction in lymphocytes but did not meet the full
Fig 2. Survival of DiSC assay-sensitive (ÐÐ) and assay-resistant
response criteria before withdrawing from the study. The
(± ± ±) patients from beginning of cladribine treatment. Difference in
remainder (6/34, 18%; CI 5±30%) had no response.
survival was not signi®cant (P 0´12).
Response rates strati®ed by Binet stage and W.H.O.
performance status at baseline are shown in Table I.
Median remission duration of the responders was
Fourteen patients had received ¯udarabine; 12 responded
1´50 years (range 0±3´23) for the PR patients and 2´30
but later relapsed. The two other patients who had not
years (range 0´71±3´74) for the CR patients. Three of the
responded to previous ¯udarabine treatment were resistant
CR patients and one of the PR patients remain in remission
to cladribine by DiSC assay and withdrew from the study
q 1999 Blackwell Science Ltd, British Journal of Haematology 106: 474±476
476 Andrew G. Bosanquet et albecause of persistent thrombocytopenia after one or two
Bath); Dr A. G. Prentice, Dr M. D. Hamon, Sister E. Leppard
cycles of cladribine. Another patient developed autoimmune
(Derriford Hospital, Plymouth); Dr M. J. Phillips, Sister C.
haemolytic anaemia during cladribine treatment and
Ibberson (Taunton and Somerset Hospital); Dr A. S.
therefore withdrew, having previously stopped ¯udarabine
Duncombe, Mrs S. M. Course (Royal Southants Hospital,
treatment for the same condition.
Southampton). The study was funded by Janssen-Cilag Ltd
and we acknowledge the contributions of Elizabeth Hope
(study monitoring), Lorna Legg (statistical analysis) and
Five patients developed severe haemolysis shortly after
Elizabeth Wager (manuscript preparation).
treatment; 4/5 had positive direct antiglobulin tests
(DAGT), two subsequently died, all four had previously
received chlorambucil and ¯udarabine. Haemolysis occurred
during the ®rst (one), second (three) or third (one) cycle of
cladribine, from 5±24 (median 18) days after cladribine
Bosanquet, A.G. (1991) Correlations between therapeutic response
administration. All four patients who received more than
of leukaemias and in-vitro drug sensitivity assay. Lancet, 337,
one cycle of cladribine achieved a partial response. These
Bosanquet, A.G. & Bell, P.B. (1996) Enhanced ex vivo drug
cases have been reported by Chasty et al (1998). Six patients
sensitivity testing of chronic lymphocytic leukemia using re®ned
experienced persistent thrombocytopenia. Two other
DiSC assay methodology. Leukemia Research, 20, 143±153.
patients died during the course of treatment (from a
Bosanquet, A.G., Johnson, S.A. & Richards, S.M. (1999) Prognosis
myocardial infarct and cerebrovascular accident). Three
for ¯udarabine therapy of chronic lymphocytic leukaemia based
patients developed further malignancies or disease
on ex vivo drug response by DiSC assay. British Journal of
Haematology, 106, 71±77.
Bromidge, T.J., Turner, D.L., Howe, D.J., Johnson, S.A. & Rule, S.A.J.
(1998) In vitro chemosensitivity of chronic lymphocytic leukae-
mia to purine analogues: correlation with clinical course.
In this study the DiSC assay identi®ed resistance to
Leukemia, 12, 1230±1235.
Chasty, R.C., Myint, H., Oscier, D.G., Orchard, J.A., Bussutil, D.P.,
cladribine, having previously been shown to detect ex vivo
Hamon, M.D., Prentice, A.G. & Copplestone, J.A. (1998)
sensitivity and resistance accurately in both solid and
Autoimmune haemolysis in patients with B-CLL treated with
haematological tumours (Bosanquet, 1991; Bosanquet &
chlorodeoxyadenosine (CDA). Leukaemia and Lymphoma, 29, 391±
Bell, 1996; Bosanquet et al, 1999). Other groups have
published preliminary results using ex vivo drug sensitivity
Cheson, B.D., Bennett, J.M., Grever, M., Kay, N., Keating, M.J.,
tests in CLL with mixed success (Bromidge et al, 1998; Liu et
O'Brien, S. & Rai, K.R. (1996) National Cancer Institute-
al, 1997; Lambert et al, 1992; Hansen et al, 1991). However,
sponsored Working Group guidelines for chronic lymphocytic
the results of this work and the powerful prognostic factor of
leukemia: revised guidelines for diagnosis and treatment. Blood,
the ¯udarabine DiSC assay sensitivity for response and
survival after ¯udarabine (Bosanquet et al, 1999) suggest
Delannoy, A., Martiat, P., Gala, J.L., Deneys, V., Ferrant, A., Bosly, A.,
Schieff, J.M. & Michaux, J.L. (1995) 2-Chlorodeoxyadenosine for
that the DiSC assay should be used routinely before purine
patients with previously untreated chronic lymphocytic leukemia.
analogues are administered to patients.
Leukemia, 9, 1130±1135.
Knowledge of ex vivo resistance enables patients who are
Hanson, J.A., Bentley, D.P., Bean, E.A., Nute, S.R. & Moore, J.L.
unlikely to respond to avoid ineffective and potentially
(1991) In vitro chemosensitivity testing in chronic lymphocytic
debilitating treatment. Patients with good performance
leukemia patients. Leukemia Research, 15, 565±569.
status and in an early stage of their disease (i.e. W.H.O.
Juliusson, G., Christiansen, I., Hansen, M.M., Johnson, S., Kimby, E.,
grade 1 or Binet stage A) are more likely to achieve a CR or
Elmhorn-Rosenborg, A. & Liliemark, J. (1996) Oral cladribine as
PR with cladribine (Table I).
primary therapy for patients with B-cell chronic lymphocytic
The overall response rate observed in this study (76%) is
leukemia. Journal of Clinical Oncology, 14, 2160±2166.
similar to that reported for untreated patients (73±87´5%)
Lambert, E., Rees, J.K. & Twentyman, P.R. (1992) Resistance
circumvention strategies tested in clinical leukemia specimens
(Mulligan et al, 1996; Saven et al, 1995; Delannoy et al,
using the MTT colorimetric assay. Leukemia, 6, 1063±1071.
1995; Juliusson et al, 1996). This group is therefore more
Liu, K.Z., Schultz, C.P., Johnston, J.B., Lee, K. & Mantsch, H.H.
sensitive than most previously treated patients ± indeed
(1997) Comparison of infrared spectra of CLL cells with their ex
many patients had responded to their previous treatments.
vivo sensitivity (MTT assay) to chlorambucil and cladribine.
The median response duration compares favourably with
Leukemia Research, 21, 1125±1133.
Mulligan, S.P., Gill, D., Eliadis, P., Dale, B., Bunce, I., Bashford, J.,
In conclusion, the DiSC assay can delineate CLL patients
Page, F., Matthews, J. & Bradstock, K. (1996) 2-Chlorodeoxy-
who could, or who will not, bene®t from cladribine
adenosine by shortened infusion results in high response rate in
previously untreated chronic lymphocytic leukemia: an Austra-
lian Leukemia Study Group Trial. British Journal of Haematology,
93, (Suppl. 2), 116.
Saven, A., Lemon, R.H., Kosty, M., Beutler, E. & Piro, L.D. (1995)
2-Chlorodeoxyadenosine activity in patients with untreated
We thank the following for their help and collaboration with
chronic lymphocytic leukemia (CLL). Journal of Clinical Oncology,
this study: Mrs A. Burlton, Mr P. Bell (Royal United Hospital,
q 1999 Blackwell Science Ltd, British Journal of Haematology 106: 474±476
Sudden Hearing Loss: Audiological Diagnosis Ali A. Danesh, PhD. Associate Professor, Department of Communication Sciences & Disorders and Department of Biomedical Sciences, Florida Atlantic University Adjunct Professor, Audiology Department, Nova Southeastern University William D. Andreassen, B.S.
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