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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: a.g.bosanquet@ (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 ˆ 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. >ˆ2, 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 ˆ 0´12).
Response rates strati®ed by Binet stage and W.H.O.
performance status at baseline are shown in Table I.
Previous treatment 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 87, 4990±4997.
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.
previous reports.
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, 13, 570±574.
q 1999 Blackwell Science Ltd, British Journal of Haematology 106: 474±476


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