Ann Hematol (2003) 82:759–765DOI 10.1007/s00277-003-0710-5 Patrick D. Thornton · Estella Matutes ·Andrew G. Bosanquet · Anil K. Lakhani ·Henri Grech · Janet E. Ropner · Rajeev Joshi ·Peter H. Mackie · Ian D. C. Douglas ·Stella J. Bowcock · Daniel Catovsky High dose methylprednisolone can induce remissions in CLL patientswith p53 abnormalities Received: 2 April 2003 / Accepted: 3 June 2003 / Published online: 10 October 2003 Springer-Verlag 2003 Abstract Abnormalities of the p53 gene are known to apoptotic drug sensitivity index (DSI). HDMP was given confer detrimental effects in chronic lymphocytic leukae- alone or in combination with other drugs: vincristine, mia (CLL) and are associated with short survival. We CCNU, Ara-C, doxorubicin, mitoxantrone and chloram- have used high dose methylprednisolone (HDMP) to treat bucil, according to the results of DSI. Three patients were 25 patients with advanced refractory CLL of whom 45% treated twice and each treatment was analysed separately.
had p53 abnormalities shown by one or more methods: The overall response rate was 77% with a median flow cytometry, fluorescent in situ hybridisation and duration of 12 months (range 7 –23+). Responders direct DNA sequencing. Fifteen were resistant to fludara- included 5/10 with abnormal p53, of which two achieved bine and 16 were non-responders to their most recent nodular PR. Patients with p53 abnormalities fared worse therapy. Methylprednisolone had a cytotoxic effect on than those with normal p53. There were no differences in lymphocytes from 95% of cases assessed by an ex vivo response according to whether HDMP was used alone orin combination. Nine of the 22 evaluable patients (3 NRand 6 PR) have died from progressive disease or P. D. Thornton · E. Matutes · D. Catovsky ())Academic Department of Haematology and Cytogenetics, transformation. Main toxicity was infection in 7/25 patients. Event free and overall survival were significant- ly better in responders vs non-responders (P>0.0001 and P=0.04 respectively). Patients with a DSI of 100% to steroids had a better overall and event free survival, but this was not statistically significant. This study demon- strates that HDMP alone or in combination with other agents is a useful treatment strategy in refractory CLL including patients with p53 abnormalities.
Keywords CLL · p53 gene · Methyl prednisolone · DisC Farnborough Hospital, Kent, Orpington, UK assay · Refractory CLL · Resistance to fludarabine · p53 Royal Berkshire Hospital, London Road, Reading, Berkshire, UK J. E. RopnerGloucestershire Royal Hospital, Cheltenham, Gloucester, UK Chronic lymphocytic leukaemia (CLL) usually follows a St. Mary’s Hospital, Parkhurst Road, Newport, Isle of Wight relatively benign course with affected individuals surviv- ing in excess of ten years, while in others, the disease Wexham Park Hospital, Slough, Berkshire, UK follows an aggressive course with a shorter survival.
Initial management of symptomatic or progressive CLL frequently involves alkylating agents, however resistance Royal Surrey County Hospital, Egerton Road, frequently develops. An important improvement in ther- apy during the 1980’s was the introduction of the nucleoside analogue fludarabine. Overall response rates Queen Mary’s Sidcup NHS Trust, Frognal Avenue, of 78% have been reported with fludarabine as first line therapy, yet the response rate in previously treated tance conferred by mutant p53 has been shown to be patients is variable (12–94%) [1].
entirely independent of MDR1 or MDR3 [16, 17].
Moreover, fludarabine is not curative and most We describe the response rate and outcome of patients will eventually relapse after therapy. There are refractory/advanced CLL patients (half of them refractory a number of salvage regimens for relapsed or resistant to fludarabine) in which a high percentage had abnor- CLL, but few of these studies were aimed specifically at malities of p53 determined by a variety of techniques. We patients resistant to purine analogues. Furthermore, the also ascertained by the drug sensitivity assay as a myelotoxicity of anthracycline containing regimens and predictor of response and showed that steroids, and the increased susceptibility to opportunistic infections and frequently vincristine, killed CLL cells with abnormal autoimmune problems following purine analogues [2], p53 function. This is most likely due to the fact that may cause morbidity without the benefit of disease steroids and vincristine can cause apoptosis independent of the p53 pathway [11, 12]. We conclude that in vitro The ex-vivo apoptotic drug sensitivity assay, previ- testing and treatments with cellular killing mechanisms ously known as differential staining cytotoxicity (DiSC) independent of a normally functioning p53 is a rational assay, has been shown to have a 92% predictive accuracy and effective approach to the management of this poor in identifying drug resistance with a sensitivity of 77% [3]. Knowledge of ex-vivo resistance may enable us todetect patients who are unlikely to respond to certaincytotoxics thus avoiding unnecessary treatment and morbidity. Cells from previously treated patients havebeen found to be more sensitive to methylprednisolone This study included 25 patients with advanced CLL. Diagnosis was than cells from untreated patients, and a good correlation confirmed by morphology and immunophenotyping of peripheralblood lymphocytes using a CLL scoring system as previously with ex-vivo responses and response to steroid treatment described [18]. All the patients had received a median of three as well as purine analogues has been reported [3, 4, 5].
previous treatments (range 1–6) and 16 were non-responders to There are a number of chromosomal and immunophe- their last therapy (Table 1) Out of 25, 24 patients had fludarabine notypic markers of worse prognosis and treatment alone, or in combination with other drugs, and 15 had no response resistance in CLL [6] and it has recently been shown to fludarabine, when last used. Fludarabine was not repeated due toautoimmune haemolytic anaemia following a previous course in that the feature conferring worse prognosis involves the tumour suppressor gene P53 [7, 8, 9, 10]. Döhner et al. [8] HDMP was given intravenously at a dose of 1gm/m2 daily for 5 found that patients with p53 deletion failed to respond to days repeated at four weekly intervals. All patients were given H2 either fludarabine or pentostatin, compared with a 36% antagonists and low dose oral antifungal prophylaxis. Patients whowere neutropenic were given prophylactic antibiotics, and acyclovir response rate in patients without deletion. Similarly, was used in those with a previous history of herpes infection.
Wattel et al. [7] documented a 12.5% vs 80% response Blood pressure was monitored prior to starting treatment and rate to chlorambucil in cases with p53 deletion vs cases half-hourly during treatment. Urine sugar was monitored daily while having the first course of HDMP and oral hypoglycaemicagents were given if the blood sugar was sustained above The mechanism of action of fludarabine involves the 12 mmols/l. Patients received a median of 4.5 courses of HDMP incorporation of its metabolite F-Ara-A (9-b-D arabino- (range 1–8); three patients (cases 10, 12 and 15) were treated on furanosyl-2-fluroadenine) triphosphate into elongating two different occasions and each treatment has been analysed nucleic acid chains resulting in termination of DNA and RNA synthesis [1]. Chlorambucil and other nitrogen High dose methylprednisolone (HDMP) was used, alone in 13 of the treatment courses or in combination with other agents in 15, mustards produce DNA cross-links and monoadducts according to the results of the ex vivo apoptotic drug sensitivity inhibiting transcription [11]. All of these agents induce assay: vincristine in seven patients and other drugs in eight others apoptosis, which is most likely dependant on normally (Table 1). The drug sensitivity assay was performed on 20/25 of functioning or wild-type p53. This was demonstrated by patients studied as described elsewhere [3].
Where peripheral blood or bone marrow was available, patients’ Johnston et al. [12] who measured increased levels of the cells were tested for abnormalities of the p53 gene. Fluorescent in p53 protein and mdm2 following treatment of CLL cells situ hybridization (FISH) was performed in 22/25 to detect with fludarabine, cladribine and chlorambucil; and deletions and; in 21 of these, flow cytometry was carried out for Gartenhaus et al. [13] who measured increased expression detection of an abnormal p53 protein expression. Flow cytometrywas also performed in one patient’s sample where FISH data is of p53 and its downstream target WAF1/CIP1 following unavailable. In total, 23/25 were tested for p53 abnormalities by treatment with cladribine. P53 independent mechanisms either method. Cells from patients with abnormal p53 by the above for purine analogue induced apoptosis are described methods, and available DNA, were sequenced for mutations.
including NAD+/ATP depletion by poly ADP-ribose FISH analysis was performed using standard methods [19] with polymerase. However, this mechanism appears to accel- a p53 locus specific probe (LSI p53 Spectrum Orange, Vysis,Richmond, UK) in combination with a probe specific for the erate the late stages of apoptosis rather than initiate the chromosome 17 centromere (CEP17 Spectrum Green, Vysis) to exclude the possibility of monosomy 17 causing loss of hybridiza- Resistance to these treatments, both in vitro and in tion signal for the p53 specific probe. Flow cytometry was carried vivo, correlates with abnormal p53 [12, 15]. Wild-type out on fixed mononuclear cells with the monoclonal antibody DO1(Novocastra, Newcastle upon Tyne, UK) recognising amino acids p53 has been shown to repress the activity of the multi- 11–25 of both wild-type and mutant p53. Cells were analyzed on a drug resistance gene, MDR1, however treatment resis- FACScan flow cytometer (Becton Dickinson, Oxford, UK) [19].
Direct sequencing was conducted in seven samples with either expression by flow cytometry and 4/7 had mutations by deletion by FISH and/or protein expression by flow cytometry, direct sequencing. The remaining three patients had obtained at the same time as those used for FISH experiments.
normal sequencing of exons 4 –9. A summary of the Sequencing of exons 4 –9 of p53 was performed by polymerasechain reaction (PCR) amplification of exonic sequences from genomic DNA followed by fluorescent automated cycle sequencingof both DNA strands. All mutations were re-sequenced from adifferent PCR product. Obtained DNA sequences were analysed using Sequence Analysis software (version 3.0) (ABI) and alignedand compared to published p53 sequence using Sequence Navigatorsoftware (ABI).
Twenty-five patients have been treated, three patients The ex vivo drug sensitivity assay was carried out on peripheral have been treated twice and are analysed separately blood mononuclear cells incubated with drugs for 94 h as described giving a total of 28 treatment courses. Twenty-two by Bosanquet [20]. Cells from untreated patients were incubatedwith 7–10 standard CLL drugs: chlorambucil, cyclophosphamide patients (25 treatments) were evaluable and responses (mafosfamide in vitro), prednisolone, vincristine, doxorubicin, are summarised in Table 1. Three patients were unevalu- epirubicin, fludarabine, cladribine, pentostatin and methylprednis- able for response as they developed serious infection olone. Previously treated patients’ cells were incubated with up to following the first course of treatment.
25 other agents. At the end of incubation, fixed duck erythrocytes Seventeen of the 22 (77%) evaluable patients respond- (as an internal standard) and fast green/nigrosin (to stain dead cellsblack) were added and the cells cytocentrifuged. Counterstaining ed to treatment, two of these had a nodular PR with a Romanowsky stain allowed identification of remaining live documented by BM trephine biopsy. Even of the five cells which were evaluated morphologically to determine LC90 s non-responders (NR), four had a significant reduction of — the lowest concentration of drug to produce a 90% reduction in the lymphocyte count and decrease in lymph node size tumour cell survival compared with control cells [3]. The drug DSIwas determined by comparison with all previous assay results with but short of PR; the other NR progressed while on the drug so that 0% is the most resistant and 100% the most treatment. Five of the ten patients with p53 abnormalities responded (2 nodular PR, 3 PR). However patients withp53 abnormalities still fared worse than those with normalp53 (Table 3).
Two out of three patients treated twice (cases 10, 12 and 15) responded a second time to HDMP giving a total of 19/25 (76%) responses. Of these 19, the median eventfree survival (EFS) from start of treatment was signifi- Ten out of 22 patients (45%) had hemizygous p53 cantly longer than in the non-responders (Table 3).
deletion by FISH, 7/9 with deletion had also p53 Table 3 Survival and EFS by response and p53 status a Three patients were treated twice: for overall survival only the earlier of the two treatments was analysed The remission duration from the end of treatment of 100% (Table 1). All responders had values equal to or ranged from 2.2 to 18.9+ months with median of 6.6 months. Five patients remain in remission with a range of In all 20 cases analysed, methylprednisolone alone or 4.7+ –18.9+ months (median 9.4+ months). Nine of the 22 in combination with another drug showed the greatest evaluable patients have died; three were non-responders who died of progressive CLL. The six others hadresponded to treatment, but four died of progressivedisease and two of them with large cell lymphoma.
A summary of patients’ responses, previous treatments and p53 status are displayed in Table 1. Responders to The treatment of refractory CLL remains disappointing HDMP had a better overall survival and event free and there are several reports of salvage regimens. Keating survival from start of treatment as determined by log rank et al. [21] reported a 56% response rate with fludarabine statistics. (Table 3). Other chemotherapy agents were in 68 previously treated patients, with a mean of two added in 15 cases where they showed a good ex vivo previous treatments, including 13% complete remissions.
sensitivity. Twelve achieved PR including one nodular However Monserrat et al. used fludarabine in 68 heavily PR (case 17). Comparison of HDMP alone vs in treated CLL patients and documented an overall response combination with other therapies shows no advantage in rate and improved survival in only 28% [22].De Rossi et the addition of other drugs in event free survival. It is of al. combined fludarabine with prednisolone in 22 pre- interest that of the three patients treated on two separate treated, chlorambucil refractory patients and reported occasions, two (cases 10 and 12) had a longer duration of 36% responses [23]. Marotta et al. [24] combined low response when HDMP was given with another drug.
dose fludarabine with cyclophosphamide in 20 patients,refractory to conventional therapy achieving an overallresponse rate of 85%. Tallman et al. [25] used cladribine in 26 relapsed refractory patients and attained a 31%remission rate. Bowen et al. [26] described a 50% The most common side effect observed was insomnia or response rate using subcutaneous Campath 1-H in hyperactivity and the most serious side effect was fludarabine resistant CLL with a median survival of 11 infection, which was recorded in seven of the 25 patients.
These were pneumonia (3), herpes zoster (1), E.coli In our study, all patients were refractory to alkylating septicaemia (1), oral candida (1), tuberculous osteomy- agents and 72% were resistant to fludarabine. Ex vivo elitis (1) and pyrexia of unknown origin. Treatment was drug sensitivity suggested sensitivity to steroids (40– stopped in three patients after one course due to infection 100%) in all cases where it was performed. Resistance to and one of these died from intractable pneumonia. Two fludarabine with this assay was 20/26 and resistance to all patients required oral hypoglycaemic agents and one nucleoside analogues was found in 11/26. Treatment with patient required insulin due to reversible steroid induced HDMP produced a 76% response rate in these refractory diabetes. One patient had a spontaneous wedge collapse patients, including half of those with p53 abnormalities.
of his L2 vertebrae, four months following completion of This compares favourably with previous responses to fludarabine (0/12) and Chlorambucil (1/8) in patients withp53 abnormalities [7, 8]. Vincristine is also noted to causeapoptosis in a p53 independent fashion and notably is the Correlation with the drug sensitivity assay most commonly used drug with HDMP in this series.
Although HDMP used showed no survival advantage over Ex vivo apoptotic drug sensitivity assay results were HDMP, in combination therapy with other drugs the 4/5 available in 22/25 evaluable cases and showed 40 –100% patients who remain in remission had an additional drug – ex vivo sensitivity of CLL lymphocytes to methyl- so this may prove to be an advantage with time. In prednisolone. Eleven out of 17 responders had a DSI of addition, two of the three patients treated twice with 100% whereas only one out of five of the NRs had a DSI HDMP had a longer response when another agent wasused.
Abnormalities of the p53 gene whether detected by 2. O’Brien S, Kantarjian H, Beran H, Smith M, Koller T, Estey C, protein expression [9], FISH [8] or direct DNA sequenc- Robertson E, Lerner LE, Keating M (1993) Results offludarabine and prednisolone therapy in 264 patients with ing [27] all confer worse prognosis and drug resistance in chronic lymphocytic leukaemia with multivariate analysis- CLL and in the hierarchical model described by Döhner et derived prognostic model for response to treatment. Blood al. [6], p53 deletion by FISH is the abnormality, conveying the worst prognosis. The generally accepted 3. Bosanquet A, Cann SRM, Mills M, Catovsky D (1995) mode of drug resistance is felt to be an impairment of the Methylprednisolone in advanced chronic lymphocytic leukae-mia: Rationale for and effectiveness of treatment suggested by apoptotic process upon which most of the cytotoxics rely [17]. Other studies have demonstrated that steroid and 4. Bosanquet A, Copplestone JA, Johnson SAN, Smith AG, Povey vincristine cytotoxicity is independent of the p53 pathway SJ, Orchard JA, Oscier DG (1999) Response to cladrabine in [12]. Although the association of drug resistance and poor previously treated patients with chronic lymphocytic leukaemiaidentified by ex vivo assessment of drug sensitivity by DiSC prognosis with abrogation of normal p53 function is well known, there is little in the literature to suggest a way 5. Bosanquet AG, Johnson SA, Richards SM (1999) Prognosis for forward in overcoming this problem. Some experimental fludarabine therapy of chronic lymphocytic leukaemia based on work and early clinical studies suggest that proteosome ex-vivo drug response by DiSC assay. Br J Haemat 106:71–77 6. Döhner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, inhibitors may offer an alternative apoptotic pathway to Bullinger L, Döhner K, Bentz M, Lichter P (2000) Genomic p53 dependent mechanisms [28, 29]; however, these aberrations and survival in chronic lymphocytic leukemia. N drugs are not readily available in practice. It has been previously shown that in vitro sensitivity to steroids 7. Wattel E, Preudhomme C, Hequet B, Vanrumbeke M, Quesnel correlates with clinical outcome, and nucleoside resis- B, Dervite I, Morel P, Fenaux P (1994) p53 Mutations areassociated with resistance to chemotherapy and short survival tance ex vivo has a similar in vivo correlation [3]. The in haematological malignancies. Blood 84:3148–3157 role of steroids in lymphocyte induced apoptosis is well 8. Döhner H, Fischer K, Bentz M, Hansen K, Benner A, Cabot G, known. These agents induce apoptosis by a number of Diehl D, Schlenk R, Coy J, Stilgenbauer S, Volkmann M, Galle mechanisms including inhibition of cytokine production, PR, Poustka A, Hunstein W, Lichter P (1995) p53 Genedeletion predicts for poor survival and non-response to therapy alteration of mitochondrial membrane potential causing with purine analogues in chronic B-cell leukaemias. Blood caspase activation, down regulation of cyclinD3 causing G1 arrest and inhibition of NF-kB, a transcription factor 9. Cordone I, Masi S, Mauro FR, Soddu S, Morsilli O, Valentini complex important in cell survival [28, 29, 30, 31, 32, T, Vegna ML, Guglielmi C, Manchini F, Giuliacci S, Sacchi A,Mandelli F, Foa R (1998) p53 expression in B-Cell Chronic Lymphocytic Leukaemia: A marker of disease progression and In summary, CLL patients with abnormal p53 are unlikely to respond to standard therapy. Treatments 10. Lens D, Dyer MJ, Garcia-Marco JM, Schouwer PJ de, Hamoudi directed outside p53 dependent apoptotic pathways, such RA, Jones D, Farahat N, Matutes E, Catovsky D (1997) p53 as steroids or vincristine, have a greater chance of abnormalities in CLL are associated with excess of prolym-phocytes and poor prognosis. Br J Haemat 99:848–857 success. We have demonstrated that these agents are 11. Begleiter A, Mowat M, Israels LG, Johnston J (1996) effective in resistant patients where abnormalities of p53 Chlorambucil in chronic lymphocytic leukaemia: Mechanism are identified by current methods. This treatment causes little or no myelosupression, so it can be used in stage C 12. Johnston JB, Daeninck P, Verberg L, Lee K, Williams G, Israels LG, Mowat MRA, Begleiter A (1997) p53, MDM 2, patients with profound cytopenias. Although almost one BAX and BCL-2 and drug resistance in chronic lymphocytic third of patients had infective episodes following treat- ment and the immunosuppressive effects of high dose 13. Gartenhaus RB, Wang P, Hoffman M, Janson D, Rai KR (1996) steroids are well recognised, HDMP may not be the sole The induction of p53 and WAF1/CIP1 in chronic lymphocyticleukaemia cells treated with 2-chlorodeoxyadenosine. J Mol factor involved as CLL itself has a higher rate of infection due to its intrinsic immunodeficiency [34]. We conclude 14. Pettitt AR, Sherrington PD, Cawley JC (2000) Role of that HDMP alone or in combination with other drugs, Poly(ADP-ribosylation) in the killing of chronic lymphocytic guided by an ex vivo drug sensitivity assay, is a logical leukaemia cells by purine analogues. Cancer Res 60:4187–4193 and effective treatment strategy in resistant CLL includ- 15. Silber R, Degar B, Costin D, Newcomb EW, Mani M, Rosenberg CR, Morse L, Drygas JC, Canellakis ZN, Potmesil M (1994) Chemosensitivity of lymphocytes from patients withB-cell chronic lymphocytic leukaemia to chlorambucil, flu- Acknowledgements This work was supported by a clinical darabine and camptothecin analogs. Blood 84:3440–3446 fellowship from the The Leukaemia Research Fund, London. We 16. Rouby SE, Thomas A, Costin D, Rosenberg CR, Potmesil M, would also like to thank Rifat Hamoudi, Institute of Cancer Silber R, Newcomb EW (1993) p53 Gene mutation in B-cell Research, for his help with the p53 gene sequencing.
chronic lymphocytic leukaemia is associated with drug resis-tance and is independent of MDR1/MDR3 gene expression.
Blood 82:3452–3459 17. Newcomb EW (1995) p53 Gene mutations in lymphoid diseases and their possible relevance to drug resistance. LeukLymphoma 17:211–221 1. Adkins J, Peters D, Markham A (1997) Fludarabine an update 18. Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, of its pharmacology and use in the treatment of haematological Houlihan A, Que TH, Catovsky D (1994) The immunological profile of B-cell disorders and proposal of a scoring system forthe diagnosis of CLL. Leukemia 8:1640–1645 19. Gruszka-Westwood AM, R.A. Hamoudi, Matutes E, Tuset E, 27. Fenaux P, Preudhomme C, Lai JL, Quiquandon I, Jonveaux P, Catovsky D (2001) p53 abnormalities in splenic lymphoma Vanrumbeke M, Sartiaux C, Morel P, Loucheux-Lefebvre MH, with villous lymphocytes. Blood 97:3552–3558 Bauters F, Berger R, Kerckaert JP (1992) Mutations of the p53 20. Bosanquet A, Bell P (1996) Enhanced ex vivo drug sensitivity gene in B-cell chronic lymphocytic leukaemia: A report on 39 testing of chronic lymphocytic leukaemia using refined DiSC cases with cytogenetic analysis. Leukaemia 6:246–250 28. McConkey D, Chandra J (1992) Protease activation and 21. Keating MJ, Kantarjian H, Talpaz M, Redman J, Credie KB glucocorticoid- induced apoptosis in chronic lymphocytic (1988) Fludarabine therapy in chronic lymphocytic leukaemia 29. Masdehors P, Omura S, Merle-Beral H, Mentz F, Cosset J-M, 22. Monserrat E, Lopez-Lorenzo JL, Manso F, Martin A, Prieto E, Dumont J, Magdelenat H, Delic J (1999) Increased sensitivity Arais-Sampedro J, Fernandez MN, Oyarzabal FJ, Odriozola J, of CLL-derived lymphocytes to apoptotic death activation by Alcala A, Garcia-Conde J, Conde E, Guardia R, Bosch. F the proteosome-specific inhibitor lactacystin. Br J Haemat (1996) Fludarabine in resistant or relapsing B-cell chronic lymphocytic leukaemia. The Spanish group experience. Leuk 30. King KL, Cidlowski JA (1998) Cell cycle regulation and 23. Rossi GD de, Mauro FR, Caruso R, Monarca B, Mandelle F 31. Mori N, Yamate J, Stassen APM, Oka S, Okumoto M, Tsubura (1993) Fludarabine and prednisolone in pretreated and refrac- A, Akamtsu T, Sakuma S, Demant P (1999) Modulations of tory B-chronic lymphocytic leukaemia (B-CLL) in advanced Glucorticoid-induced apoptosis linked to the p53 deletion and to the apoptosis susceptibility gene Rapop1 ( radiation-induced 24. Marotta G, Bigazzi C, Lenoci M, Tozzi M, Bocchia M, Lauria F (2000) Low-dose fludarabine and cyclophosphamide in 32. Smets L, Salomons G, van den Berg J (1999) Glucorticoid elderly patients with B-cell chronic lymphocytic leukaemia induced apotosis in leukaemia. Adv Exp Med Biol 457:607– refractory to conventional therapy. Haematologica 85:1268– 33. Riccardi C, Zollo O, Nocentini G, Bruscoli S, Bartoli A, 25. Tallman MS, Hakiaman D, Zanzig C, Hogan DK, Rademaker D’Adamio F, Cannarile L, Delfino D, Ayroldi E, Migliorati G A, Rose E, Variakojis D (1995) Cladrabine in the treatment of (2000) Glucocorticoid hormones in the regulation of cell death.
relapsed or refractory chronic lymphocytic leukaemia. J Clin 34. Bartik MM, Welker D, Kay NE (1998) Impairments in immune 26. Bowen AL, Zomas A, Emmett E, Matutes E, Dyer MJS, cell function in B cell chronic lymphocytic leukemia. Semin Catovsky D (1997) Subcutaneous Campath-1H in fludarabine resistant/relapsed chronic lymphocytic leukaemia and B-pro-lymphocytic leukaemia. Br J Haemat 96:617–619


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