Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial.

BACKGROUND: Previous studies of patients with chronic lymphocytic leukaemia reported high response rates to fludarabine combined with cyclophosphamide. We aimed to establish whether this treatment combination provided greater survival benefit than did chlorambucil or fludarabine. METHODS: 777 patients with chronic lymphocytic leukaemia requiring treatment were randomly assigned to fludarabine (n=194) or fludarabine plus cyclophosphamide (196) for six courses, or chlorambucil (387) for 12 courses. The primary endpoint was overall survival, with secondary endpoints of response rates, progression-free survival, toxic effects, and quality of life. Analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number NCT 58585610. FINDINGS: There was no significant difference in overall survival between patients given fludarabine plus cyclophosphamide, fludarabine, or chlorambucil. Complete and overall response rates were better with fludarabine plus cyclophosphamide than with fludarabine (complete response rate 38%vs 15%, respectively; overall response rate 94%vs 80%, respectively; p<0.0001 for both comparisons), which were in turn better than with chlorambucil (complete response rate 7%, overall response rate 72%; p=0.006 and 0.04, respectively). Progression-free survival at 5 years was significantly better with fludarabine plus cyclophosphamide (36%) than with fludarabine (10%) or chlorambucil (10%; p<0.00005). Fludarabine plus cyclophosphamide was the best combination for all ages, including patients older than 70 years, and in prognostic groups defined by immunoglobulin heavy chain gene (V(H)) mutation status and cytogenetics, which were tested in 533 and 579 cases, respectively. Patients had more neutropenia and days in hospital with fludarabine plus cyclophosphamide, or fludarabine, than with chlorambucil. There was less haemolytic anaemia with fludarabine plus cyclophosphamide (5%) than with fludarabine (11%) or chlorambucil (12%). Quality of life was better for responders, but preliminary analyses showed no significant difference between treatments. A meta-analysis of these data and those of two published phase III trials showed a consistent benefit for the fludarabine plus cyclophosphamide regimen in terms of progression-free survival. INTERPRETATION: Fludarabine plus cyclophosphamide should now become the standard treatment for chronic lymphocytic leukaemia and the basis for new protocols that incorporate monoclonal antibodies.

Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy.

Inactivation of p53 has been shown to correlate with poor prognosis and drug resistance in malignant tumors. Nevertheless, few reports have directly shown such effects in primary tumor cells. Here, we investigated the p53 mutational status in 138 B-CLL samples and compared these findings with drug and gamma-irradiation sensitivity profiles. p53 mutations resulted not only in a shorter survival but, notably also in selective resistance to alkylating agents, fludarabine and gamma-irradiation. In contrast, no such effect was observed for vincristine, anthracyclines and glucocorticoids. Thus, these latter compounds induce cell death at least in part by p53-independent pathways. Interestingly, p53 mutations clustered in patients who had received prior chemotherapy. In fact, we show for the first time that treatment with DNA-damaging alkylating agents correlates with occurrence of p53 mutations in a clinical setting. This finding may explain at least to some extent the development of resistance to second-line anticancer chemotherapy.

Ex vivo assessment of drug response by differential staining cytotoxicity (DiSC) assay suggests a biological basis for equality of chemotherapy irrespective of age for patients with chronic lymphocytic leukaemia.

With a mean age at diagnosis for chronic lymphocytic leukaemia (CLL) of 65 years, development of optimal therapeutic regimens has been hampered by the advanced age of patients. In general, because of comorbidity older patients are not treated with the intent of achieving a complete response and so do not attain the quality of response of younger patients and do not survive as long. We have investigated whether or not ex vivo cellular sensitivity to cytotoxic drugs could be an underlying biological basis for this age differential in response and survival by comparing ex vivo drug response with age in untreated CLL patients. Cells from 365 untreated CLL patients aged 31.1-87.1 years (average 65.3 years) were tested for drug response by differential staining cytotoxicity (DiSC) assay with a panel of 10 drugs. An average of 280 results (range 196-361) obtained for each drug was compared with patient age. For chlorambucil, cyclophosphamide, prednisolone, vincristine, doxorubicin, epirubicin, fludarabine, cladribine and methylprednisolone, no relationship was found between ex vivo drug response and age (r<0.12). For pentostatin, a possible but very weak relationship (r = 0.18; n = 210; P = 0.06) was found. We conclude that cellular sensitivity to cytotoxic drugs does not support the differential treatment of older and younger CLL patients.

Bax expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine or corticosteroids in B cell chronic lymphocytic leukemia.

In B-CLL, non-proliferating B cells accumulate due to defective apoptosis. Cytotoxic therapies trigger apoptosis and deregulation of apoptotic pathways contributes to chemoresistance. Loss of the apoptosis-promoting Bax has been implicated in resistance to cytotoxic therapy. We therefore evaluated ex vivo drug sensitivity of CLL, producing chemoresponse data which are prognostic indicators for B-CLL, in particular in the case of purine nucleoside analogs. To analyze the underlying mechanisms of drug resistance, we compared endogenous Bax and Bcl-2 expression to ex vivo response to eight drugs, and to survival in 39 B-CLL patients. We found that reduced Bax levels correlated well with ex vivo resistance to traditional B-CLL therapies - anthracyclines, alkylating agents and vincristine (all P < 0.04). Surprisingly, no such relationship was observed for the purine nucleoside analogs or corticosteroids (all P > 0.5). Mutational analysis of p53 could not explain the loss of Bax protein expression. Levels of Bcl-2 were not associated with sensitivity to any drug. In contrast to the ex vivo data, neither Bax or Bcl-2 expression nor doxorubicin sensitivity were associated with increased survival whereas sensitivity to fludarabine correlated with better overall survival (P = 0.031). These findings suggest that the resistance to purine nucleoside analogs and corticosteroids in B-CLL is due to inactivation of pathways different from those activated by anthracyclines, vinca alkaloids and alkylating agents and may be the molecular rationale for the efficacy of purine analogs in this disease.

Pharmacokinetics of 2,5-diaziridinyl-3,6-bis(2-hydroxyethylamino)-1,4-benzoquinone (BZG, NSC 224070) during a phase I clinical trial.

Plasma levels of 2,5-diaziridinyl-3,6-bis(2-hydroxyethylamino)-1,4-benzoquinone (BZQ, NSC 224070) were measured in nine patients after i.v. administration of the drug during a Phase I trial. Our own isocratic high performance liquid chromatographic (HPLC) method with a sensitivity of 3 ng/ml was used to quantify BZQ. Patients receiving 18-60 mg BZQ i.v. showed alpha and beta plasma decays with half-lives of 6.2 +/- 1.5 (mean +/- S.D.) and 24 +/- 4 min respectively. The apparent volume of the central compartment was 12.2 +/- 4.6 l, and the total volume of distribution was 33.6 +/- 11.3 l. The calculated plasma AUCs were linearly related to dose. A marked similarity in kinetic parameters was found for BZQ and diaziquone (AZQ, NSC 182986), another diaziridinylbenzoquinone that has recently completed phase II clinical trials.

Filtration of chylomicrons by the liver may influence cholesterol metabolism and atherosclerosis.

A fenestrated endothelial lining of sinusoids in rat liver has been shown to separate chylomicrons of different sizes following their injection into the portal vein. This sieving may have physiological importance, since during low dietary fat intake some intestinal lipoproteins are probably small enough to contact liver cells, but during high dietary fat loads most chylomicrons are too large to pass through the filter and must first be degraded to smaller remnants. The liver plays a central role in cholesterol metabolism since it catabolises dietary cholesterol which inhibits synthesis of cholesterol to be circulated as liver-derived very low density lipoproteins (VLDL) and low density lipoproteins. The sieving of chylomicrons, remnants and other lipoproteins by the sinusoidal endothelium of the liver may thus play an important role in lipid transport, affecting the balance of various lipoprotein moieties which in turn may affect the relationship of dietary lipids to various hyperlipidaemias and atherosclerosis.

The effect of alkylating agents and other drugs on the accumulation of melphalan by murine L1210 leukaemia cells in vitro.

The effect of cytotoxic and other drugs on the accumulation of melphalan by L1210 murine leukaemia cells was studied. We have confirmed that uptake is an active process competitively inhibited by L-leucine. In 36 experiments in amino acid-free medium the mean concentration of melphalan taken up was 225 pmoles/10(6) cells. High pressure liquid chromatographic analysis showed that the majority of the drug is present as free native melphalan. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was the only drug that stimulated accumulation, but without significant effect on influx or efflux rates. Busulphan, chlorambucil, cyclophosphamide, interferon, methotrexate and prednisolone had no effect on accumulation after 30 min melphalan transport. Adriamycin, CCNU, methyl CCNU, mustine and vincristine all impaired melphalan accumulation as did the non-cytotoxic drugs aminophylline, chlorpromazine and ouabain. Adriamycin, aminophylline, chloropromazine, indomethacin and ouabain all reduced melphalan influx.

Enhanced ex vivo drug sensitivity testing of chronic lymphocytic leukaemia using refined DiSC assay methodology.

Ex vivo drug sensitivity testing is of considerable benefit in aiding the choice of optimum chemotherapy for leukaemia patients, especially when several therapeutic options exist, e.g. for relapsed chronic lymphocytic leukaemia (CLL). We have used the Differential Staining Cytotoxicity (DiSC) assay to assess drug sensitivity in CLL for over a decade and here present many methodological improvements, including depositing multiple samples per microscope slide and performing a rapid LC90 evaluation. Using these improvements, 412/450 specimens were successfully tested. Failures were mainly due to extended specimen transit time. All 38 drugs tested exhibited dose-dependent cell kill and broad ranges of resultant LC90S were observed. Comparison of 2- and 4-day incubations underscored a requirement for 4-day incubation with pentostatin and steroids. The rapid, simple and streamlined DiSC assay presented here can aid choice of optimum therapy, identify novel anticancer agents and be used to study drug resistance.

Semi-micro adaptation of a 4-day differential staining cytotoxicity (DiSC) assay for determining the in-vitro chemosensitivity of haematological malignancies.

A semi-micro differential staining cytotoxicity (micro-DiSC) assay has been developed to determine the in-vitro chemosensitivity of haematological cancers. The method comprised isolation of leukocytes from blood or bone marrow, drug exposure and culture for 4 days in 1 ml tubes arranged in the microtitre format. Drug-induced tumour cell kill was determined by differential staining of live and dead cells, such that the former could be morphologically identified. Tumour cell viability was calculated by reference to an internal standard of fixed duck red blood cells. Up to 15 drugs at 5-6 concentrations each could be set up at a time in the assay within one hour of receipt of a sample, using only 10(7) viable cells. A result was obtained in 38 of 40 samples received. The assay is of potential use for the routine prediction of clinical response to cytotoxic drugs in haematological cancers and warrants wider investigation.

Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. III. Antimetabolites, tubulin-binding agents, platinum drugs, amsacrine, L-asparaginase, interferons, steroids and other miscellaneous antitumor agents.

The stability of solutions of the antitumour antimetabolites, vinca alkaloids, podophyllotoxins, interferons, steroids and platinum drugs as well as maytansine, asparaginase, amsacrine, flavone-8-acetic acid, mitoguazone, and N-phosphonoacetyl-L-aspartate (PALA) is reviewed. Much of the published work has been done with biological, not stability-indicating, assays; thus, the relevant results should be used with caution. With this proviso, almost all of these drugs can be stored in solution for several days at room temperature or 4 degrees C. Most reports also suggest that the drugs that have been tested are stable when frozen in solution. For a number of the drugs, particular precautions are required; for instance, amsacrine should not be mixed with chloride-containing solutions, whereas cisplatin is most stable in solutions containing greater than 0.1 M chloride.

Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. General considerations, the nitrosoureas and alkylating agents.

In vitro drug sensitivity of tumour biopsies is currently being determined using a variety of methods. For these chemosensitivity assays many drugs are required at short notice, and this in turn means that the drugs must generally be stored in solution. There are, however, a number of potential problems associated with dissolving and storing drugs for in vitro use, which include (a) drug adsorption; (b) effects of freezing; (c) drug stability under the normal conditions of dilution and setting up of an in vitro assay; and (d) insolubility of drugs in normal saline (NS) or phosphate-buffered saline (PBS). These problems are considered in general, and some recommendations for use of solutions of drugs in in vitro assays are suggested. The nitrosoureas and alkylating agents are also investigated in greater detail in this respect. The nitrosoureas are found to be very labile in PBS at pH 7, with 5% degradation (t0.95) occurring in 10-50 min at room temperature. These values are increased about 10-fold on refrigeration and about 5- to 10-fold on reduction of the pH of the medium to pH 4-5. At pH 7 and room temperature, t0.95 is observed in under 1 h with the alkylating agents nitrogen mustard, chlorambucil, melphalan, 2,5-diaziridinyl-3,6-bis(2-hydroxyethylamino)-1,4-benzoquinone (BZQ), dibromodulcitol, dibromomannitol, treosulphan, and procarbazine. Of the other alkylating agents, 4-hydroperoxycylophosphamide (sometimes used in vitro in place of cyclophosphamide), busulphan, dianhydrogalactitol, aziridinylbenzoquinone (AZQ), and dacarbazine have a t0.95 of between 2 and 24 h, while ifosfamide and pentamethylmelamine are both stable in aqueous solution for greater than 7 days. About half the drugs studied in detail have been stored frozen in solution for in vitro use, although very little is known about their stability under these conditions.

Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays. II. Assay methods, adriamycin and the other antitumour antibiotics.

The methods used to test drug stability are discussed in the light of two recent publications using biological assays. It is concluded that, as far as possible, stability-indicating assays should be used so that possible false results do not lead to erroneous conclusions. Many of the results of the stability studies with adriamycin were found to be at variance with each other, with a 20-fold difference in stability being reported in one case by different groups from virtually identical experiments. Definitive statements about adriamycin stability are therefore impossible, but it is clear that it is sensitive to light, adsorbs to membrane filters and containers (except polypropylene and siliconised glass), chelates metal ions and probably degrades rapidly in medium. Adriamycin's analogues may well have the same spectrum of sensitivity. Bleomycin, actinomycin D and neocarzinostatin were found to be stable for greater than or equal to 2 weeks at room temperature. All the other antitumour antibiotics investigated (except rubidazone) are stable for greater than or equal to 24 h at room temperature and longer at 5 degrees C. Almost all of them are sensitive to light and are most stable in neutral or slightly acid media, and many of them adsorb to membrane filters. They can probably all be stored frozen in solution.

Chlorambucil: stability of solutions during preparation and storage.

A stability-indicating high-performance liquid chromatographic method has been used to investigate the stability of chlorambucil in solution. Dissolved in ethanol and diluted with 150 mM NaCl (NS), the drug was found to be very much more stable frozen, with a value for 5% degraded (t0.95) of approximately 8 months at -70 degrees C. The presence of agar or cells, and freezing and thawing the solution did not reduce the concentration of chlorambucil significantly. The drug seemed to adsorb to polyvinyl chloride (PVC) infusion bags (but to no other container material) and also to all three filtration units tested. Intense light increased the degradation of the drug, whilst the addition of 10% serum to chlorambucil in medium increased the drug's stability four fold. Dilution with NS of stock solutions of chlorambucil in ethanol resulted in supersaturated solutions of the drug. For preparation of the drug for in vitro drug sensitivity assays, the results suggest that care should be taken in the initial dilution of the drug; not to use filtration units to sterilize solutions; to anticipate different degradation rates (and therefore cytotoxicity) if the serum concentration in medium is altered; and to avoid the use of PVC.

Degradation of melphalan in vitro: rationale for the use of continuous exposure in chemosensitivity assays.

The hydrolysis of melphalan in cell culture medium at 37 degrees C has been studied. Degradation of melphalan proceeded via monohydroxy-melphalan (MOH) to dihydroxymelphalan [M(OH)2] with a half-life of 66 min for melphalan and 58 min for MOH. The half-life for melphalan was similar to the terminal half-life of the drug in vivo. The effect of the two metabolites, MOH and M(OH)2, on the chemosensitivity of K562 leukaemia cells during continuous exposure to melphalan was also examined. M(OH)2 had no potentiating effect on melphalan cytotoxicity at concentrations up to 100 micrograms/ml. MOH also had little effect on cell kill at concentrations higher than those commonly achieved during in vitro chemosensitivity assays. The LD50 for 1 h exposure to melphalan was twice that for continuous exposure: this also suggests no interference by MOH and M(OH)2. These data suggest that continuous exposure of melphalan in in vitro chemosensitivity assays is probably preferable to the arbitrary 1 h drug exposure time commonly employed.

Comparison of the fed and fasting states on the absorption of melphalan in multiple myeloma.

Melphalan absorption was studied over three consecutive days in five patients with multiple myeloma. On 1 day melphalan (approximately 7 mg/m2 = 10-12 mg) was administered IV, on 1 day PO fasting, and on 1 day PO after a standard breakfast. The order was different for each patient to minimise trends that might affect absorption. Melphalan concentrations were determined by high-pressure liquid chromatography and fitted to biexponential equations by computer. The parameters of these equations were in broad agreement with previously published data, and melphalan absorption varied between patients. Considerable differences were observed in the melphalan concentration curves between the 'PO fed' and 'PO fasting' days: on the PO fed days the delay before absorption started was longer (1.1 +/- 0.5 h as against 0.3 +/- 0.1 h); peak plasma levels were one-third the value (65 +/- 15 ng/ml; 195 +/- 80 ng/ml) and occurred at twice the time after administration (2.8 +/- 0.8 h; 1.3 +/- 0.3 h); and areas under the curve were smaller 10.8 +/- 4.7 min X micrograms/ml; 23.8 +/- 13.8 min X micrograms/ml). There was a significant difference between the fraction of the dose of melphalan absorbed on the PO fed day (0.49 +/- 0.20) and on the PO fasting day (0.93 +/- 0.22), with P less than 0.005. This work suggests that melphalan should be taken first thing in the morning to obtain greatest absorption.

Correlation of bcl-2 with P-glycoprotein expression in chronic lymphocytic leukaemia and other haematological neoplasms but of neither marker with ex vivo chemosensitivity or patient survival.

We compared bcl-2 with P-glycoprotein expression (C494 and JSB1), and both with ex vivo chemosensitivity by Differential Staining Cytotoxicity (DiSC) assay (25 cytotoxic drugs), in 76 fresh haematological specimens, including 51 chronic lymphocytic leukaemias (CLL). Strong correlations were seen between bcl-2 and Pgp expression in both CLL (r = 0.5; p < 0.001) and AML (r = 0.9; p < 0.001) although bcl-2 expression was only raised in Pgp positive cells. However, there was no correlation between high or low marker levels and either ex vivo drug sensitivity (-0.30 < r < 0.37; p all > 0.1) or patient survival (chi 2 < or = 0.1; p > 0.7). One B-CLL, one PLL and one hairy cell leukaemia were negative for both bcl-2 and Pgp, whilst 3 T-cell specimens were bcl-2 negative but strongly positive for Pgp. These results suggest that the expression of Pgp and bcl-2 may be interlinked and related to immunophenotype and that clinical sensitivity to MDR-inducing and/or apoptosis-inducing drugs is best determined by ex vivo chemosensitivity testing rather than measurement of Pgp or bcl-2 expression.

Effect of interleukin-2 on CD95 (Fas/APO-1) expression in fresh chronic lymphocytic leukaemia and mantle cell lymphoma cells: relationship to ex vivo chemoresponse.

Cells from B-cell chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL) rarely express the CD95 (Fas/APO-1) antigen. Our aims were to determine whether CD95 levels (assessed by flow cytometry) in fresh neoplastic CD19+/CD5+ B-lymphocytes from CLL and MCL could be upregulated using clinically relevant doses of interleukin-2 (IL-2), and to compare this with their ex vivo cytotoxic drug sensitivity (assessed by DiSC assay). CD95-expression was absent/negligible in 13/14 CLL and 7/7 MCL specimens. Following culture (without IL-2) CD95 was expressed on dying CD5+ B-lymphocytes but only on live cells in 2/15 cases. In live cells from 2 CLL specimens, IL-2 caused up-regulation of CD95 and was associated with ex vivo drug resistance. Clinically relevant doses of IL-2 had pleiotropic effects on CD95-levels in fresh CLL cells, but did not induce CD95 in MCL cells. The link between CD95-induction and ex vivo drug resistance may point to a clinically resistant subset of CLL patients.

The in vitro radiosensitivity of lymphocytes from chronic lymphocytic leukaemia using the differential staining cytotoxicity (DiSC) assay. I--Investigation of the method.

A tumour sensitivity assay, the differential staining cytotoxicity (DiSC) assay, which has been shown to have potential in predicting tumour response to cytotoxic drugs, has been developed to investigate the radiosensitivity of peripheral blood lymphocytes isolated from patients with chronic lymphocytic leukaemia (CLL). The method involved irradiating isolated lymphocytes (0.64-20.48 Gy at 8 x 10(5) cells/ml) and incubating for 4 days. Subsequent radiation-induced cell kill was assessed by differential staining of dead and live cells and calculation of tumour cell viability. Factors affecting assay success rate and in vitro radioresponse were investigated. Results were shown to be reproducible both when repeat assays were performed on the same specimen on the same day, and on different specimens from the same patient after intervening periods of time. Greater than 1 day transit time (whole blood) or storage (separated cells in medium) was found to be detrimental to assay success, but pH of the medium (either pH 7.3 or 8) had little effect. An increased incubation period (from 4 to 7 days) slightly reduced cell survival, but the underlying radioresponsiveness of the cells did not change.

The in vitro radiosensitivity of lymphocytes from chronic lymphocytic leukaemia using the differential staining cytotoxicity (DiSC) assay. II--Results on 40 patients.

A tumour sensitivity assay, the differential staining cytotoxicity (DiSC) assay, which has been shown to have potential in predicting tumour response to cytotoxic drugs, has been used to investigate the radiosensitivity of peripheral blood lymphocytes isolated from patients with chronic lymphocytic leukaemia (CLL). The isolated lymphocytes were irradiated and incubated for 4 days. Radiation-induced cell kill was assessed by differential staining of dead and live cells with subsequent calculation of tumour cell viability. The results of 61 CLL specimens from 40 patients are reported, showing profound inter-patient differences in the sensitivity of cells to radiation. Five patient specimens, which were radioresistant in vitro, were from patients who were also resistant clinically to irradiation. Another patient who responded very well clinically was found to be extremely sensitive in the assay.

Stability of melphalan solutions during preparation and storage.

A stability-indicating high-performance liquid chromatographic assay has been used to investigate the stability of solutions of melphalan under conditions that pertain to setting up in vitro chemosensitivity assays. Melphalan (1 and 20 micrograms/mL) was found to be 30% more stable in 150 mM NaCl solution (normal saline) than in Dulbecco's phosphate-buffered saline. The drug stored at 20 micrograms/mL in normal saline at room temperature (21.5 degrees C) lost 5% of its activity (t0.95) in 1.5 h; at 5 degrees C the t0.95 was 20 h. From an Arrhenius plot of the results, the activation energy of the hydrolysis reaction in normal saline was 100.2 kJ/mol. Less than 5% of drug activity was lost when solutions of melphalan were (a) stored at -20 degrees C and -35 degrees C for 7 months, (b) frozen and thawed up to four times, or (c) filtered through various membranes. Drug degradation was not affected by storage-container material. The half-life of melphalan in a Roswell Park Memorial Institute medium containing 10% fetal bovine serum at 37 degrees C was 1.13 +/- 0.10 h; the presence of 0.3% agar had no effect. It is suggested that melphalan be handled at temperatures greater than 5 degrees C for a minimum of time, but solutions of the drug can be stored for at least 6 and possibly up to 12 months at -20 degrees C without significant deterioration taking place.