Andrew G. Bosanquet

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.

Pharmacokinetics of oral and intravenous melphalan during routine treatment of multiple myeloma

Plasma melphalan levels have been measured in nine (mostly stage IIIA) multiple myeloma patients after therapeutic doses of drug had been given p.o. and i.v. A new isocratic high-pressure liquid chromatographic (HPLC) method with a sensitivity limit o 5 ng/ml was used to quantify the melphalan. Patients receiving 8-28.5 mg melphalan i.v. showed alpha and beta plasma decays with half-lives of 7.7 +/- 3.3 (mean +/- S.D.) and 83 +/- 14 min respectively. The apparent volume of the central compartment was 12.8 +/- 4.3 1, and the total volume of distribution was 0.62 +/- 0.21 l/kg. Very variable absorption was seen in the same patients after receiving 5-12 mg melphalan p.o. The half-life of the absorption phase varied from 2.1 to 62.1 min (22.8 +/- 18.1 min) with delays (before absorption started) of 0-113 min. The fraction of dose absorbed varied from 0.32 to 1.03 (0.72 +/- 0.23), and the half-life of the beta phase was 92 +/- 27 min. The type of breakfast eaten before p.o. melphalan was found to correlate with the fraction of drug absorbed.

Stability of solutions of antineoplastic agents during preparation and storage for in vitro assays

SummaryThe 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°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 >0.1 M chloride.

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.

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.

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

SummaryMelphalan 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 occured 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 μg/ml; 23.8±13.8 min x μg/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 P0.005. This work suggests that melphalan should be taken first thing in the morning to obtain greatest absorption.

Prognosis for fludarabine therapy of chronic lymphocytic leukaemia based on ex vivo drug response by DiSC assay

The cytotoxic antimetabolite fludarabine is a widely used active agent in chronic lymphocytic leukaemia (CLL). However, cost and occasional adverse side‐effects necessitate careful use. Identifying before treatment patients not likely to benefit from fludarabine could advance disease management both clinically and financially.

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.

Long-term comparison of results of a drug sensitivity assay in vitro with patient response in lymphatic neoplasms.

A short‐term differential staining cytotoxicity (DiSC) assay was used to assess the sensitivity of tumor cells in vitro from patients with chronic lymphocytic leukemia (CLL) and non‐Hodgkins lymphoma to various cytotoxic drugs. The results have been correlated with drug sensitivities of the tumors in vivo. The chemosensitivity in vitro of eight patients with CLL was observed for 12 to 42 months. In 44 cases the assay correctly predicted seven sensitive and 30 resistant tumors (84% positive correlations). There were six false predictions of sensitivity and one false prediction of resistance. Repeated testing of patients receiving treatment revealed significant and progressive development of drug resistance, while serial tests on untreated patients with CLL gave unaltered results. The development of cross‐resistance to structurally related drugs was observed after treatment and many samples showed a high level of cross‐resistance. However, teniposide showed greater activity than etoposide, and mitoxantrone showed greater activity than the anthracyclines. The high level of agreement between laboratory and clinical results suggests that the DiSC assay may have a useful place (1) in guiding the clinician in the selection of drugs for chemotherapy and (2) in giving an added indication of prognosis for an individual with a lymphatic neoplasm.

An assessment of a short-term tumour chemosensitivity assay in chronic lymphocytic leukaemia.

A 4-day tumour sensitivity assay of potential use in predicting tumour response to cytotoxic drugs has been investigated in patients with chronic lymphocytic leukaemia. The method comprised isolation of white cells from peripheral blood, drug exposure and incubation for 4 days. Drug-induced tumour cell kill was assessed by differential staining of dead and live cells such that the latter could be morphologically identified, with subsequent calculation of tumour cell viability. Concentrations of drug for use in the assay were chosen for chlorambucil (2 micrograms ml-1), 4-hydroperoxy-cyclophosphamide (2 micrograms ml-1)--which was used in vitro in place of cyclophosphamide--prednisolone (0.5 microgram ml-1) and vincristine (0.1 microgram ml-1), to give a scatter of values which was in good agreement with clinical expectations. In 21 cases where the in vitro result could be compared with the in vivo response, there were 4 true positive comparisons (sensitive in vitro, sensitive in vivo), 15 true negative comparisons (resistant both in vitro and in vivo) and 2 false positive comparisons (sensitive in vitro, resistant in vivo). A result was obtained in 86% (65/76) of samples received. The assay appears to show considerable promise as a tumour chemosensitivity test and warrants wider investigation, including prospective in vivo/in vitro correlations that could be based on the results presented here.