Leukemia Research Vol. 15, No. 7, pp. 565-569, 1991. Printed in Great Britain.
0145-2126/91 $3.00 + .DO Pergamon Press plc
IN VITRO CHEMOSENSITIVITY TESTING IN CHRONIC LYMPHOCYTIC LEUKAEMIA PATIENTS JANE A. HANSON, D. PAUL BENTLEY,* ELIZABETH A. BEAN, SARAH R. NUTE and JOHN L. MOORE
Radiation Science Laboratory, Velindre Hospital, Whitchurch, Cardiff CF4 7XL, U.K. and * Department of Haematology, Llandough Hospital, Penarth, Cardiff, U.K. (Received 31 July 1990. Revision accepted 8 December 1990)
AbstractnTwenty-nine samples from eighteen patients with chronic lymphocytic leukaemia (CLL) were used in a direct comparison of in vitro response to chlorambucil measured in a metabolic (MTI') and dye exclusion (D.Ex) assay. Reduced ability to produce formazan corresponded to a reduced number of dye-excluding viable cells and a significant correlation was found between dose-response measured in the two assays. Initial low absorbance values obtained with untreated control cells in the M'I'T assay were effectively overcome by increasing both the cell seeding density and M'IT exposure time with the consequent increase in assay sensitivity. The MTI" assay provided qualitatively similar dose-response data to that obtained in the D.Ex assay. A wide range in in vitro response was seen for both pretreatment and treatment patient groups. In vitro dose-responses were seen to coincide with decreasing or stable white cell counts, taken around the time of sampling, in samples from 4 patients. Less marked dose-responses were observed for 2 patients considered clinically resistant to chlorambucil. The MTT assay would seem, therefore, to be applicable to in vitro assay of cell response in CLL. Key words: CLL, dye exclusion, MTI" assay, in vitro cell-response.
INTRODUCTION
exclusion (D.Ex) and MTI" assays for chemosensitivity testing in leukaemia have reported that qualitatively similar dose-response data are obtained [4-6]. In vitro dose-response data, obtained with a D.Ex assay, have been shown to correlate with clinical response in 44 cases of CLL and nonHodgkins lymphoma [7]. Highly significant clinical correlations have also recently been reported for chemosensitivity testing of various leukaemias with the MTT assay [2, 6]. Chronic lymphocytic leukaemia is a B-cell disease in more than 95% of patients and, in the western hemisphere, it is the most common type of leukaemia in over 50-year-olds [8]. The clinical course is variable and patients are treated palliatively when diseaserelated symptoms arise [8]. This study was initiated to determine whether chemosensitivity of essentially non-proliferating CLL lymphocytes following in vitro exposure to chlorambucil, the most effective agent for this disease, could be measured in an MTT assay. For comparison, cell response was also measured in a D.Ex assay. A major problem in chemosensitivity testing in general is to achieve realistic drug exposures in vitro. This is particularly difficult with multi-agent chemotherapy. No less a problem is the subsequent correlation of in vivo and in vitro data. In the present
THE MTT assay with its technical simplicity, speed and reproducibility has become increasingly used to measure in vitro cell response. The basic parameter measured, namely cellular reduction of a tetrazolium salt, results in the assay of all metabolising cells. This effectively limits its application in studies of solid tumours to work with cultured cell lines derived from biopsy samples where growth of normal fibroblasts can be avoided. This lack of specificity for the tumour cell population is less of a limitation with leukaemias with readily available samples predominantly ( = > 7 0 % ) comprised of tumour cells [1, 2]. The more labour intensive dye exclusion assays measuring cell membrane integrity, with cytologic evaluation of cells, do however allow for the specific quantitation of response in the tumour cell population [3]. Comparative studies investigating both dye Abbreviations: CLL, chronic lymphocytic leukaemia; CHO-K1, Chinese hamster ovary cells; D.Ex, dye exclusion; DMSO, dimethyl sulfoxide; DRBC, duck red blood cells; IDSO, drug concentration reducing response to 50% of control value; MTT, 3-(4,5 dimethylthiazolyl)2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline. 565
566
J.A.
HANSON et al.
study, as chlorambucil was often given as a single agent, we also attempted to correlate in vitro data with clinical response.
2.0
MATERIALS AND METHODS
1.o
Separation of leucocytes Heparinised blood samples (coded) were usually received within 3 h and leucocytes were separated by velocity sedimentation through a Histopaque gradient (Sigma). Gradient (5 ml) was carefully layered under 14 ml of blood previously diluted (1/2) in RPMI 1640 culture medium (Gibco) containing 15% foetal calf serum (Gibco), penicillin (Glaxo) and streptomycin (Evans). The samples were centrifuged in a bench top Mistral centrifuge at 2500 rpm (500 g) for 30 min. The resultant leucocyte layer was collected and washed twice with culture medium. Cells were counted using a haemocytometer. For the D.Ex assay, cells were diluted in culture medium to 2 × 10S/ml and dispensed at 0.9 ml per tube (Falcon 2054). Cells were diluted to 2.8 × 106/ml for the MTI" assay and dispensed at 180 ~tl per multiwell (Falcon 3054). Linearity between the parameter measured (dye excluding cells or absorbance) and cell number was not assumed and, in both assays, control cell cultures were set up with at least 2 further cell dilutions. Patient samples were decoded and correlated with clinical data on completion of the study.
Drug storage and in vitro exposure Pure chlorambucil was stored as a dessicated crystalline powder at 4°C. Batches of drug were made by dissolving it in ethanol (Burrough's), diluting to 100 ~tg/ml with phosphate-buffered saline (PBS) and dispensing 0.5 ml aliquots in tubes (Falcon 2054) which were capped, wrapped in foil and frozen (-20°C) without delay. Cells in culture medium were dispensed in tubes and multiwells prior to addition of drug. An aliquot of drug was thawed immediately before use and dilutions made with PBS to × 10 the final concentration and 100 ~tl or 20 Ixl were dispensed into tubes (D.Ex assay) or multiwells (MTT assay) respectively. Drug was left in continuous contact with the cells which were incubated at 37°C in a humidified CO2/air incubator. Cell response was measured after 4 or 6 days of culture with extended assay times of up to 14 days undertaken in 11 samples. In D.Ex experiments, cells were fixed in 2.5% glutaraldehyde (BDH, GPR grade) in Millonig's buffer and differentially stained with alcian blue (Gurr C.I.74240) at a final concentration of 0.017% [9]. In the MTT assay, MTT (4 h incubation at 0.45 ~tg/mi final concentration) was added to multiwells with subsequent removal of culture medium, solubilisation of formazan in dimethyl sulfoxide (DMSO) and measurement of optical density (570 nm) [10]. To check for loss of activity upon storage, aliquots of drug were periodically tested against Chinese hamster ovary (CHO-K1) cells in the MTT assay. RESULTS
Patient samples We received 35 samples from 21 patients and 29 (18 patients) were assessed for in vitro response to chlorambucil in one or both assays. O f the 6 samples
1.5
0.5
_
0.0
0
,
~
•
•
,
,
,
20
40
60
,
80 % v i a b i l i t y D.Ex assay
,
100
FIG. 1. The relationship between control viability in the D.Ex assay and the control rate of formazan production in the M T r assay. Each data point is the mean % viability from at least 2 replicate control slides plotted against the mean control absorbance of 4 replicate multiwells for individual samples determined on day 4 or 6 of culture. A significant correlation was found (r = 0.665, p
0145-2126/91 $3.00 + .DO Pergamon Press plc
IN VITRO CHEMOSENSITIVITY TESTING IN CHRONIC LYMPHOCYTIC LEUKAEMIA PATIENTS JANE A. HANSON, D. PAUL BENTLEY,* ELIZABETH A. BEAN, SARAH R. NUTE and JOHN L. MOORE
Radiation Science Laboratory, Velindre Hospital, Whitchurch, Cardiff CF4 7XL, U.K. and * Department of Haematology, Llandough Hospital, Penarth, Cardiff, U.K. (Received 31 July 1990. Revision accepted 8 December 1990)
AbstractnTwenty-nine samples from eighteen patients with chronic lymphocytic leukaemia (CLL) were used in a direct comparison of in vitro response to chlorambucil measured in a metabolic (MTI') and dye exclusion (D.Ex) assay. Reduced ability to produce formazan corresponded to a reduced number of dye-excluding viable cells and a significant correlation was found between dose-response measured in the two assays. Initial low absorbance values obtained with untreated control cells in the M'I'T assay were effectively overcome by increasing both the cell seeding density and M'IT exposure time with the consequent increase in assay sensitivity. The MTI" assay provided qualitatively similar dose-response data to that obtained in the D.Ex assay. A wide range in in vitro response was seen for both pretreatment and treatment patient groups. In vitro dose-responses were seen to coincide with decreasing or stable white cell counts, taken around the time of sampling, in samples from 4 patients. Less marked dose-responses were observed for 2 patients considered clinically resistant to chlorambucil. The MTT assay would seem, therefore, to be applicable to in vitro assay of cell response in CLL. Key words: CLL, dye exclusion, MTI" assay, in vitro cell-response.
INTRODUCTION
exclusion (D.Ex) and MTI" assays for chemosensitivity testing in leukaemia have reported that qualitatively similar dose-response data are obtained [4-6]. In vitro dose-response data, obtained with a D.Ex assay, have been shown to correlate with clinical response in 44 cases of CLL and nonHodgkins lymphoma [7]. Highly significant clinical correlations have also recently been reported for chemosensitivity testing of various leukaemias with the MTT assay [2, 6]. Chronic lymphocytic leukaemia is a B-cell disease in more than 95% of patients and, in the western hemisphere, it is the most common type of leukaemia in over 50-year-olds [8]. The clinical course is variable and patients are treated palliatively when diseaserelated symptoms arise [8]. This study was initiated to determine whether chemosensitivity of essentially non-proliferating CLL lymphocytes following in vitro exposure to chlorambucil, the most effective agent for this disease, could be measured in an MTT assay. For comparison, cell response was also measured in a D.Ex assay. A major problem in chemosensitivity testing in general is to achieve realistic drug exposures in vitro. This is particularly difficult with multi-agent chemotherapy. No less a problem is the subsequent correlation of in vivo and in vitro data. In the present
THE MTT assay with its technical simplicity, speed and reproducibility has become increasingly used to measure in vitro cell response. The basic parameter measured, namely cellular reduction of a tetrazolium salt, results in the assay of all metabolising cells. This effectively limits its application in studies of solid tumours to work with cultured cell lines derived from biopsy samples where growth of normal fibroblasts can be avoided. This lack of specificity for the tumour cell population is less of a limitation with leukaemias with readily available samples predominantly ( = > 7 0 % ) comprised of tumour cells [1, 2]. The more labour intensive dye exclusion assays measuring cell membrane integrity, with cytologic evaluation of cells, do however allow for the specific quantitation of response in the tumour cell population [3]. Comparative studies investigating both dye Abbreviations: CLL, chronic lymphocytic leukaemia; CHO-K1, Chinese hamster ovary cells; D.Ex, dye exclusion; DMSO, dimethyl sulfoxide; DRBC, duck red blood cells; IDSO, drug concentration reducing response to 50% of control value; MTT, 3-(4,5 dimethylthiazolyl)2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline. 565
566
J.A.
HANSON et al.
study, as chlorambucil was often given as a single agent, we also attempted to correlate in vitro data with clinical response.
2.0
MATERIALS AND METHODS
1.o
Separation of leucocytes Heparinised blood samples (coded) were usually received within 3 h and leucocytes were separated by velocity sedimentation through a Histopaque gradient (Sigma). Gradient (5 ml) was carefully layered under 14 ml of blood previously diluted (1/2) in RPMI 1640 culture medium (Gibco) containing 15% foetal calf serum (Gibco), penicillin (Glaxo) and streptomycin (Evans). The samples were centrifuged in a bench top Mistral centrifuge at 2500 rpm (500 g) for 30 min. The resultant leucocyte layer was collected and washed twice with culture medium. Cells were counted using a haemocytometer. For the D.Ex assay, cells were diluted in culture medium to 2 × 10S/ml and dispensed at 0.9 ml per tube (Falcon 2054). Cells were diluted to 2.8 × 106/ml for the MTI" assay and dispensed at 180 ~tl per multiwell (Falcon 3054). Linearity between the parameter measured (dye excluding cells or absorbance) and cell number was not assumed and, in both assays, control cell cultures were set up with at least 2 further cell dilutions. Patient samples were decoded and correlated with clinical data on completion of the study.
Drug storage and in vitro exposure Pure chlorambucil was stored as a dessicated crystalline powder at 4°C. Batches of drug were made by dissolving it in ethanol (Burrough's), diluting to 100 ~tg/ml with phosphate-buffered saline (PBS) and dispensing 0.5 ml aliquots in tubes (Falcon 2054) which were capped, wrapped in foil and frozen (-20°C) without delay. Cells in culture medium were dispensed in tubes and multiwells prior to addition of drug. An aliquot of drug was thawed immediately before use and dilutions made with PBS to × 10 the final concentration and 100 ~tl or 20 Ixl were dispensed into tubes (D.Ex assay) or multiwells (MTT assay) respectively. Drug was left in continuous contact with the cells which were incubated at 37°C in a humidified CO2/air incubator. Cell response was measured after 4 or 6 days of culture with extended assay times of up to 14 days undertaken in 11 samples. In D.Ex experiments, cells were fixed in 2.5% glutaraldehyde (BDH, GPR grade) in Millonig's buffer and differentially stained with alcian blue (Gurr C.I.74240) at a final concentration of 0.017% [9]. In the MTT assay, MTT (4 h incubation at 0.45 ~tg/mi final concentration) was added to multiwells with subsequent removal of culture medium, solubilisation of formazan in dimethyl sulfoxide (DMSO) and measurement of optical density (570 nm) [10]. To check for loss of activity upon storage, aliquots of drug were periodically tested against Chinese hamster ovary (CHO-K1) cells in the MTT assay. RESULTS
Patient samples We received 35 samples from 21 patients and 29 (18 patients) were assessed for in vitro response to chlorambucil in one or both assays. O f the 6 samples
1.5
0.5
_
0.0
0
,
~
•
•
,
,
,
20
40
60
,
80 % v i a b i l i t y D.Ex assay
,
100
FIG. 1. The relationship between control viability in the D.Ex assay and the control rate of formazan production in the M T r assay. Each data point is the mean % viability from at least 2 replicate control slides plotted against the mean control absorbance of 4 replicate multiwells for individual samples determined on day 4 or 6 of culture. A significant correlation was found (r = 0.665, p
