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In vitro chemosensitivity testing in acute non lymphocytic leukemia using the bioluminescence ATP assay
Leukemia Research 24 (2000) 445 – 452 www.elsevier.com/locate/leukres
In vitro chemosensitivity testing in acute non lymphocytic leukemia using the bioluminescence ATP assay Lars Mo¨llga˚rd a,*, Ulf Tidefelt b, Britt Sundman-Engberg a, Christina Lo¨fgren a, Christer Paul a b
a Department of Hematology, Huddinge Uni6ersity Hospital, S-141 86 Huddinge, Stockholm, Sweden Department of Hematology, O8 rebro Medical Centre Hospital, O8 rebro and Karolinska Institute, Stockholm, Sweden
Received 8 July 1999; accepted 18 December 1999
Abstract The ATP assay is a short term in vitro chemosensitivity assay where the amount of viable cells are determined by their content of ATP. The aim of the study was to compare the in vitro results of six cytostatic drugs to the clinical outcome in 83 acute non-lymphocytic leukemia (ANLL) patients. The secondary ANLL at diagnosis showed an in vitro resistance to daunorubicin that was significantly higher compared to de novo ANLL at diagnosis (P B0.003). De novo ANLL at diagnosis that achieved complete remission (CR) were significantly more sensitive to daunorubicin compared to those who didn’t achieve CR (PB0.05). There was an vitro correlation between topoisomerase II active drugs but not between these drugs and ara-C. In vitro ara-C sensitivity ( 5 the median of the de novo ANLL at diagnosis) was correlated to poor overall survival (P = 0.02). In vitro sensitivity to daunorubicin and mitoxantrone was associated with prolonged disease free survival (P= 0.03 and P = 0.04). We conclude that despite significant correlation to clinical parameters for daunorubicin and mitoxantrone the predictive value of the ATP assay in this material was insufficient for directing therapy. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Bioluminiscence ATP assay; Chemosensitivity; Cytotoxicity; Drug resistance; Myeloid leukemia
1. Introduction Many attempts have been made to develop methods for in vitro chemosensitivity testing in different tumors. The aim has been to find a reliable test with high predictive value that could be a useful instrument in the choice of treatment both at diagnosis and in resistant disease. The clonogenic assays and assays based on incorporation of DNA precursors have been used to predict response to chemotherapy but technical difficulties and
Abbre6iations: ANLL, acute non lymphocytic leukemia; ara-C, cytarabine; ATP, adenosine 5%-triphophate; CR, complete remission; DiSC, differential staining cytotoxicity; FAB, French American British; LC50, the drug concentration lethal to 50% of the leukemic cells; MTT, 3-[4,5-dimethylthiazol-2,5-diphenyl] tetrazolium bromide; Pgp, P-glycoprotein; TCA, trichloracetic acid. * Corresponding author. Tel.: + 46-8-58580000; fax: +46-858582525. E-mail address: [email protected] (L. Mo¨llga˚rd)
long culturing time have limited the application of the methods [1–3]. The differential staining cytotoxicity assay (DiSC) measures total cell kill by microscopic evaluation of dye exclusion by viable cells and is capable of discriminating between effects on tumor and normal cells [4,5]. Studies have shown correlation, both to the initial response to chemotherapy and to the long term outcome [6–9]. The DiSC is a short time assay (96 h) but labor-intensive and relies on the subjective assessment by a skilled observer. In the MTT-assay surviving cells convert MTT into formazan which can be quantified by spectrophotometry. The method have shown good correlation with the DiSC and correlation to clinical outcome in both leukemias and solid tumors [10–15]. Alternatively, as in the FMCA assay, fluorescein diacetate can be used as a marker of cell viability [16–18]. Another possibility is the bioluminescence ATP assay based on metabolic activity measured as cellular ATP content [19,20]. The amount of ATP in a specific cell
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type is relatively constant [21,22]. ATP is rapidly degraded by ATP-ases leading to prompt depletion if the respiratory cycle is disturbed in aerobic cells. Since the ATP levels are constant in a given healthy cell it can be used as an indirect method for measuring cell growth or death. The bioluminescence ATP assay has shown good correlation with the DiSC and clonogenic assays and has been used in different tumors [2,23 – 26]. The objective of this study was to evaluate the feasibility and the predictive value of the bioluminescence ATP chemosensitivity assay in acute non-lymphocytic leukemia (ANLL).
2. Materials and methods
2.1. Patients A total of 94 samples with ANLL cells were separated from bone marrow or peripheral blood from 86 patients. Eighty-three of these samples, from 77 patients, were technically successful and diagnosis and stage at inclusion are shown in Table 1. Seventy-four of the 77 patients were classified according to the FAB criteria’s [27]: 14 M1; 24 M2; 3 M3; 17 M4; 13 M5; 2 M6 and 1 M7.
2.2. Sample collection Peripheral blood and/or bone marrow was collected in heparinized tubes before start of treatment. The leukemic cells were separated by centrifugation (400×g for 20 min) on metrizoate-dextran (Lymphoprep, Nyegaard and Co., AS, Oslo, Norway) as previously described [28,29]. The blast cells (80–90% pure) were then washed twice in PBS (phosphate buffered 0.9% saline, pH 7.4).
2.3. Incubations and culturing Only cells from fresh samples were used. The cells (1.0× 105 cells/ml) were incubated in a medium consisting of 1.8 ml RPMI 1640 supplemented with 1% L-glutamine and 10% fetal calf serum and 0.2 ml of the cytostatic drug at final drug concentrations as follows: daunorubicin 0.2 mM for 1 h, Ara–C 0.5 mM continuously, mitoxantron 0.05 mM for 1 h, idarubicin 0.05 mM for 1 h, amsacrine 1.0 mM continuously and etoposide 20 mM for 1 h. All incubations were performed in duplicate and with a drug-free control. After the short time incubations the cells were spun down (400× g for 10 min) and the supernatant removed. A volume of 2 ml of fresh medium as described above was added. All the samples were then cultured for 4 days in a humidified incubator (37°C, 5% CO2).
2.4. Extraction of ATP Table 1 Diagnosis and stage at inclusion for all ANLL samples and clinical outcome in de novo ANLL Diagnosis and stage at inclusion De no6o ANLL Diagnosis CR after one to two courses No CR after two courses No CR after only one course Early deaths Low dose treatment Response to therapy not assessed Relaps Resistant disease
Myelodysplastic syndrome CML in blastcrisis Other myeloproliferative disease Other cytostatic treatment
Total
46 19 10 4 8 4 1 11 9
Secondary ANLL Diagnosis
Resistant disease Methodological failure
No. of samples
14 8 2 2 2 3 11 94
Extraction of ATP in leukemic cells was performed by mixing equal volumes (100 ml) of cell-suspension and 2.5% TCA (trichloracetic acid). The extracts were assayed immediately or stored in a freezer (− 20°C) until analysis.
2.5. ATP assay The bioluminescence assay was performed automatically in a Bio Orbit photometer (Turku, Finland) as previously described [23]. The ATP monitoring reagent and the ATP standard used were both supplied by Bio Orbita (Turku, Finland). The ATP standard was reconstituted in 10 ml distilled water giving a 10 mM solution. The ATP monitoring reagent was reconstituted with 5 ml Tris-EDTA buffer at pH 7.75 (100 mM Tris and 2 mM EDTA, pH adjusted with acetic acid). A volume of 20 ml of the sample was added to 900 ml Tris-EDTA buffer. The cuvette was placed in the photometer. Automatically 100 ml ATP monitoring reagent was dispensed in a cuvette placed in the photometer and the resulting light emission was measured. The ATP standard was then automatically added (10 ml) and the emitted light remeasured. The amount of ATP was calculated with correction for the blanks. With this
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Fig. 1. In vitro results in de novo and secondary ANLL. The symbol indicates the median. The box encompasses 50% and the box-whiskers 90% of the observations. ** P B0.01 compared to de novo ANLL at diagnosis.
procedure the light emitted is proportional to the amount of ATP in the sample. The results were given as nmol ATP/sample. The percentage ATP in a sample when compared to the drug-free control was then calculated.
2.6. Drug therapy Thirty-seven de novo ANLL and 11 secondary ANLL all at diagnosis received one or two intensive induction courses according to different protocols. Most of the patients received ara-C (n =47) and one anthraquinone, e.g. mitoxantrone (n = 23) and daunorubicin (n =12). Twenty-six patients also received etoposide and 11 thioguanine. Four patients received the resistance modifying cyclosporine analogue PSC 833. One patient with ANLL M3 also received all-trans retinoic acid (ATRA). Two patients underwent an autologous bone marrow transplantation and three patients underwent an allogeneic bone marrow transplantation
2.8. In 6itro–in 6i6o comparison and sur6i6al analysis In the patients with ANLL at diagnosis who achieved induction therapy the result of the single in vitro most active drug that the patient received was compared to the clinical outcome. In the survival analysis the median for each drug from the 46 de novo ANLL at diagnosis was used as a cut off level to separate in vitro sensitivity from in vitro resistance (Fig. 1.). In the overall survival analysis the ANLL patients at diagnosis were included on an intention to treat basis. Disease free survival was defined as the time from complete remission to the date of relapse, death or last follow up.
2.9. Statistical analysis The differences in cytotoxic effect in vitro between different groups of patients were evaluated with t-test for independent samples. The Kaplan–Meier method and the log-rank test were used to estimate differences in survival.
2.7. Clinical e6aluation Complete remission (CR) was defined as = 5% blast cells for M1 and M5a and for the other FAB-groups also = 10% leukemic cells (blastcells, promyelocytes and promonocytes), absence of Auer rods and absence of leukemic cell clusters in a bone marrow aspirate. In vivo sensitivity was defined as CR after one to two induction courses and in vivo resistance as no CR after two induction courses.
3. Results
3.1. Methodological considerations Assays from 11/94 patients could not be evaluated, 10 due to failure in the control (six samples had too low levels of ATP in the drug free control (B20 nM) and in four samples only one of the duplicates in the control
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was successful) and one due to extremely high levels of ATP compared to the drug free control. In 33 cases one single drug sample had to be excluded mainly because of failure in one of the duplicates. Totally 443 out of 542 individual drug samples (82%) were successful.
3.2. Clinical outcome The clinical outcome for the 33 evaluable de novo ANLL patients at diagnosis who achieved induction treatment is shown in Table 1. The eight early deaths were due to infections or haemorrhages. Four of these patients died before treatment had started and four died after the first course but before the response could be evaluated.
3.3. In 6itro –in 6i6o comparison in different groups of patients In Fig. 1 the in vitro results from the de novo ANLL at diagnosis, the de novo ANLL at relapse/resistant disease and secondary ANLL at diagnosis are shown. There was a wide distribution of the results for each drug and an obvious overlapping when comparing the separate drugs in the different groups. Although the mean values were higher in the de novo ANLL at relapse/resistant disease group compared to the de novo ANLL at diagnosis group for daunorubicin (48 and 43%), mitoxantrone (50 and 46%) and idarubicin (46 and 43%) these differences were not significant. The in
vitro effect of daunorubicin was significantly higher at diagnosis in the de novo ANLL group compared to the secondary ANLL (43 and 66%, P= 0.003). The mean effect of mitoxantrone, idarubicin, amsacrine and etoposide was also higher at diagnosis in the de novo ANLL group compared to the secondary ANLL group but the differences were not significant. The effect of daunorubicin was significantly lower in the group of patients who did not achieve CR after two courses compared to those who achieved CR after one to two courses (56 and 38%, P 5 0.05), (Fig. 2). The mean values for ara-C (60 and 48%), mitoxantrone (55 and 40%) idarubicin (44 and 38%) and amsacrine (40 and 35%) showed the same tendency but the differences were not significant. In the patients with ANLL at diagnosis who achieved induction therapy and who were evaluable for clinical response, the single in vitro most active drug of the drugs that the patient actually received could not predict the short term clinical outcome.
3.4. In 6itro correlation between different drugs In vitro cross resistance can be indicated by the correlation between different drugs. Results from all ANLL samples which were included in this study are shown in Table 2. There was a clear correlation between the in vitro effect of daunorubicin, idarubicin, mitoxantrone, etoposide and amsacrine but not between these drugs and ara-C.
Fig. 2. In vitro results and clinical outcome in previously untreated de novo ANLL. The symbol indicates the median. The box encompasses 50% and the box-whiskers 90% of the observations. * P5 0.05 compared to the CR group.
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Table 2 Relationship between the in vitro effect of different drugs in all ANLL patients expressed as correlation coefficients
Daunorubicin Idarubicin Mitoxantrone Ara-C Amsacrine
Idarubicin
Mitoxantron
Ara-C
Amsacrine
Etopside
0.74 – – – –
0.74 0.84 – – –
0.20 0.31 0.32 – –
0.77 0.80 0.81 0.19 –
0.65 0.77 0.74 0.22 0.64
3.5. In 6itro results and sur6i6al analysis Patients who were in vitro sensitive to daunorubicin showed a tendency towards better over all survival (P = 0.14). In vitro sensitivity to the other drugs was not associated with better overall survival. Patients who were in vitro sensitive to ara-C had a poor over all survival (P= 0.02). Patients who were in vitro sensitive to daunorubicin (Fig. 3a) or mitoxantrone showed a prolonged disease free survival that was significant (P= 0.03 and P=0.04). Idarubicin showed the same tendency (P=0.06). In vitro sensitivity to amsacrine or etoposide could not predict the length of disease free survival. Patients that were in vitro sensitive to ara-C did not differ from in vitro resistant patients in disease free survival (P = 0.99, Fig. 3b).
incubation corresponds better to in vivo continuos infusion of the drug than higher doses administered as short infusion which most of the patients in our study received. The ara-C results are in contrast to a previous study with the DiSC assay [8] but in accordance with another study using the MTT assay where non-responders were in vitro more resistant to daunorubicin but not to ara-C compared to responders [15].
4. Discussion In this study with samples from patients with de novo ANLL we showed that in vitro sensitivity to daunorubicin, using the short term bioluminescence ATP-assay, was associated with response to induction therapy in previously untreated patients. In vitro sensitivity to daunorubicin and mitoxantrone was associated with prolonged disease free survival but not overall survival. As expected, in vitro resistance to daunorubicin was more common in the group of secondary ANLL at diagnosis. In the analysis of different drugs’ correlation in vitro, the results corresponded well to what is known about these drugs mechanism of action and cross resistance. The significantly higher in vitro effect of daunorubicin in patients who entered a CR compared to patients who did not, is in accordance with other studies [8,15]. Ara-C is another important drug in the treatment of ANLL, but here we found no significant in vitro differences between responders and non responders. One reason could be that ara-C has another mechanism of action affecting the cell in the S-phase [30]. In the short term incubations, contrary to clonogenic assays, the cells are just kept alive and further growth and cell-divisions are not required. Another reason could be that the in vitro continuous ara-C
Fig. 3. (a) Disease free survival in de novo and secondary ANLL. The median for daunorubicin is used to distinguish in vitro sensitivity from in vitro resistance. (b) Disease free survival in de novo and secondary ANLL. The median for ara-C is used to distinguish in vitro sensitivity from in vitro resistance.
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In the survival analysis sensitivity to both daunorubicin and mitoxantrone was associated with prolonged disease free survival. There is no obvious explanation of the shorter overall survival in patients who were in vitro sensitive to ara-C. The specific problem with ara-C incubations has already been discussed and in addition there are reports showing that a high number of S-phase cells in ANLL is associated with a poor survival [31]. Thus one could theoretically assume that a high proportion of cells in S-phase makes the cells more sensitive to ara-C in the in vitro incubation. In the in vivo situation a high proportion of cells in S-phase instead indicates a highly proliferative disease with a high potential of regrowth and consequently a poor survival. This can illustrate the difficulties in translating in vitro results to the in vivo situation but due to the limited material for the survival analysis in our study and a high proportion of early deaths in the ara-C sensitive group no further conclusions can be drawn. In a previous study, using the DiSC assay, we showed that in vitro sensitivity to anthracyclines and/or ara-C predicted overall survival (P 5 0.01) [8]. Another study that used the MTT assay showed that in vitro sensitivity to both daunorubicin and ara-C predicted disease free survival (P = 0.02) and that in vitro sensitivity to ara-C could predict continuous complete remission (P =0.02) [15]. Interestingly, six out of seven patients who were in vitro sensitive to daunorubicin and resistant to ara-C entered a complete remission. The study of in vitro correlation between different drugs illustrates cross resistance and how different mechanisms are involved in the drug resistance. The Pgp mediated multidrug resistance (MDR) is associated with resistance mainly to anthracyclines, vinca alkaloids and epipodophyllotoxins [32]. Topoisomerase II mediated drug resistance affects important drugs used for treatment of ANLL, e.g. anthracyclines, anthracendions, acridines and epipodophyllotoxins. In our study these mechanisms were illustrated by the strong correlation between these drugs. The nucleoside analogue araC is not affected by these two mechanisms and there was no cross resistance between ara-C and the other drugs in our study. In the survival analysis the definition of ‘in vitro sensitive’ had to be stated. One way is just to arbitrarily define a cut-off level (often 30%) between in vitro sensitive and resistant [33]. Others used each drug in several concentrations and defined sensitive/resistant using the median LC50 (the drug concentration lethal to 50% of the leukemic cells) as cut-off point [15]. Another way is to find the cut-off level that best separates in vivo sensitive and resistant patients [8]. In one study Kristenssen et al. divided the results for each drug, from all patients, into quartiles considering the lower quartile as in vitro sensitive. This made it possible to find an individual cut-off level for each drug [17]. We
chose this last method in our survival analysis but instead of the lower quartile we used the median which separated the material in groups where the number of patients were equal. For most of the cytostatic drugs the level for in vitro sensitivity was about 40% compared to the arbitrarily 30% limit (see Fig. 1). Compared to Tidefelt et al our ara-C level was higher, 49% compared to 35%, and the daunorubicin level lower, 39 compared to 60% [8]. In our study, patients with de novo ANLL at diagnosis who achieved induction therapy, the single in vitro most active drug of the drugs that the patient actually received could not predict the short term clinical outcome. The majority of the patients in our study received ara-C in their induction treatment but the other drugs varied. Some of the patients were included in protocols where the induction course had a more or less novel design and in some cases the bioluminescence ATP-assay did not include all drugs the patient received. On the other hand all patients got an anthracycline or anthracendion derivate in addition to ara-C and the correlation study suggested that in vitro sensitivity to daunorubicin corresponded to the in vitro sensitivity to other anthracyclines and anthracendions. A possible explanation to the discrepancy between our in vitro and in vivo data could be, as mentioned above, that short term assays may not be appropriate in the in vitro testing of ara-C. We have not estimated the blast percentage at the end of the test and there is a possibility that non malignant cells present at this stage may have decreased the predictive power of the test. The short term assays which are used today are in many aspects comparable, e.g. the culture procedure. The main difference is the various techniques that are used to estimate viable cells after the incubation. The bioluminescence ATP assay has been in use for a long time within the fields of biochemistry and microbiology, and a previous study has shown that it correlates satisfactorily to the DiSC assay (r= 0.8) [21–23]. The use of drug concentrations mimicking in vivo conditions, in combination with the DiSC assay, have shown good correlations to clinical outcome [8]. We have only used fresh samples, in contrast to other studies, where cryopreserved samples also have been analyzed [15,17]. One crucial point is the amount of leukemic cells after the density gradient centrifugation. In this aspect the DiSC assay has an advantage in the possibility to morphologically distinguish leukemic cells from other cells. Even if that procedure is very time consuming it may contribute to the good correlation between in vitro and in vivo data [8]. The success rate in our study was 82%. Four samples were excluded because of failure in one of the duplicates in the drug free control. If that could have been avoided by using three instead of two drug free controls the success rate would have been 86%.
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Even if our study, as well as other previous studies, has shown correlation’s to the clinical outcome in ANLL, more convincing results are needed before these short term assays will be accepted for tailoring chemotherapy treatment in clinical practice [8,14,15,17]. Another possible application of the in vitro chemosensitivity data is in the risk group stratification of ANLL patients, but also in this aspect further prospective studies are necessary to confirm that, e.g. in vitro resistance to daunorubicin is an independent risk factor in ANLL. In all different total cell kill assays there is always the risk of contamination of non malignant cells. Even if the proportion of blasts so far generally exceeded 80–90%, the remaining normal cells may affect the result. In acute lymphocytic leukemia there have been attempts to overcome this problem by the use of flow cytometry were the blast population can be separated from non malignant cells [34]. We have established a similar method for ANLL that currently is evaluated.
Acknowledgements This work was supported by grants from the Swedish Cancer Society. The authors thank Sofia Bengtsson, Ulrika Broberg and Malin Prenkert for their technical assistance.
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In vitro chemosensitivity testing in acute non lymphocytic leukemia using the bioluminescence ATP assay Lars Mo¨llga˚rd a,*, Ulf Tidefelt b, Britt Sundman-Engberg a, Christina Lo¨fgren a, Christer Paul a b
a Department of Hematology, Huddinge Uni6ersity Hospital, S-141 86 Huddinge, Stockholm, Sweden Department of Hematology, O8 rebro Medical Centre Hospital, O8 rebro and Karolinska Institute, Stockholm, Sweden
Received 8 July 1999; accepted 18 December 1999
Abstract The ATP assay is a short term in vitro chemosensitivity assay where the amount of viable cells are determined by their content of ATP. The aim of the study was to compare the in vitro results of six cytostatic drugs to the clinical outcome in 83 acute non-lymphocytic leukemia (ANLL) patients. The secondary ANLL at diagnosis showed an in vitro resistance to daunorubicin that was significantly higher compared to de novo ANLL at diagnosis (P B0.003). De novo ANLL at diagnosis that achieved complete remission (CR) were significantly more sensitive to daunorubicin compared to those who didn’t achieve CR (PB0.05). There was an vitro correlation between topoisomerase II active drugs but not between these drugs and ara-C. In vitro ara-C sensitivity ( 5 the median of the de novo ANLL at diagnosis) was correlated to poor overall survival (P = 0.02). In vitro sensitivity to daunorubicin and mitoxantrone was associated with prolonged disease free survival (P= 0.03 and P = 0.04). We conclude that despite significant correlation to clinical parameters for daunorubicin and mitoxantrone the predictive value of the ATP assay in this material was insufficient for directing therapy. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Bioluminiscence ATP assay; Chemosensitivity; Cytotoxicity; Drug resistance; Myeloid leukemia
1. Introduction Many attempts have been made to develop methods for in vitro chemosensitivity testing in different tumors. The aim has been to find a reliable test with high predictive value that could be a useful instrument in the choice of treatment both at diagnosis and in resistant disease. The clonogenic assays and assays based on incorporation of DNA precursors have been used to predict response to chemotherapy but technical difficulties and
Abbre6iations: ANLL, acute non lymphocytic leukemia; ara-C, cytarabine; ATP, adenosine 5%-triphophate; CR, complete remission; DiSC, differential staining cytotoxicity; FAB, French American British; LC50, the drug concentration lethal to 50% of the leukemic cells; MTT, 3-[4,5-dimethylthiazol-2,5-diphenyl] tetrazolium bromide; Pgp, P-glycoprotein; TCA, trichloracetic acid. * Corresponding author. Tel.: + 46-8-58580000; fax: +46-858582525. E-mail address: [email protected] (L. Mo¨llga˚rd)
long culturing time have limited the application of the methods [1–3]. The differential staining cytotoxicity assay (DiSC) measures total cell kill by microscopic evaluation of dye exclusion by viable cells and is capable of discriminating between effects on tumor and normal cells [4,5]. Studies have shown correlation, both to the initial response to chemotherapy and to the long term outcome [6–9]. The DiSC is a short time assay (96 h) but labor-intensive and relies on the subjective assessment by a skilled observer. In the MTT-assay surviving cells convert MTT into formazan which can be quantified by spectrophotometry. The method have shown good correlation with the DiSC and correlation to clinical outcome in both leukemias and solid tumors [10–15]. Alternatively, as in the FMCA assay, fluorescein diacetate can be used as a marker of cell viability [16–18]. Another possibility is the bioluminescence ATP assay based on metabolic activity measured as cellular ATP content [19,20]. The amount of ATP in a specific cell
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type is relatively constant [21,22]. ATP is rapidly degraded by ATP-ases leading to prompt depletion if the respiratory cycle is disturbed in aerobic cells. Since the ATP levels are constant in a given healthy cell it can be used as an indirect method for measuring cell growth or death. The bioluminescence ATP assay has shown good correlation with the DiSC and clonogenic assays and has been used in different tumors [2,23 – 26]. The objective of this study was to evaluate the feasibility and the predictive value of the bioluminescence ATP chemosensitivity assay in acute non-lymphocytic leukemia (ANLL).
2. Materials and methods
2.1. Patients A total of 94 samples with ANLL cells were separated from bone marrow or peripheral blood from 86 patients. Eighty-three of these samples, from 77 patients, were technically successful and diagnosis and stage at inclusion are shown in Table 1. Seventy-four of the 77 patients were classified according to the FAB criteria’s [27]: 14 M1; 24 M2; 3 M3; 17 M4; 13 M5; 2 M6 and 1 M7.
2.2. Sample collection Peripheral blood and/or bone marrow was collected in heparinized tubes before start of treatment. The leukemic cells were separated by centrifugation (400×g for 20 min) on metrizoate-dextran (Lymphoprep, Nyegaard and Co., AS, Oslo, Norway) as previously described [28,29]. The blast cells (80–90% pure) were then washed twice in PBS (phosphate buffered 0.9% saline, pH 7.4).
2.3. Incubations and culturing Only cells from fresh samples were used. The cells (1.0× 105 cells/ml) were incubated in a medium consisting of 1.8 ml RPMI 1640 supplemented with 1% L-glutamine and 10% fetal calf serum and 0.2 ml of the cytostatic drug at final drug concentrations as follows: daunorubicin 0.2 mM for 1 h, Ara–C 0.5 mM continuously, mitoxantron 0.05 mM for 1 h, idarubicin 0.05 mM for 1 h, amsacrine 1.0 mM continuously and etoposide 20 mM for 1 h. All incubations were performed in duplicate and with a drug-free control. After the short time incubations the cells were spun down (400× g for 10 min) and the supernatant removed. A volume of 2 ml of fresh medium as described above was added. All the samples were then cultured for 4 days in a humidified incubator (37°C, 5% CO2).
2.4. Extraction of ATP Table 1 Diagnosis and stage at inclusion for all ANLL samples and clinical outcome in de novo ANLL Diagnosis and stage at inclusion De no6o ANLL Diagnosis CR after one to two courses No CR after two courses No CR after only one course Early deaths Low dose treatment Response to therapy not assessed Relaps Resistant disease
Myelodysplastic syndrome CML in blastcrisis Other myeloproliferative disease Other cytostatic treatment
Total
46 19 10 4 8 4 1 11 9
Secondary ANLL Diagnosis
Resistant disease Methodological failure
No. of samples
14 8 2 2 2 3 11 94
Extraction of ATP in leukemic cells was performed by mixing equal volumes (100 ml) of cell-suspension and 2.5% TCA (trichloracetic acid). The extracts were assayed immediately or stored in a freezer (− 20°C) until analysis.
2.5. ATP assay The bioluminescence assay was performed automatically in a Bio Orbit photometer (Turku, Finland) as previously described [23]. The ATP monitoring reagent and the ATP standard used were both supplied by Bio Orbita (Turku, Finland). The ATP standard was reconstituted in 10 ml distilled water giving a 10 mM solution. The ATP monitoring reagent was reconstituted with 5 ml Tris-EDTA buffer at pH 7.75 (100 mM Tris and 2 mM EDTA, pH adjusted with acetic acid). A volume of 20 ml of the sample was added to 900 ml Tris-EDTA buffer. The cuvette was placed in the photometer. Automatically 100 ml ATP monitoring reagent was dispensed in a cuvette placed in the photometer and the resulting light emission was measured. The ATP standard was then automatically added (10 ml) and the emitted light remeasured. The amount of ATP was calculated with correction for the blanks. With this
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Fig. 1. In vitro results in de novo and secondary ANLL. The symbol indicates the median. The box encompasses 50% and the box-whiskers 90% of the observations. ** P B0.01 compared to de novo ANLL at diagnosis.
procedure the light emitted is proportional to the amount of ATP in the sample. The results were given as nmol ATP/sample. The percentage ATP in a sample when compared to the drug-free control was then calculated.
2.6. Drug therapy Thirty-seven de novo ANLL and 11 secondary ANLL all at diagnosis received one or two intensive induction courses according to different protocols. Most of the patients received ara-C (n =47) and one anthraquinone, e.g. mitoxantrone (n = 23) and daunorubicin (n =12). Twenty-six patients also received etoposide and 11 thioguanine. Four patients received the resistance modifying cyclosporine analogue PSC 833. One patient with ANLL M3 also received all-trans retinoic acid (ATRA). Two patients underwent an autologous bone marrow transplantation and three patients underwent an allogeneic bone marrow transplantation
2.8. In 6itro–in 6i6o comparison and sur6i6al analysis In the patients with ANLL at diagnosis who achieved induction therapy the result of the single in vitro most active drug that the patient received was compared to the clinical outcome. In the survival analysis the median for each drug from the 46 de novo ANLL at diagnosis was used as a cut off level to separate in vitro sensitivity from in vitro resistance (Fig. 1.). In the overall survival analysis the ANLL patients at diagnosis were included on an intention to treat basis. Disease free survival was defined as the time from complete remission to the date of relapse, death or last follow up.
2.9. Statistical analysis The differences in cytotoxic effect in vitro between different groups of patients were evaluated with t-test for independent samples. The Kaplan–Meier method and the log-rank test were used to estimate differences in survival.
2.7. Clinical e6aluation Complete remission (CR) was defined as = 5% blast cells for M1 and M5a and for the other FAB-groups also = 10% leukemic cells (blastcells, promyelocytes and promonocytes), absence of Auer rods and absence of leukemic cell clusters in a bone marrow aspirate. In vivo sensitivity was defined as CR after one to two induction courses and in vivo resistance as no CR after two induction courses.
3. Results
3.1. Methodological considerations Assays from 11/94 patients could not be evaluated, 10 due to failure in the control (six samples had too low levels of ATP in the drug free control (B20 nM) and in four samples only one of the duplicates in the control
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was successful) and one due to extremely high levels of ATP compared to the drug free control. In 33 cases one single drug sample had to be excluded mainly because of failure in one of the duplicates. Totally 443 out of 542 individual drug samples (82%) were successful.
3.2. Clinical outcome The clinical outcome for the 33 evaluable de novo ANLL patients at diagnosis who achieved induction treatment is shown in Table 1. The eight early deaths were due to infections or haemorrhages. Four of these patients died before treatment had started and four died after the first course but before the response could be evaluated.
3.3. In 6itro –in 6i6o comparison in different groups of patients In Fig. 1 the in vitro results from the de novo ANLL at diagnosis, the de novo ANLL at relapse/resistant disease and secondary ANLL at diagnosis are shown. There was a wide distribution of the results for each drug and an obvious overlapping when comparing the separate drugs in the different groups. Although the mean values were higher in the de novo ANLL at relapse/resistant disease group compared to the de novo ANLL at diagnosis group for daunorubicin (48 and 43%), mitoxantrone (50 and 46%) and idarubicin (46 and 43%) these differences were not significant. The in
vitro effect of daunorubicin was significantly higher at diagnosis in the de novo ANLL group compared to the secondary ANLL (43 and 66%, P= 0.003). The mean effect of mitoxantrone, idarubicin, amsacrine and etoposide was also higher at diagnosis in the de novo ANLL group compared to the secondary ANLL group but the differences were not significant. The effect of daunorubicin was significantly lower in the group of patients who did not achieve CR after two courses compared to those who achieved CR after one to two courses (56 and 38%, P 5 0.05), (Fig. 2). The mean values for ara-C (60 and 48%), mitoxantrone (55 and 40%) idarubicin (44 and 38%) and amsacrine (40 and 35%) showed the same tendency but the differences were not significant. In the patients with ANLL at diagnosis who achieved induction therapy and who were evaluable for clinical response, the single in vitro most active drug of the drugs that the patient actually received could not predict the short term clinical outcome.
3.4. In 6itro correlation between different drugs In vitro cross resistance can be indicated by the correlation between different drugs. Results from all ANLL samples which were included in this study are shown in Table 2. There was a clear correlation between the in vitro effect of daunorubicin, idarubicin, mitoxantrone, etoposide and amsacrine but not between these drugs and ara-C.
Fig. 2. In vitro results and clinical outcome in previously untreated de novo ANLL. The symbol indicates the median. The box encompasses 50% and the box-whiskers 90% of the observations. * P5 0.05 compared to the CR group.
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Table 2 Relationship between the in vitro effect of different drugs in all ANLL patients expressed as correlation coefficients
Daunorubicin Idarubicin Mitoxantrone Ara-C Amsacrine
Idarubicin
Mitoxantron
Ara-C
Amsacrine
Etopside
0.74 – – – –
0.74 0.84 – – –
0.20 0.31 0.32 – –
0.77 0.80 0.81 0.19 –
0.65 0.77 0.74 0.22 0.64
3.5. In 6itro results and sur6i6al analysis Patients who were in vitro sensitive to daunorubicin showed a tendency towards better over all survival (P = 0.14). In vitro sensitivity to the other drugs was not associated with better overall survival. Patients who were in vitro sensitive to ara-C had a poor over all survival (P= 0.02). Patients who were in vitro sensitive to daunorubicin (Fig. 3a) or mitoxantrone showed a prolonged disease free survival that was significant (P= 0.03 and P=0.04). Idarubicin showed the same tendency (P=0.06). In vitro sensitivity to amsacrine or etoposide could not predict the length of disease free survival. Patients that were in vitro sensitive to ara-C did not differ from in vitro resistant patients in disease free survival (P = 0.99, Fig. 3b).
incubation corresponds better to in vivo continuos infusion of the drug than higher doses administered as short infusion which most of the patients in our study received. The ara-C results are in contrast to a previous study with the DiSC assay [8] but in accordance with another study using the MTT assay where non-responders were in vitro more resistant to daunorubicin but not to ara-C compared to responders [15].
4. Discussion In this study with samples from patients with de novo ANLL we showed that in vitro sensitivity to daunorubicin, using the short term bioluminescence ATP-assay, was associated with response to induction therapy in previously untreated patients. In vitro sensitivity to daunorubicin and mitoxantrone was associated with prolonged disease free survival but not overall survival. As expected, in vitro resistance to daunorubicin was more common in the group of secondary ANLL at diagnosis. In the analysis of different drugs’ correlation in vitro, the results corresponded well to what is known about these drugs mechanism of action and cross resistance. The significantly higher in vitro effect of daunorubicin in patients who entered a CR compared to patients who did not, is in accordance with other studies [8,15]. Ara-C is another important drug in the treatment of ANLL, but here we found no significant in vitro differences between responders and non responders. One reason could be that ara-C has another mechanism of action affecting the cell in the S-phase [30]. In the short term incubations, contrary to clonogenic assays, the cells are just kept alive and further growth and cell-divisions are not required. Another reason could be that the in vitro continuous ara-C
Fig. 3. (a) Disease free survival in de novo and secondary ANLL. The median for daunorubicin is used to distinguish in vitro sensitivity from in vitro resistance. (b) Disease free survival in de novo and secondary ANLL. The median for ara-C is used to distinguish in vitro sensitivity from in vitro resistance.
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In the survival analysis sensitivity to both daunorubicin and mitoxantrone was associated with prolonged disease free survival. There is no obvious explanation of the shorter overall survival in patients who were in vitro sensitive to ara-C. The specific problem with ara-C incubations has already been discussed and in addition there are reports showing that a high number of S-phase cells in ANLL is associated with a poor survival [31]. Thus one could theoretically assume that a high proportion of cells in S-phase makes the cells more sensitive to ara-C in the in vitro incubation. In the in vivo situation a high proportion of cells in S-phase instead indicates a highly proliferative disease with a high potential of regrowth and consequently a poor survival. This can illustrate the difficulties in translating in vitro results to the in vivo situation but due to the limited material for the survival analysis in our study and a high proportion of early deaths in the ara-C sensitive group no further conclusions can be drawn. In a previous study, using the DiSC assay, we showed that in vitro sensitivity to anthracyclines and/or ara-C predicted overall survival (P 5 0.01) [8]. Another study that used the MTT assay showed that in vitro sensitivity to both daunorubicin and ara-C predicted disease free survival (P = 0.02) and that in vitro sensitivity to ara-C could predict continuous complete remission (P =0.02) [15]. Interestingly, six out of seven patients who were in vitro sensitive to daunorubicin and resistant to ara-C entered a complete remission. The study of in vitro correlation between different drugs illustrates cross resistance and how different mechanisms are involved in the drug resistance. The Pgp mediated multidrug resistance (MDR) is associated with resistance mainly to anthracyclines, vinca alkaloids and epipodophyllotoxins [32]. Topoisomerase II mediated drug resistance affects important drugs used for treatment of ANLL, e.g. anthracyclines, anthracendions, acridines and epipodophyllotoxins. In our study these mechanisms were illustrated by the strong correlation between these drugs. The nucleoside analogue araC is not affected by these two mechanisms and there was no cross resistance between ara-C and the other drugs in our study. In the survival analysis the definition of ‘in vitro sensitive’ had to be stated. One way is just to arbitrarily define a cut-off level (often 30%) between in vitro sensitive and resistant [33]. Others used each drug in several concentrations and defined sensitive/resistant using the median LC50 (the drug concentration lethal to 50% of the leukemic cells) as cut-off point [15]. Another way is to find the cut-off level that best separates in vivo sensitive and resistant patients [8]. In one study Kristenssen et al. divided the results for each drug, from all patients, into quartiles considering the lower quartile as in vitro sensitive. This made it possible to find an individual cut-off level for each drug [17]. We
chose this last method in our survival analysis but instead of the lower quartile we used the median which separated the material in groups where the number of patients were equal. For most of the cytostatic drugs the level for in vitro sensitivity was about 40% compared to the arbitrarily 30% limit (see Fig. 1). Compared to Tidefelt et al our ara-C level was higher, 49% compared to 35%, and the daunorubicin level lower, 39 compared to 60% [8]. In our study, patients with de novo ANLL at diagnosis who achieved induction therapy, the single in vitro most active drug of the drugs that the patient actually received could not predict the short term clinical outcome. The majority of the patients in our study received ara-C in their induction treatment but the other drugs varied. Some of the patients were included in protocols where the induction course had a more or less novel design and in some cases the bioluminescence ATP-assay did not include all drugs the patient received. On the other hand all patients got an anthracycline or anthracendion derivate in addition to ara-C and the correlation study suggested that in vitro sensitivity to daunorubicin corresponded to the in vitro sensitivity to other anthracyclines and anthracendions. A possible explanation to the discrepancy between our in vitro and in vivo data could be, as mentioned above, that short term assays may not be appropriate in the in vitro testing of ara-C. We have not estimated the blast percentage at the end of the test and there is a possibility that non malignant cells present at this stage may have decreased the predictive power of the test. The short term assays which are used today are in many aspects comparable, e.g. the culture procedure. The main difference is the various techniques that are used to estimate viable cells after the incubation. The bioluminescence ATP assay has been in use for a long time within the fields of biochemistry and microbiology, and a previous study has shown that it correlates satisfactorily to the DiSC assay (r= 0.8) [21–23]. The use of drug concentrations mimicking in vivo conditions, in combination with the DiSC assay, have shown good correlations to clinical outcome [8]. We have only used fresh samples, in contrast to other studies, where cryopreserved samples also have been analyzed [15,17]. One crucial point is the amount of leukemic cells after the density gradient centrifugation. In this aspect the DiSC assay has an advantage in the possibility to morphologically distinguish leukemic cells from other cells. Even if that procedure is very time consuming it may contribute to the good correlation between in vitro and in vivo data [8]. The success rate in our study was 82%. Four samples were excluded because of failure in one of the duplicates in the drug free control. If that could have been avoided by using three instead of two drug free controls the success rate would have been 86%.
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Even if our study, as well as other previous studies, has shown correlation’s to the clinical outcome in ANLL, more convincing results are needed before these short term assays will be accepted for tailoring chemotherapy treatment in clinical practice [8,14,15,17]. Another possible application of the in vitro chemosensitivity data is in the risk group stratification of ANLL patients, but also in this aspect further prospective studies are necessary to confirm that, e.g. in vitro resistance to daunorubicin is an independent risk factor in ANLL. In all different total cell kill assays there is always the risk of contamination of non malignant cells. Even if the proportion of blasts so far generally exceeded 80–90%, the remaining normal cells may affect the result. In acute lymphocytic leukemia there have been attempts to overcome this problem by the use of flow cytometry were the blast population can be separated from non malignant cells [34]. We have established a similar method for ANLL that currently is evaluated.
Acknowledgements This work was supported by grants from the Swedish Cancer Society. The authors thank Sofia Bengtsson, Ulrika Broberg and Malin Prenkert for their technical assistance.
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