ANTICANCER RESEARCH 33: 1189-1194 (2013)

Individual Tumor Response Testing in Multiple Relapsed Acute Myeloid Leukemia in Children

JAN STYCZYNSKI*, MAGDALENA PIATKOWSKA*, KRZYSZTOF CZYZEWSKI, MONIKA POGORZALA and MARIUSZ WYSOCKI

Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland

Abstract. Aim: The analysis of the individualized tumor in therapy is dependent on three factors: pharmacokinetic response testing (ITRT) at first and subsequent relapse in resistance, cellular drug resistance and residual disease (4). children with acute myeloid leukemia (AML). Patients and Cellular drug resistance can be defined as cellular Methods: A total of 76 pediatric AML samples underwent insensitivity to drugs reaching the cell. ITRT for up to 21 drugs. Results: No significant differences Leukemia cells of children with de novo AML show between ITRT at first and subsequent relapse were found, higher ex vivo resistance (called individual tumor resistance and no drug was found, for which significantly higher testing, ITRT) to most drugs, when compared to cells of ALL resistance of myeloblasts was observed at subsequent at diagnosis (5, 6). However, little is known regarding drug relapse, when compared to first relapse of AML. For most resistance in relapse of AML in children. There is only a tested drugs, patients with relapse had higher IRTR than limited number of studies published to date (7, 8). It has those with de novo AML. The median relative resistance been shown that children with relapsed AML were ex vivo, a value between patients with relapse and those with de novo median of 3-fold more resistant to than the initial diagnosis for all 21 drugs tested was 1.6. Samples of AML group, however the group of patients was relatively relapsed AML samples were significantly more resistant to: small; in the group of poor responders to , 3- (1.8-fold), (5.9-fold), cytarabine (1.7- fold higher resistance to cytarabine was observed in fold), (3.7-fold) and (4.3-fold). comparison to the group of good responders (5). In our Conclusion: ITRT in relapsed AML is higher in study, we aimed to compare ex vivo drug resistance (ITRT) at comparison to that at initial diagnosis, while no diagnosis and at first and subsequent relapses in a group of differences in ITRT between first and subsequent relapse patients with AML. of AML were found. Patients and Methods The current cure rate achieves 80% of long-term survival in Patient samples. childhood acute lymphoblastic leukemia (ALL) and 50-60% A total number of 76 leukemia samples were included in the study, including 44 samples obtained from children in acute myeloblastic leukemia (AML) (1-3). Inspite of at initial AML diagnosis, 22 at first relapse of leukemia, and 10 continuous progress in therapy of acute leukemia, relapses obtained at subsequent leukemic relapse. Detailed patient still occur frequently, both in children and adults. The results characteristics, with respect to the phase of the disease, are of therapy, in relapse of AML in childhood, do not exceed presented in Table I. The distribution of patients between these three 30% and are very poor in subsequent relapses (2, 3). Failure groups was comparable.

Individual tumor response testing. ITRT was tested by 3-4,5- dimethylthiazol-2-yl-2,5-difenyl tetrazolium bromide (MTT) assay, *These Authors contributed equally to this study. as described previously (9). The drug concentration that was inhibitory to 50% of the cells (IC50) was calculated from the dose- Correspondence to: Jan Styczynski, MD, Ph.D., Department of response curve and was used as a measure of ex vivo drug resistance Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus for each sample. The relative resistance (RR) between groups Copernicus University, ul. Curie-Sklodowskiej 9, 85-094 analyzed for each drug was calculated as the ratio of the mean values Bydgoszcz, Poland. Tel: +48 525854860, Fax: +48 525854867, of the IC50 of the respective groups for this drug. Only patients who e-mail: [email protected] had a successful MTT assay at-diagnosis were included in the study.

Key Words: Ex vivo drug resistance, individualized tumor response Drugs. The following 21 drugs and concentrations were used: testing, acute myeloid leukemia, children, relapse, minimal residual Prednisolone (Fenicort; Jelfa, Jelenia Gora, Poland), tested disease. concentration range 0.007-250 μg/ml; dexamethasone (Dexamethasone;

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Table I. Patients’ characteristics.

Initial AML First AML relapse Subsequent AML relapse

Number of patients 44 22 10 Gender (male/female) 23/21 14/8 6/4 Median age, years (range) 12 (0.3-19) 12.5 (2-19) 13.5 (5-18) FAB type M0 3 1 1 M1 12 7 4 M2 20 9 5 M3 - 1 2 M4 4 1 - M5 5 2 - M6 - 1 - Down syndrome 3 - - Median WBC count (range), 109/l 20.3 (1.2-341.0) 3.5 (0.7-186.0) 6.1 (2.7-10.4)

AML: Acute myeloblastic leukemia; FAB: French-American-British classification; WBC: white blood cell count.

Jelfa), 0.0002-6 μg/ml; (Vincristine; Eli-Lilly, Indianapolis, on subsequent relapse) and for another 11 drugs, the RR value IN, USA), 0.019-20 μg/ml; idarubicin (Zavedos; Farmitalia, Milan, was greater than 1 (i.e. higher drug resistance on subsequent Italy), 0.0019-2 μg/ml; (Daunorubicin; Rhone-Poulenc- relapse). No drug showed a trend towards better cellular Rhorer, Paris, France), 0.0019-2 μg/ml; (Doxorubicin; Farmitalia, Milan, Italy), 0.0078-8 μg/ml; (Mitoxantrone; sensitivity at first versus subsequent relapse, as the differences Jelfa), 0.001-1 μg/ml; etoposide (Vepeside; Bristol–Myers Squibb, were not significant for each tested drug. Princeton, NJ, USA), 0.048-50 μg/ml; L- (Medac; Medac, Since the ex vivo drug resistance profile in children with Hamburg, Germany), 0.0032-10 IU/ml; cytarabine (Cytosar; Pharmacia first versus subsequent relapsed AML was comparable, we & Upjohn, Kalamazoo, MI, USA), 0.0097-10 μg/ml; fludarabine pooled all patients into one group for further analysis, in (Fludara; Schering, Berlin, Germany), 0.019-20 μg/ml; order to compare drug resistance between relapse and (Biodribin; Bioton, Warsaw, Poland), 0.0004-40 μg/ml; initially diagnosed AML. For most tested drugs, patients with (Ovastat; Medac, Hamburg, Germany), 0.0005-1 μg/ml; (Thiotepa; Lederle, Greifswald, Germany), 0.032-100 μg/ml; relapse had higher IC50 in their ITRT profile (Table III). The (Alkeran; Glaxo, Parma, Italy), 0.038-40 μg/ml; 4-HOO- median RR of all tested drugs was 1.6. For five drugs, RR (Asta Medica; Hamburg, Germany), 0.096-100 was significantly higher at relapse: idarubicin (1.8-fold), μg/ml; 4-HOO- (Asta Medica), 0.096-100 μg/ml; etoposide (5.9-fold), cytarabine (1.7-fold), fludarabine (3.7- (Velcade; Janssen Pharmaceutica N.V., Beerse, Belgium), 19-2000 nM; fold) and busulfan (4.3-fold). busulfan (Busilvex; Pierre-Fabre Medicament, Boulogne, France), 1.17- 1200 μg/ml; 6- (Sigma, St. Louis, MO, USA), 15.6-500 Discussion μg/ml; 6-Thioguanine (Sigma), 1.56-50 μg/ml.

Statistical methods. Observed differences in proportions were tested In the present study, we have shown that drug resistance of for statistical significance using the appropriate chi-square test. For myeloblasts in patients with relapsed AML is higher than that of small sample sizes, the Fisher’s exact test was used. Differences in de novo ones. Still, relapse remains a significant problem for all the distribution of the IC50 values between two groups were children with AML. In the study of the Dutch-German group, analyzed using the Mann-Whitney U-test. Using the two-tailed test, no significant differences in drug resistance were reported in a p<0.05 was considered statistically significant. The study was large cohort of childhood AML samples taken at diagnosis approved by the Local Bioethical Committee. between patients remaining in continuous complete remission Results versus those with refractory/relapsed AML (10). In general, relapsed AML has a dismal prognosis, mainly related to the No significant differences between ITRT at first and subsequent time interval between initial diagnosis and relapse, and cellular relapse of childhood AML were found (Table II). Out of the 21 drug resistance can play a key role in therapy failure of relapsed drugs analyzed, no drug was found for which significantly higher childhood AML. This is important, as patients with relapse had resistance of myeloblasts was observed at subsequent relapse, myeloblasts more resistant to basic drugs used in the therapy of when compared to first relapse of AML. The median RR value childhood AML, such as: cytarabine, idarubicin, daunorubicin, between second and first relapse of all tested drugs was 1.0; for mitoxantrone and etoposide. Relapsed leukemia blasts were also 10 drugs the RR was less than 1 (i.e. assumed better sensitivity more resistant to drugs commonly used in therapy of relapsed

1190 Styczynski et al: Multiple Relapsed Acute Myeloid Leukemia

Table II. Comparison of the ex vivo drug resistance profile (median and range of inhibitory concentrations) between first and subsequent relapse of acute myeloblastic leukemia.

Drug (μg/ml) First relapse Subsequent relapse RR p-Value

N Median Range N Median Range

Prednisolone 17 95.10 3.40- >250.00 7 112.36 36.07-147.50 1.2 0.924 Dexamethasone 12 >6.00 0.03- >6.00 6 >6.00 >6.00- >6.00 1.0 0.303 Vincristine 17 4.27 0.13- >20.00 7 2.59 0.57-10.47 0.6 0.775 Idarubicin 17 0.39 0.03- >2.00 9 0.26 0.12- >2.00 0.7 0.725 Daunorubicin 17 0.55 0.03- >2.00 7 0.55 0.24-1.59 1.0 0.727 Doxorubicin 13 5.00 0.34- >8.00 6 1.06 0.64- >8.00 0.2 0.472 Mitoxantrone 12 0.55 0.01- >1.00 6 0.61 0.10- >1.00 1.1 0.772 Etoposide 18 20.14 0.30- >50.00 6 22.03 15.75- >50.00 1.1 0.662 L-Asparaginase (IU/ml) 15 1.40 0.01- >10.00 7 1.49 0.20- >10.00 1.2 0.800 Cytarabine 16 0.81 0.22- >10.00 8 0.64 0.14- >10.00 0.8 0.478 Fludarabine 13 1.46 0.06- >20.00 6 1.19 0.17- >20.00 0.8 0.929 Cladribine 17 10.00 0.01- >40.00 8 0.09 0.01- >40.00 0.1 0.438 Treosulfan 9 0.60 0.01- >1.00 6 0.58 0.01-2.11 1.0 0.903 Thiotepa 9 1.59 0.03-12.11 5 1.96 0.59- >4.00 1.2 0.947 Melphalan 8 5.27 0.91-34.45 7 6.65 1.35-15.06 1.3 0.838 4-HOO-Cyclophosphamide 10 2.74 0.38-17.41 6 1.29 0.39-3.13 0.5 0.193 4-HOO-Ifosfamide 7 16.82 8.17-96.90 6 9.72 1.19-32.05 0.6 0.513 Bortezomib (nM) 8 1044.27 261.82- >2000.00 7 1199.43 358.13- >2000.00 1.1 0.767 Busulfan 7 64.65 33.53- >1200.00 7 488.06 95.34-952.00 7.5 0.564 6-Thioguanine 10 21.25 1.56- >50.00 5 6.25 4.42-18.95 0.3 0.141 6-Mercaptopurine 9 308.72 141.01- >500.00 7 63.55 31.25-81.39 0.2 0.105

N: Number of patients; RR: relative resistance=median IC50 (subsequent relapse)/median IC50 (first relapse).

Table III. Comparison of the ex vivo drug resistance profile (median and range of inhibitory concentrations) between relapsed and de novo acute myeloblastic leukemia (AML).

Drug (μg/ml) Initial AML Relapsed AML RR p-Value

N Median Range N Median Range

Prednisolone 38 94.65 0.40- >250.00 24 100.65 3.40- >250.00 1.1 0.295 Dexamethasone 18 6.00 0.01- >8.00 18 6.00 0.03- >6.00 1.0 0.664 Vincristine 38 2.73 0.02-16.09 24 4.08 0.13- >20.00 1.5 0.435 Idarubicin 40 0.22 0.01- >2.00 26 0.38 0.03- >2.00 1.8 0.041 Daunorubicin 37 0.27 0.01- >2.00 24 0.55 0.03- >2.00 2.0 0.052 Doxorubicin 33 1.69 0.24- >8.00 19 1.41 0.34- >8.00 0.8 0.870 Mitoxantrone 34 0.23 0.01-13.28 18 0.61 0.01- >1.00 2.6 0.077 Etoposide 36 3.44 0.05- >50.00 24 20.14 0.30- >50.00 5.9 0.007 L-Asparaginase (IU/ml) 33 0.68 0.03- >10.00 22 1.35 0.01- >10.00 2.0 0.058 Cytarabine 40 0.47 0.01-12.19 24 0.78 0.14- >10.00 1.7 0.050 Fludarabine 35 0.40 0.02-15.54 19 1.46 0.06- >20.00 3.7 0.022 Cladribine 32 0.04 0.01- >40.00 25 0.75 0.01- >40.00 21.2 0.072 Treosulfan 31 0.32 0.01- >1.00 15 0.60 0.01-2.11 1.9 0.572 Thiotepa 31 1.88 0.12- >100.00 14 1.94 0.03-12.11 1.0 0.787 Melphalan 25 4.65 0.10- >40.00 15 6.57 0.91-34.45 1.4 0.973 4-HOO-Cyclophosphamide 30 1.68 0.24-9.35 16 2.16 0.38-17.41 1.3 0.890 4-HOO-Ifosfamide 13 1.98 0.35-34.74 13 13.27 1.19-96.90 6.7 0.136 Bortezomib (nM) 16 353.74 191.50-1096.83 15 1044.27 261.82- >2000.00 3.0 0.137 Busulfan 14 15.19 1.17-42.30 14 64.65 24.12- >1200.00 4.3 0.004 6-Thioguanine 17 14.63 1.36- >50.00 15 14.79 1.56- >50.00 1.0 0.533 6-Mercaptopurine 18 106.15 15.63- >500.00 16 229.25 31.25- >500.00 2.2 0.118

N: Number of patients; RR: relative resistance=median IC50 (relapsed AML)/median IC50 (initial AML).

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AML: fludarabine, cytarabine and idarubicin. High ex vivo Perot C, Reinhardt D, Rubnitz JE, Smith FO, Stary J, Stasevich drug-resistance in childhood AML might partially explain worse I, Strehl S, Taga T, Tomizawa D, Webb D, Zemanova Z, Pieters clinical results of therapy, when compared to ALL. It is R, Zwaan CM and van den Heuvel-Eibrink MM: Prognostic significance of additional cytogenetic aberrations in 733 de novo commonly assumed that patients with relapse are more drug pediatric 11q23/MLL-rearranged AML patients: Results of an resistant than those diagnosed de novo, and this was confirmed international study. Blood 117: 7102-7111, 2011. in the present analysis of samples of relapsed AML. 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Acknowledgements 9 Styczynski J, Piatkowska M, Jaworska-Posadzy A, Czyzewski K, Kubicka M, Kolodziej B, Kurylo-Rafinska B, Debski R, The study was supported by grant MNiSW N407 541339. The Pogorzala M and Wysocki M: Comparison of prognostic value Authors thank Beata Kolodziej, Beata Kurylo-Rafinska and of in vitro drug resistance and bone marrow residual disease on Malgorzata Kubicka for technical assistance. day 15 of therapy in childhood acute lymphoblastic leukemia. Anticancer Res 32: 5495-5500, 2012. References 10 Zwaan CM, Kaspers GJ, Pieters R, Hahlen K, Huismans DR, Zimmermann M, Harbott J, Slater RM, Creutzig U and Veerman 1 Pui CH, Robison LL and Look AT: Acute lymphoblastic AJ: Cellular drug resistance in childhood acute myeloid leukaemia. Lancet 371: 1030-1043, 2008. leukemia is related to chromosomal abnormalities. Blood 100: 2 Coenen EA, Raimondi SC, Harbott J, Zimmermann M, Alonzo 3352-3360, 2002. 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