Background: The prognostic role of the ex vivo drug resistance profile has not yet been proved in childhood acute myeloid leukemia (AML). The aim of the study was the analysis of the impact of the ex vivo drug resistance profile in a cohort of 44 children with AML undergoing hematopoietic stem cell transplantation (HSCT). Patients and Methods: Myeloblasts for drug resistance testing were obtained from the bone marrow either on diagnosis or at relapse, before the HSCT procedure and were tested by the MTT assay. Results: Children who relapsed after transplantation showed higher ex vivo resistance of the leukemic blasts to etoposide, mercaptopurine, thioguanine, fludarabine, mitoxantrone and treosulfan than those who stayed in remission. Despite being nondiscriminative, the combined ex vivo drug resistance profile to fludarabine, treosulfan and etoposide (FTE score) was the strongest prognostic factor by multivariate analysis. Conclusion: The combined drug resistance profile to fludarabine, treosulfan and etoposide may be useful for better stratification of children with AML undergoing stem cell transplantation or to indicate the necessity for additional post-transplant therapy

Balcerska, Anna

Balwierz, Walentyna

Boruczkowski, Dariusz

Chybicka, Alicja

Dębski, Robert

Gil, Lidia

Kowalczyk, Jerzy

Krawczuk-Rybak, Maryn

Matysiak, Michał

Sońta-Jakimczyk, Danuta

Styczyński, Jan

Toporski, Jacek

Wachowiak, Jacek

Wysocki, Mariusz

Wójcik, Beata

Anticancer Research

English

Jagiellonian Univeristy Repository

Abstract. Background: The prognostic role of the ex vivo drugresistance profile has not yet been proved in childhood acutemyeloid leukemia (AML). The aim of the study was theanalysis of the impact of the ex vivo drug resistance profile in acohort of 44 children with AML undergoing hematopoietic stemcell transplantation (HSCT). Patients and Methods:Myeloblasts for drug resistance testing were obtained from thebone marrow either on diagnosis or at relapse, before the HSCTprocedure and were tested by the MTT assay. Results: Childrenwho relapsed after transplantation showed higher ex vivoresistance of the leukemic blasts to etoposide, mercaptopurine,thioguanine, fludarabine, mitoxantrone and treosulfan thanthose who stayed in remission. Despite being nondiscriminative,the combined ex vivo drug resistance profile to fludarabine,treosulfan and etoposide (FTE score) was the strongestprognostic factor by multivariate analysis. Conclusion: Thecombined drug resistance profile to fludarabine, treosulfan andetoposide may be useful for better stratification of children withAML undergoing stem cell transplantation or to indicate thenecessity for additional post-transplant therapy.The combined ex vivo drug resistance profile to prednisolone,vincristine and L-asparaginase (PVA score) has prognosticvalue in childhood de novo acute lymphoblastic leukemia(ALL) (1, 2). The results of therapy for childhood acutemyeloid leukemia (AML) differ from those of ALL. Thedevelopment of drug resistance is the limiting factor in thetherapy of AML (3). In pediatric AML, in spite of severalreports (3-5), the prognostic role of the ex vivo drugresistance profile has not yet been proved, with one possibleexception of the prognostic role of sensitivity to cytarabine(6), while this relationship has been confirmed in adult AML(7). Apart from one study of children and adults with acuteleukemia (8), no data are available regarding the possiblerole of drug resistance in children undergoing hematopoieticstem cell transplantation (HSCT). In this study, theprognostic value of the ex vivo drug resistance profile inpediatric AML patients undergoing HSCT was analyzed. 1547Correspondence to: Jan Styczynski, MD, Ph.D., Department ofPediatric Hematology and Oncology, Collegium Medicum,Nicolaus Copernicus University, ul. Curie-Sklodowskiej 9, 85-094Bydgoszcz, Poland. Tel: +48 601 222 131, Fax: +48 52 585 4867, e-mail: jstyczynski@cm.umk.plKey Words: Drug resistance, drug sensitivity, acute myeloidleukemia, hematopoietic stem cell transplantation.ANTICANCER RESEARCH 27: 1547-1552 (2007)Fludarabine, Treosulfan and Etoposide Sensitivity and theOutcome of Hematopoietic Stem Cell Transplantation in Childhood Acute Myeloid LeukemiaJAN STYCZYNSKI1, JACEK TOPORSKI2, MARIUSZ WYSOCKI1, ROBERT DEBSKI1, ALICJA CHYBICKA2, DARIUSZ BORUCZKOWSKI3, JACEK WACHOWIAK3, BEATA WOJCIK4,JERZY KOWALCZYK4, LIDIA GIL5, WALENTYNA BALWIERZ6, MICHAL MATYSIAK7, MARYNA KRAWCZUK-RYBAK8, ANNA BALCERSKA9 and DANUTA SONTA-JAKIMCZYK101Department of Pediatric Hematology and Oncology, Medical College, Nicolaus Copernicus University, ul. Curie-Sklodowskiej 9, 85-094 Bydgoszcz; 2Department of Pediatric Transplantology, Hematology and Oncology, Medical University, ul. Bujwida 44, 50-345 Wroclaw; 3Department of Pediatric Transplantology, Hematology and Oncology, Medical University, ul. Szpitalna 27/33, 60-572 Poznan;4Department of Pediatric Hematology and Oncology, Medical University, ul. Chodzki 2, 20-093 Lublin; 5Department of Hematology, Medical University, ul. Szamarzewskiego 84, 60-569 Poznan; 6Department of Pediatric Oncology/Hematology, Medical College, Jagiellonian University, ul. Wielicka 265, 30-663 Krakow; 7Department of Pediatric Hematology and Oncology, Medical University, ul. Marszalkowska 24, 00-576 Warsaw; 8Department of Pediatric Hematology and Oncology, Medical University, ul. Waszyngtona 17, 15-274 Bialystok; 9Department of Pediatric Hematology, Oncology and Endocrinology, Medical University, ul. Debinki 7, 80-210 Gdansk;10Department of Pediatric Hematology and Oncology, Medical University, ul. 3 Maja 13/15, 41-800 Zabrze, Poland0250-7005/2007 $2.00+.40Patients and MethodsPatients. Fourty-four children (28 male, 16 female) with AML,including 20 in complete remission (CR1) and 24 relapsed patients,aged 0.7-17 years (median 10 years), who underwent HSCT, wereincluded in the study. Patients with favourable cytogenetics of theleukemic blasts and those suffering early transplant-relatedmortality were not included in the study. The patients receivedmyeloablative conditioning based on busulfan (n=32), treosulfan(n=8), fractionated total body irradiation (FTBI, n=3), orcyclophosphamide (n=1). Thymoglobulin was administered beforeunrelated or haploidentical HSCT. The source of the graft wasmatched sibling donor (n=20), matched unrelated donor (n=9),autologous (n=12) or mismatched family donor (n=3). Thepatients were followed up for a median of 2.5 (range 0.2-4.3) years.Fresh myeloblasts for drug resistance testing were obtained fromthe bone marrow either on diagnosis or at relapse, before HSCT,and were processed as described previously (9). Only samples withat least of 70% of myeloblasts were included in the study. Thestudy was approved by the local Bioethical Committee and writteninformed consent was obtained from all patients and their parents.Drug resistance profile. The drugs used in the study are listed inTable I. The cytotoxicity of the tested compounds to leukemic cellswas measured by the MTT assay, as described previously (10). Allexperiments were performed in duplicate. The cytotoxicity wasexpressed as IC50, the inhibitory concentration for 50% of the cells.According to the median cytotoxicity for each tested drug, all thepatients were scored as resistant (score 2) or sensitive (score 1) tothis drug. The FTE score was defined as the sum of threerespective score values for fludarabine, treosulfan and etoposide;thus the FTE score ranged from 3 to 6. An FTE score of 3-4 wasregarded as sensitive and 5-6 as resistant.Statistical analysis. The Mann-Whitney U-test was used forunpaired comparisons. Survival curves were calculated by theKaplan-Meier method and compared by the log-rank test. The Coxproportional hazards regression model was used in univariateanalysis. The significantly important factors were fitted together inmultivariate analysis in a backward stepwise manner using thelikelihood ratio test until all factors in the model were significant.All reported p-values are two-sided, p<0.05 was considered asstatistically significant.ResultsRelapses after HSCT occurred in 15/44 children. The childrenwho relapsed after HSCT showed higher ex vivo resistance oftheir leukemic blasts to fludarabine, treosulfan, etoposide,mercaptopurine, thioguanine and mitoxantrone (Table I). Nosignificant differences were found for the other drugs. The overall probability of disease-free survival (pDFS) forall 44 patients was 0.64±0.07, with a mean survival time of2.7 years [95% confidence interval (CI)=2.1-3.3]. The type ofHSCT, disease status and age at transplantation had noinfluence on the pDFS (Figure 1A-C). Better pDFS wasobserved for patients ex vivo sensitive to fludarabine(0.80±0.10 vs. 0.41±0.12, p=0.0184), thioguanine (0.81±0.09vs. 0.44±0.11, p=0.0090), treosulfan (0.85±0.09 vs.0.46±0.13, p=0.0368), etoposide (0.89±0.07 vs. 0.47±0.10,p=0.0038), and for patients with a sensitive combined drugresistance profile (FTE score) (0.91±0.08 vs. 0.46±0.12,p=0.0221) (Figure 1D). Cell-biological features (age, sex, blast morphology, initialleukocytosis, cytogenetics), response to therapy and drugresistance profile parameters were taken together in the Coxmodel. Out of all tested factors, those predicting positiveoutcome by Cox univariate analysis were: ex vivo sensitivity tofludarabine [p=0.036, hazard ratio (HR)=0.50, 95%CI=0.26-0.95], etoposide (p=0.016, HR=0.40, 95%CI=0.19-0.85),treosulfan (p=0.018, HR=0.46, 95%CI=0.24-0.87),thioguanine (p=0.048, HR=0.59, 95%CI=0.21-0.97), and asensitive combined drug resistance profile discriminated byFTE score (p=0.014, HR=0.44, 95%CI=0.18-0.83). Nofactors showed prognostic value by multivariate analysis,however the sensitive combined drug resistance profilediscriminated by FTE score was the strongest prognosticfactor (p=0.076, HR=0.39, 95%CI=0.13-1.10), though it waspossible to define the FTE score in only 33 out of 44 patients.DiscussionThe drug resistance profile identifies patients at higher riskof treatment failure. In the study of Miller et al., whichincluded children and adults with ALL and AML, thesensitivity of the occult leukemia colony-forming units to 4-hydroperoxycyclophosphamide was the only factor thatpredicted relapse following HSCT (8). In the present study,the combined ex vivo drug resistance profile, although beingnondiscriminative, was the strongest prognostic factor in amultivariate analysis for AML children undergoing HSCT.No firm conclusions regarding the necessity for the use offludarabine, treosulfan and etoposide in the treatment ofAML with HSCT can be drawn from our study; howeverthese results might indicate that patients whose myeloblastsare sensitive to fludarabine, treosulfan and etoposide wouldbenefit from the use of these drugs during conditioningbefore HSCT, and possibly at the earlier stages of therapy.Resistance to these drugs might suggest the necessity ofimplementation of post-transplant procedures, such as closemonitoring of minimal residual disease, reduction ofimmunosuppressive therapy, immunotherapy based on donorlymphocyte infusion, or administration of interleukin-2. TheFTE score, however, could not prevent the overtreatment ofthe patients with sensitive FTE scores, as all patientsqualifying for HSCT need very intensive therapy. AML is a relatively rare disease in children, however, theresults of therapy for this disease are still not satisfactory,as only 50%-60% remission has been reported (11-14). Thevalue of testing sensitivity to fludarabine, treosulfan andANTICANCER RESEARCH 27: 1547-1552 (2007)1548etoposide in pediatric AML is related to the use of thesedrugs which so far have not been tested (treosulfan) or notwidely tested (fludarabine). It has shown that these drugsmight have a role in the therapy of childhood AML (15),especially treosulfan and fludarabine which have not reallybeen used as cytotoxic agents for AML but more asbiochemical modulators for cytarabine (16). In conclusion, ex vivo drug resistance to fludarabine,treosulfan and etoposide is of predictive value in childhoodAML undergoing HSCT. Therefore, the drug resistanceprofile may be used for better stratification of children withAML indicating those patients who may be cured bychemotherapy based on these drugs and to identify thosepatients who are at high risk of treatment failure and whoStyczynski et al: Fludarabine, Treosulfan and Etoposide Sensitivity in AML1549Table I. Comparison of ex vivo drug resistance in patients staying in remission and relapsing after HSCT. The MTT assay was performed at AMLdiagnosis, before HSCT was planned.Drug (Company) Concentration IC50 (median and quartiles) RR p-value[ÌM]Remission Relapse Cytarabine 0.04-41 2.38 (n=28) 2.71 (n=15) 1.13 0.838(Pharmacia Upjohn, Bentley, Australia) 0.57-9.66 1.35-5.13Cladribine 0.001-140 0.26 (n=28) 0.24 (n=15) 0.94 0.508(Bioton, Warsaw, Poland) 0.04-2.32 0.12-4.44Fludarabine phosphate 0.05-54 2.16 (n=20) 8.68 (n=13) 4.01 0.036*(Schering AG, Berlin, Germany) 0.87-8.15 3.25-33.1Daunorubicin 0.002-3.5 1.01 (n=28) 1.25 (n=15) 1.24 0.177(Rhone-Poulenc-Rhorer, Paris, France) 0.54-1.50 0.95-2.16Doxorubicin 0.01-13.8 4.61 (n=22) 11.2 (n=12) 2.32 0.720(Farmitalia, Milan, Italy) 1.31-13.8 1.56-13.8Epirubicin 0.003-3.4 1.79 (n=20) 1.19 (n=11) 0.66 0.495(Farmitalia, Milan, Italy) 0.93-3.13 0.75-2.51Idarubicin 0.003-3.7 0.82 (n=27) 0.80 (n=15) 0.97 0.609(Zavedos, Pharmacia, Milan, Italia) 0.35-1.53 0.39-1.12Mitoxantrone 0.002-1.9 0.67 (n=18) 1.33 (n=11) 1.97 0.045*(Jelfa, Jelenia Gora, Poland) 0.42-1.02 0.34-1.9Etoposide 0.08-85 23.6 (n=28) 47.6 (n=14) 2.01 0.018*(Bristol–Myers Squibb, Princeton, NJ, USA) 4.12-43.2 34.7-856-Thioguanine 9.3-299 45.9 (n=21) 130.9 (n=14) 2.84 0.006*(Sigma, A4882, St. Louis, MO, USA) 25.6-103.8 79.2-2996-Mercaptopurine 91-2937 418 (n=19) 1328 (n=14) 3.17 0.012*(Sigma, M7000, St. Louis, MO, USA) 206-1038 629-19814-HOO-cyclophosphamide 0.3-341 6.85 (n=23) 10.6 (n=10) 1.55 0.153(Asta Medica AG, Frankfurt/Main, Germany) 3.65-14.0 5.66-33.14-HOO-ifosfamide 0.33-341 59.0 (n=9) 64.9 (n=6) 1.10 0.679(Asta Medica AG, Frankfurt/Main, Germany) 21.4-130.8 14.9-116.4Mafosfamide 0.19-200 6.41 (n=23) 17.8 (n=10) 2.78 0.357(Asta Medica AG, Frankfurt/Main, Germany) 2.37-17.6 4.49-18.9Glufosfamide 0.5-522 66.8 (n=8) 51.3 (n=6) 0.76 0.697(Asta Medica AG, Frankfurt/Main, Germany) 17.7-96.9 19.9-69.7Treosulfan 0.002-3.6 0.006 (n=23) 0.077 (n=10) 12.6 0.020*(Medac, Hamburg, Germany) 0.002-1.31 0.012-3.6Melphalan 0.12-131 49.4 (n=14) 32.7 (n=5) 0.66 0.459(Glaxo Wellcome, Parma, Italy) 20.0-58.6 12.6-74.7Thiotepa Lederle 0.16-528 30.6 (n=18) 29.1 (n=7) 0.95 0.785(Riemser, Greifswald, Germany) 11.4-47.8 18.3-42.9Prednisolone 0.02- 694 303 (n=28) 304 (n=15) 1.00 0.452(Jelfa, Jelenia Gora, Poland) 171-375 165-449Vincristine 0.02-21 4.33 (n=28) 7.38 (n=15) 1.70 0.236(Oncovin, Eli-Lilly, Indianapolis, IN, USA) 1.28-8.12 2.80-10.44L-asparaginase 0.003-10 1.17 (n=29) 1.21 (n=15) 1.03 0.380(Medac, Hamburg, Germany) 0.33-8.20 0.50-10.0IC50: value of in vitro resistance, given in U/L for L-asparaginase and in mM for other drugs; RR: relative resistance = median IC50 (relapsedAML) / median IC50 (remission AML); n: the number of patients; p-value: Mann-Whitney U-test; * significant differences between remission andrelapse.therefore may benefit from more intensive treatment atinitial diagnosis, or require additional post-transplanttherapeutic strategies.AcknowledgementsThe authors thank Beata Kolodziej, Beata Rafinska and MalgorzataKubicka for their technical support. In addition to the list ofauthors, this study was performed with the significant contributionof the following researchers who provided patient samples and theirdata: Igor Olejnik, Marta Kuzmicz, Jolanta Stefaniak, TomaszSzczepanski, Elzbieta Stanczak, Iwona Malinowska, BenignaKonatkowska and Lucyna Kapuscinska. This study was supportedby grants KBN 6 PO5E 082 21 and N407 078 32/2964. References1 Den Boer ML, Harms DO, Pieters R, Kazemier KM, Gobel U,Korholz D, Graubner U, Haas RJ, Jorch N, Spaar HJ, KaspersGJ, Kamps WA, Van der Does-Van den Berg A, Van WeringER, Veerman AJ and Janka-Schaub GE: Patient stratificationbased on prednisolone-vincristine-asparaginase resistanceprofiles in children with acute lymphoblastic leukemia. J ClinOncol 21: 3262-3268, 2003.2 Styczynski J and Wysocki M: Is the in vitro drug resistanceprofile the strongest prognostic factor in childhood acutelymphoblastic leukemia? J Clin Oncol 22: 963-964, 2004.3 Zwaan CM, Kaspers GJ, Pieters R, Hahlen K, Huismans DR,Zimmermann M, Harbott J, Slater RM, Creutzig U andVeerman AJ: Cellular drug resistance in childhood acutemyeloid leukemia is related to chromosomal abnormalities.Blood 100: 3352-3360, 2002.4 Yamada S, Hongo T, Okada S, Watanabe C, Fujii Y and OhzekiT: Clinical relevance of in vitro chemoresistance in childhoodacute myeloid leukemia. Leukemia 15: 1892-1897, 2001.5 Palle J, Frost BM, Forestier E, Gustafsson G, Nygren P,Hellebostad M, Jonsson OG, Kanerva J, Schmiegelow K,Larsson R and Lonnerholm G: Cellular drug sensitivity in MLL-rearranged childhood acute leukaemia is correlated to partnergenes and cell lineage. Br J Haematol 129: 189-198, 2005.6 Klumper E, Pieters R, Kaspers GJ, Huismans DR, Loonen AH,Rottier MM, van Wering ER, van der Does-van den Berg A,Hahlen K and Creutzig U: In vitro chemosensitivity assessedwith the MTT assay in childhood acute non-lymphoblasticleukemia. Leukemia 9: 1864-1869, 1995.7 Staib P, Staltmeier E, Neurohr K, Cornely O, Reiser M andSchinkothe T: Prediction of individual response to chemotherapyin patients with acute myeloid leukaemia using thechemosensitivity index Ci. Br J Haematol 128: 783-791, 2005.ANTICANCER RESEARCH 27: 1547-1552 (2007)1550Figure 1. Impact of (A) type of transplantation (matched unrelated donor, MUD; matched family donor, MFD; mismatched family donor, MMFD;autologous, AUTO), (B) disease status (first complete emission CR1 vs. CR≥2), (C) patient age, (D) combined ex vivo drug resistance to fludarabine,treosulfan, and etoposide (FTE score) on pDFS after HSCT in childhood AML.8 Miller CB, Zehnbauer BA, Piantadosi S, Rowley SD and JonesRJ: Correlation of occult clonogenic leukemia drug sensitivitywith relapse after autologous bone marrow transplantation.Blood 78: 1125-1131, 1991.9 Styczynski J and Wysocki M: Ex vivo drug resistance inchildhood acute myeloid leukemia on relapse is not higher thanat first diagnosis. Pediatr Blood Cancer 42: 195-199, 2004.10 Styczynski J, Kurylak A and Wysocki M: Cytotoxicity ofcortivazol in childhood acute lymphoblastic leukemia.Anticancer Res 25: 2253-2258, 2005.11 Creutzig U, Zimmermann M, Ritter J, Reinhardt D, HermannJ, Henze G, Jurgens H, Kabisch H, Reiter A, Riehm H, GadnerH and Schellong G: Treatment strategies and long-term resultsin paediatric patients treated in four consecutive AML-BFMtrials. Leukemia 19: 2030-2042, 2005.12 Ravindranath Y, Chang M, Steuber CP, Becton D, Dahl G,Civin C, Camitta B, Carroll A, Raimondi SC and Weinstein HJ:Pediatric Oncology Group (POG) studies of acute myeloidleukemia (AML): a review of four consecutive childhood AMLtrials conducted between 1981 and 2000. Leukemia 19: 2101-2116, 2005.13 Ribeiro RC, Razzouk BI, Pounds S, Hijiya N, Pui CH andRubnitz JE: Successive clinical trials for childhood acutemyeloid leukemia at St Jude Children's Research Hospital,from 1980 to 2000. Leukemia 19: 2125-2129, 2005.14 Kardos G, Zwaan CM, Kaspers GJ, de-Graaf SS, de Bont ES,Postma A, Bokkerink JP, Weening RS, van der Does-van denBerg A, van Wering ER, Korbijn C and Hahlen K: Treatmentstrategy and results in children treated on three DutchChildhood Oncology Group acute myeloid leukemia trials.Leukemia 19: 2063-2071, 2005.15 Tallman MS: New agents for the treatment of acute myeloidleukemia. Best Pract Res Clin Haematol 19: 311-320, 2006.16 Hubeek I, Peters GJ, Broekhuizen R, Zwaan CM, Kaaijk P, vanWering ES, Gibson BE, Creutzig U, Janka-Schaub GE, denBoer ML, Pieters R and Kaspers GJ: In vitro sensitivity andcross-resistance to deoxynucleoside analogs in childhood acuteleukemia. Haematologica 91: 17-23, 2006.Received December 14, 2006Revised February 27, 2007Accepted March 9, 2007Styczynski et al: Fludarabine, Treosulfan and Etoposide Sensitivity in AML1551

Fludarabine, treosulfan and etoposide sensitivity and the outcome of hematopoietic stem cell transplantation in childhood acute myeloid leukemia

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Fludarabine, treosulfan and etoposide sensitivity and the outcome of hematopoietic stem cell transplantation in childhood acute myeloid leukemia

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