Chemotherapy in Childhood Acute Lymphoblastic Leukemia: In vitro cellular drug resistance and pharmacokinetics

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(13) Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) presented at Uppsala University in 2002. ABSTRACT Frost, BM. 2002. Chemotherapy in Childhood Acute Lymphoblastic Leukemia. In vitro cellular drug resistance and pharmacokinetics. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1189, 85pp.Uppsala. ISBN 91-554-5417-8. The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL). Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up. The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level. Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and >6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters. In vitro testing of cellular drug resistance might be useful in predicting the outcome in highrisk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children. Keywords: Acute lymphoblastic leukemia, childhood, drug resistance, in vitro assay, cytotoxicity, pharmacokinetics, vincristine, doxorubicin, Down`s syndrome, CHS 828. Britt-Marie Frost, Section of Pediatrics Department of Women's and Children's Health, S- 751 85 Uppsala, Sweden © Britt-Marie Frost 2002 ISSN 0282-7476 ISBN 91-554-5417-8 Printed in Sweden by Uppsala University, Tryck och Medier, Uppsala, 2002. 2.

(14) WHERE? Where are the fairies? Where can we find them? We’ve seen the fairy-rings They leave behind them! When they have danced all night, Where do they go? Lark, in the sky above, Say do you know? Is it a secret No one is telling? Why in your garden Surely they’re dwelling! No need for journeying, Seeking afar: Where there are flowers, There fairies are! Cicely M Barker 1944. to my Daughters: Elin, Emilia, Amanda and Lisa the fairies who put magic into my life. Cover illustration by Cicely Barker (Flower Fairies of the Garden, London, Blackie, 1944) modified by Elin Frost.. 3.

(15) Papers included in the thesis. This thesis is based on the following papers, which are referred to in the text by their Roman numerals:. I. Frost BM, Nygren P, Gustafsson G, Forestier E, Jonsson O G, Kanerva J, Nygaard R, Schmiegelow K, Larsson R, Lönnerholm G. Increased in vitro cellular drug resistance is related to poor outcome in high-risk childhood acute lymphoblastic leukemia. On behalf of the Nordic Society for Paediatric Haematology and Oncology. (Submitted). II. Frost BM, Gustafsson G, Larsson R, Nygren P, Lönnerholm G. (2000). Cellular cytotoxic drug sensitivity in children with acute leukemia and Down's syndrome: an explanation to differences in clinical outcome? Leukemia 14(4): 943-4.. III. Frost BM, Lönnerholm G, Nygren P, Larsson R, Lindhagen E. (2002). In vitro activity of the novel cytotoxic agent CHS 828 in childhood acute lymphoblastic leukemia. Anti-Cancer Drugs 13(7): 735-42.. IV. Frost BM, Eksborg S, Björk O, Abrahamsson J, Behrendtz M, Castor A, Forestier E, Lönnerholm G. (2002). Pharmacokinetics of Doxorubicin in Children with Acute Lymphoblastic Leukemia. Med Pediatr Oncol. 38(5): 32937.. V. Frost BM, Lönnerholm G, Koopmans P, Abrahamsson J, Behrendtz M, Castor A, Forestier E, Uges D R A, de Graaf S S N. Vincristine in childhood leukemia; no pharmacokinetic rationale for dose reduction in adolescents. (Submitted). 4.

(16) Contents. Papers included in the thesis ......................................................................................................... 4 Contents......................................................................................................................................... 5 Abbreviations .......................................................................................................................... 7 Background ................................................................................................................................... 8 Introduction............................................................................................................................. 8 Acute Lymphoblastic Leukemia ............................................................................................. 9 Short history ............................................................................................................. 10 Epidemiology............................................................................................................ 11 Classification ............................................................................................................ 12 Risk factors related to the prognosis ..................................................................................... 12 Chemotherapy in childhood ALL ......................................................................................... 17 The NOPHO organization..................................................................................................... 18 Development of a Nordic treatment protocol ........................................................... 18 Treatment failure................................................................................................................... 21 Cellular drug resistance ............................................................................................ 21 In vitro cytotoxicity assays ....................................................................................... 23 Differences between the MTT assay and FMCA ............................................... 25 Choice of drug concentrations............................................................................ 25 Studies of in vitro resistance in childhood ALL................................................. 26 Pharmacokinetics and clinical response ................................................................... 26 Anthracyclines.................................................................................................... 27 Vincristine .......................................................................................................... 29 Regrowth capacity of malignant cells....................................................................... 31 Development of new anticancer drugs.................................................................................. 31 Aims of the investigation ............................................................................................................ 33 Material and Methods ................................................................................................................. 34 Patients .................................................................................................................................. 34 Samples ................................................................................................................................. 36 Studies of in vitro cellular resistance (papers I-III) .................................................. 36 Pharmacokinetic studies (papers IV-V).................................................................... 37 Drugs and Reagents .............................................................................................................. 37 Methods................................................................................................................................. 39 Fluorometric Microculture Cytotoxicity Assay (FMCA) ......................................... 39 Measurement of drug concentrations........................................................................ 40 Pharmacokinetic methodology ................................................................................. 40 Statistical methods ................................................................................................................ 41 Results and Discussion................................................................................................................ 42 In vitro resistance in ALL (paper I) ..................................................................................... 42 In vitro resistance in Down´s syndrome and acute leukemia (paper II)................................ 49. 5.

(17) In vitro test of CHS 828 (paper III)....................................................................................... 50 Doxorubicin pharmacokinetics (paper IV)............................................................................ 54 Vincristine pharmacokinetics (paper V) ............................................................................... 56 Pitfalls in transportation of vincristine samples .................................................................... 59 Conclusions ................................................................................................................................. 62 Paper I ................................................................................................................................... 62 Paper II.................................................................................................................................. 62 Paper III ................................................................................................................................ 62 Paper IV ................................................................................................................................ 63 Paper V.................................................................................................................................. 63 Future directions.......................................................................................................................... 64 Acknowledgements ..................................................................................................................... 66 Swedish summary. Kort svensk sammanfattning........................................................................ 68 Målsättning ........................................................................................................................... 68 Cytostatikakänslighet in vitro (delarbete I-III) ..................................................................... 68 Farmakokinetik (delarbete IV och V) ................................................................................... 70 Betydelse............................................................................................................................... 71 References ................................................................................................................................... 72. 6.

(18) Abbreviations ALL. Acute lymphblastic leukemia. AML. Acute myeloblastic leukemia. Ara-C. Cytosine arabinoside. AUC. Area under the plasma concentration-time curve. CCR. Complete remission. CHS 828. N-(6-(4-chlorophenoxy)hexyl)-N´-cyano-N”-4-pyridylguanidine. DiSC. Differential staining cytotoxicity. DMSO. Dimethyl sulphoxide. DS. Down´s syndrome. EDTA. Ethylenediaminetetraacetic acid. FDA. Fluorescein diacetate. FMCA. Fluorometric microculture cytotoxicity assay. 4-HC. 4-hydroperoxy-cyclophosphamide. HR. High risk. IC50(70). Drug concentration lethal to 50% (70%) of leukemic cells. IR. Intermediate risk. MDR. Multi-drug resistance. MTT. Methyl-thiazol-tetrazolium. NOPHO. The Nordic Society of Paediatric Haematology and Oncology. p-DFS. Probability of disease-free survival. SI. Survival index. SR. Standard risk. TCK. Total cell kill. 6-TG. 6-thioguanine. Topo II. Topoisomerase II. WBC. White blood cell count. VP-16. Etoposide. 7.

(19) Background. Introduction Acute lymphoblastic leukemia (ALL) in children is a rare disease, but devastating when it occurs. It offers a unique treatment challenge to the pediatrician. Not long ago this diagnosis was tantamount to a death sentence, but today the disease is curable in the majority of patients treated with effective chemotherapy, in combination with intensive supportive care. As survival has improved, it has become increasingly possible to define both inherent and treatment-related adverse prognostic features. The treatment has advanced from use of single agents to combination therapy, and today it is complex and multidisciplinary. The prognosis has improved considerably in the last two decades (1). In the Nordic countries the overall event-free survival has risen from 57 to 75%. Children without unfavorable features have benefited most strikingly from the intensification of therapy (event-free survival is today 80-85%). As they constitute almost 70% of all patients, the improvement has made a great impact on the overall result. However, in children with high-risk ALL, the progress has only been modest. The relapse rate has of course decreased in parallel with the improving results, but the prognosis after relapse has not improved during the above mentioned period. Only about 25-30% of children who relapse will reach and remain in a second remission. Cytotoxic treatment in childhood ALL is divided into three phases: induction, consolidation, and maintenance. The therapy is cyclic and dependent on the outcome at the point of remission evaluation. In current protocols, presenting. 8.

(20) features such as age, white blood cell count (WBC), immunophenotype and cytogenetics are used to stratify patients, and a few are to receive extra intensive therapy including CNS radiation and/or stem-cell transplantation in their first remission. Theoretically, cancer chemotherapy would be curative if the early treatment were sufficiently intensive to eradicate all malignant cells. Failure to cure leukemia is most likely due to failure to eradicate the leukemic clones. The reasons for this are of course complex and multiple. Most prognostic factors in childhood acute lymphoblastic leukemia are informative regarding groups of patients, whereas new approaches are needed to predict the efficacy of chemotherapy in the individual patient. Future research offers perspectives to optimize the oncolytic effect and to reduce dose-limiting side effects. Overcoming resistance to chemotherapy is the aim of today of pediatric oncologists. In the studies presented in this thesis emphasis has been placed on two aspects of drug resistance mechanisms: first the conditions before the drug reaches its target (systemic drug exposure) and secondly those after the drug has interacted with its target (cellular drug resistance). The cellular drug resistance has been studied by in vitro chemosensitivity testing (papers I-II), and the systemic drug exposure by a pharmacokinetic approach (papers IV-V). Another possibility of overcoming therapy failure has been examined in a study in which a new cancer agent, CHS 828, has been tested (paper III).. Acute Lymphoblastic Leukemia ALL develops from lymphoid precursor cells in the bone marrow, sometimes from even earlier pluripotent precursor cells with mixed myeloid-lymphoid features. Differentiation and maturation are blocked and the malignant clone proliferates and expands. The normal bone marrow cells will gradually be. 9.

(21) outnumbered and the leukemic blast cells will eventually disseminate into the peripheral blood (2). Other sites can also be invaded, such as lymph nodes, the central nervous system, testis, liver, spleen, skin, kidneys, and retina. The presenting features of ALL are a result of the depressed hematopoiesis due to the overcrowded bone marrow. Fatigue and pallor due to anemia, bleeding and petechiae from thrombocytopenia, and infections from granulocytopenia are all common clinical signs. ALL is characterized by a rapid clonal expansion of poorly differentiated cells, and without treatment the disease will usually lead to death within a few months. Short history Leukemia (literally “white blood”), has been recognized as a distinct disease since 1845, when Virchow in Germany and Bennet and Craigie in Scotland described the disease in separate reports (3, 4). Virchow described two different forms of leukemia based on morphological differences namely a splenic and a lymphatic form. Neumann, in 1878, was the first to describe the splenic form as myelogenous and he also suggested a relationship to disturbances in the bone marrow. Paul Ehrlich´s introduction of staining methods in 1891 allowed differentiation of leukocytes and identification of leukemia cell type (5). Progress in the description of leukemia has continued to parallel the development of new technologies, such as special staining, immunophenotyping, chromosomal analysis and molecular genotyping. A number of the leukemia subgroups are now described. The subclassification of childhood leukemia is of great importance for the choice of treatment and the prognosis. The first therapeutic attempt was made in 1948 when Farber et al introduced the folic acid antagonist aminopterin. Remission was achieved in some patients, but the survival time was less than one year (6). It was not until the late 1960s, when it was discovered that acute myeloblastic and acute lymphoblastic leukemia responded differently to prednisolone and. 10.

(22) methotrexate, that a classification into different treatment groups was initiated. It was also in that era that Pinkel et al introduced a new therapeutic approach, which made it possible to cure the disease and not merely delay relapse (7). In the 1970s, Borella and Sen demonstrated that some leukemic lymphoblasts had a thymic origin (8). Monoclonal antibodies to leukocyte antigens were developed. Today leukemia is further classified, as acute or chronic, lymphoblastic or myeloblastic. Immunophenotyping of leukemia cells with monoclonal antibodies separates the lymphoid lineage into early and late Bprecursor, mature B-cell, and T-cell. Genotypic classification by chromosomal analysis, fluorescent in situ hybridization, DNA probing or polymerase chain reaction (PCR) techniques allows molecular definition of subgroups of leukemia. Epidemiology Leukemia accounts for one third of all pediatric cancer cases and is the second cause of death in children after traffic accidents in the western world. ALL is the single most common diagnosis in pediatric oncology. Half to two-thirds of all ALL cases occur in children. ALL comprises 75-80% of childhood acute leukemias, while 20% are acute myelocytic leukemia (AML) and the remaining cases are chronic myelocytic leukemia (CML approximately 2%) and myelodysplastic syndrome (MDS) (9). Each year 175-200 children are diagnosed with ALL in the five Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). The incidence in the Nordic countries is 3.9 per 100 000 children (<15 years of age) per year (10). ALL can occur at any age of childhood, but is most common between the ages of 2 and 3 years, with 50% of the patients <5 years of age (11, 12). Children diagnosed with ALL at <1 year of age are classified as having infant ALL and generally receive treatment according to special protocols, because of the unique biological characteristics of the disease in this age group.. 11.

(23) Several genetic syndromes have been associated with an increased risk of childhood leukemia (9). Children with constitutional trisomy 21 have a significantly higher incidence of ALL and AML (13). Other genetic syndromes associated with both childhood ALL and childhood AML include Bloom syndrome, neurofibromatosis, Schwachman syndrome and ataxia-telangiectasia (9, 14, 15). Geographical differences in the incidence, age distribution and subtypes of childhood leukemia are found among countries in different parts of the world (16-18). The reported incidence rates are highest in Costa Rica, Australia, United States (among white children) and Germany. The rates are intermediate in most European countries, and lowest in India and among black children in the United States (19, 20). Classification Accurate diagnosis is essential and the subdivision of ALL patients into groups with different prognostic features is of great importance. An important subclassification of ALL divides malignant cells into groups according to the immunological phenotype. ALL can be classified into T- or Bcell lineage on the basis of the expression or lack of particular antigens. These lineages can be further subclassified according to the maturation stage. Acute leukemia without any lineage-specific markers and mixed-lineage, atypical antigen expression are also recognized (21, 22). The immunophenotype of the leukemic cells has implications for the prognosis of the patient and thereby also for the choice of treatment.. Risk factors related to the prognosis Clinical and cell-biological prognostic factors are used today to stratify children with ALL into different therapy groups (Table 1).. 12.

(24) In patients with pre-B ALL, age and WBC at onset of disease are the strongest predictors of the outcome. It is well established that the size of the tumor burden and the curability of ALL are related (23-25). Table 1. Clinical and cell-biological features used as prognostic factors in childhood ALL.. ♦Age. Good prognostic features 1-9 years of age. ♦White blood cell count. <50 x 109/l. Unfavorable prognostic features <1 or >10 years of age >50 x 109/l. ♦Sex. Female. Male. ♦Testis involvement. No. Yes. ♦CNS involvement. No. Yes. ♦Immunophenotype. B-precursor ALL. T-ALL. Hyperdiploid >50 chromosomes per cell t(12;21)(p13;q22). Other ploidy levels. ♦Karyotype Number of chromosomes Structural rearrangements ♦Drug resistance in vivo response to corticosteroids in vitro resistance ∗ ♦Morphological remission at end of induction ♦Minimal residual disease at end of induction. 11q23 translocation t(9;22)(q34;q11) t(1;19)(q23;p13). Good response. Poor response. Relatively sensitive profile <5% leukemic cells. Resistant profile. <104 leukemic cells. >102 leukemic cells. >5% leukemic cells. ∗ Experimental. To facilitate comparison of treatment results in B-cell precursor ALL, the National Cancer Institute in 1996 adopted a uniform risk classification based on age and WBC (26). Patients aged between 1 and 9 years and with WBC <50 x. 13.

(25) 109/l were considered low-risk ALL, while all others were classified as high-risk. Around 2/3 of the patients will be considered to have low-risk ALL with an estimated survival of about 80%. The prognostic impact of age and WBC can be explained partly by their association with specific genetic abnormalities. Another known risk factor is sex. Boys have a slightly more adverse outcome, but this becomes apparent only in very large materials (27). A possible reason for this may be differences in drug metabolism, as a result of which girls receive more intensive treatment (28, 29). The drug sensitivity of leukemic cells is considered to be an important prognostic factor for the outcome of treatment. In principle, it can be assessed by the clearance rate of leukemic cells from the blood or bone marrow during early induction of remission (30-32) or by in vitro testing at onset of the disease (3335). In the Nordic countries early response to combination therapy and achievement of morphological remission is evaluated on treatment days 15 and 29 after the start of induction therapy. The Berlin-Frankfurt-Münster (BFM) group has extensively documented the prognostic value of early response to corticosteroids as assessed by the disappearance of lymphoblasts from peripheral blood (36). Results from in vitro cytotoxicity assays have so far only been applied in experimental settings. In an ongoing study run by the German CoALL group, this treatment stratification approach is now being tested (37). Cytogenetic analyses of childhood ALL have been found to be of both biological and clinical importance, and banded karyotyping is today part of the routine diagnostic procedure. Chromosomal aberrations in ALL can be divided into two groups, which can be present simultaneously: Abnormalities in the number of chromosomes (ploidy) and structural aberrations, such as translocations, deletions, partial duplications, inversions, and dendritic chromosomes. During the last three decades, numerous primary and secondary,. 14.

(26) chromosomal abnormalities, both numerical and structural, have been described in ALL and also shown to correlate to clinical parameters and outcome (38, 39). Two of the most constant findings are that hypodiploidy (<45 chromosomes per cell) and presence of Philadelphia chromosome, t(9;22)(q34;q11), are risk factors for treatment failure. Massive hyperdiploidy in ALL (>51 chromosomes per cell), a typical childhood feature in this disease (1-10 years of age), has shown different degrees of impact on the prognosis with differences in treatment intensity, although in most studies it has been shown to be an indicator of a lower risk for treatment failure. Hyperdiploidy is associated with a low WBC and a marked propensity for spontaneous apoptosis in vitro, this perhaps being one of many reasons for the favorable prognosis after cytotoxic treatment (40, 41). The genetic abnormality t(12;21)(p13;q22) is observed in approximately 1/4 of all patients with preB-cell ALL in the Nordic countries (42, 43). Several groups have found that this abnormality carries a favorable prognosis (44-46), although with longer follow-up its final impact on the outcome is still under lively debate. Forestier et al has reported cytogenetic findings in a populationbased series of 787 Nordic ALL patients (47). Among these patients 2.2 % were Ph chromosome positive, 3.7% showed 11q23 translocations and 1.3% had t(1;19)(q23;p13), all of which abnormalities are usually found to carry a more or less dismal outcome. Children with trisomy 21 syndrome (Down´s syndrome, DS) have a high incidence of leukemia, especially the acute megakaryocytic type. When treated with chemotherapy, however their cure rate is twice that of the cohort of children with acute myeloblastic leukemia without DS (48, 49). The extra chromosome in DS not only introduces increased vulnerability but also implies better curability in AML patients with DS. In ALL, on the other hand, the clinical outcome tends to be slightly worse for DS than for non-DS children (50). The reason for this difference is not known.. 15.

(27) Today flow cytometry and sensitive PCR techniques allow us to study the blood and bone marrow for signs of minimal residual disease (MRD) below the level of detection by light microscope. Depending on the method used, it is possible to detect one malignant cell in 103-105 cells. The clinical importance of. Table 2. Criteria for risk grouping of children with non-B ALL according to the NOPHO ALL-92 protocol. __________________________________________________________________________________. Risk group Age Criteria _____________________________________________________________________ Standard (SR). Age 2-<10 y. WBC <10 x 109/l No high-risk criteria. Intermediate (IR). Age 2-<10 y or Age 1-<2 y or ≥ 10 y. WBC 10-<50 x 109/l. High (HR). Age ≥ 1 y. Very High. WBC <50 x 109/l No high-risk criteria and at least one of the following: WBC ≥50 x109/l Mediastinal mass CNS or testicular involvement t(9;22), 22q-, t(4;11) Slow response: >25% leukemic cells day 15 or >5% leukemic cells day 29. Age ≥ 5 y. and at least one of the following criteria: Lymphomatous features CNS involvement T-cell leukemia with other HR criteria ___________________________________________________________________________. detecting MRD early during treatment is still being investigated. The risk grouping used in the NOPHO ALL-92 protocol is shown in Table 2. Although some factors are the same, the risk classification and treatment strategies still differ considerably between international groups (1, 10, 51).. 16.

(28) Many other factors may contribute to the outcome in the individual patient. Compliance to long and complex treatment, iatrogenic factors, treatment-related toxicity, infections, and other complications may be responsible. The treatment will last for more than 2 years of a child’s upbringing, putting a great strain physically, psychologically and socially on the child and the family.. Chemotherapy in childhood ALL Most cancer drugs have a narrow therapeutic index, making their clinical use a delicate matter with a need for exact dosage and careful monitoring of the patient. The choice, schedule and dose of the drugs depend on the patient’s risk group. Current ALL protocols typically include remission induction followed by intensification or consolidation therapy to eliminate residual leukemia, and maintenance treatment to ensure continuation of remission. Treatment aiming at eradication of CNS leukemia is also a common feature. Standard remissioninduction. therapy. for. ALL. consists. of. vincristine,. prednisolone. or. dexamethasone and asparaginase, and in most protocols also an anthracycline (doxorubicin or daunorubicin). Substituting dexamethasone for prednisolone has been a recent trend, with the aim of improving the outcome by better penetration into the central nervous system (52). The BFM group pioneered the use of the consolidation therapy as early as in the 1970s. The children were given sustained intensification immediately after induction, including at least some agents to which they had not previously been exposed. The results clearly showed the value of post-induction intensification (53, 54). Continuing or maintenance therapy has repeatedly been shown to improve the outcome (11, 55). The total treatment period is between 2 and 2 ½ years. The cytotoxic drugs used for the treatment of childhood ALL are virtually the same throughout the world.. 17.

(29) The NOPHO organization The Nordic Society of Paediatric Haematology and Oncology was established in 1981 and the first research project within the organization was to start prospective registration of all children with ALL diagnosed in the five Nordic countries. A registry was created for co-ordination of diagnostic procedures, treatment, and follow-up, giving the Nordic region unique opportunities to establish reliable data and to perform epidemiological analyses. Since 1995, joint reports from registries and from various working groups concerned with special therapeutic topics have been presented annually (43, 51). The studies presented in this thesis would not have been possible without the collaboration within the NOPHO framework. Development of a Nordic treatment protocol The first protocol for treatment of childhood ALL within NOPHO was established in 1981. It included only the patients classified as having standardrisk (SR) ALL. The next step towards a common protocol was taken 1986, when a new SR protocol and two different intermediate-risk (IR) protocols were recommended. High-risk (HR) patients were still treated according to regional or national protocols. The final step towards uniform treatment of patients in all risk groups was taken in 1992. The NOPHO ALL-92 protocol This protocol was based on stratification into three major risk groups (see Table 2). Introduction of high systemic doses of methotrexate, with high-dose cytosine arabinoside (cytarabine,Ara-C) in the HR group made it possible to reduce the number of children receiving cranial irradiation to about 10%. The results have been well comparable to the best results from other international study groups (1, 11).. 18.

(30) Table 3. Treatment protocols NOPHO ALL-92. Treatment phase/drug. Single or daily dose. Days given. Comments. 60 mg/m2/d 2 mg/m2 (max 2 mg) 40 mg/m2 (24 hrs) 2 30 000 U/m daily 10-12 mg (age adj.). 1-36/45 1, 8, 15, 22, 29, 36 1, 22, 36 36-45 1, 8, 15, 29. HR/VHR-prephase. All Risk groups Induction (w 0-7) Prednisolone (po) Vincristine (iv) Doxorubicin (iv) L-Asparaginase (iv/im) Methotrexate (it). HR/VHR-1,8,22,36. Standard Risk (SR) Consolidation-SR (w 8-12) Methotrexate (iv) 5 g/m2 (24 hrs) Methotrexate (it) 10-12 mg (age adj.) Maintenance (w 14 -) 6-MP (po) 75 mg/m2/d Methotrexate (po) 20 mg/m2/w Prednisolone (po) 60 mg/m2/dx7 Vincristine (iv) 2 mg/m2 (max 2 mg) 2 Methotrexate (iv) 5 g/m (24 hrs) Methotrexate (it) 10-12 mg (age adj.). 1, 15, 29 1, 15, 29 1 until 2,5 yrs from diagnosis 1 until 2,5 yrs from diagnosis 1, 57, 113, 169, 225 1, 57, 113, 169, 225 29, 85, 151, 207, 263 1, 29, 85, 151, 207, 263. Intermediate Risk (IR) Early intensification (w 8-14) 60 mg/m2/d 6-MP (po) Cyclophosphamide (iv) 1000 mg/m2 Cytarabine (iv) 75 mg/m2/d Methotrexate (it) 10-12 mg (age adj.) Consolidation-IR (w 15-22) 6-MP (po) 25 mg/m2/d Methotrexate (iv) 5 g/m2 (24 hrs) Methotrexate (it) 10-12 mg (age adj.) Late intensification (w 24-30) 2 Dexamethasone (po) 10 mg/m /d 2 Vincristine (iv) 2 mg/m (max 2 mg) Daunorubicine (iv) 30 mg/m2 (24 hrs) L-Asparaginase (iv/im) 30 000 IE/m2 6-Thioguanine (po) 60 mg/m2 Cyclophosphamide (iv) 1000 mg/m2 2 Cytarabine (iv) 75 mg/m /d Methotrexate (it) 10-12 mg Maintenance (w 32 - ) 6-MP (po) 75 mg/m2/d Methotrexate (po) 20 mg/m2/w Methotrexate (iv) 5 g/m2 (24 hrs) Prednisolone (po) 60 mg/m2/dx7 2 mg/m2 (max 2 mg) Vincristine (iv). 1-14, 29-42 1, 29 3-6, 10-13, 31-34, 38-41 1, 29 1-56 8, 22, 36, 50 8, 22, 36, 50 1-22/29 1, 8, 15, 22 1, 8, 15, 22 1, 8, 15, 22 29-42 29 31-34, 38-41 29, 38 1 until 2 yrs from diagnosis 1 until 2 yrs from diagnosis 1, 57, 113, 169, 225 29, 85, 141, 197, 253 29, 85, 141, 197, 253. iv=intravenous, im=intramuscular, it= intrathecal, po= orally, 6-MP = 6-mercaptopurine. 19.

(31) Table 3. Treatment protocols NOPHO ALL-92 (continued). Treatment phase/drug. Single or daily dose. Days given. Comments. High Risk (HR). Induction (w 0-7) Early intensification (w 8-14) Consolidation-1 HR (w 16-26) Methotrexate (iv) 8 g/m2 (24 hrs) Cytarabine (iv) 2 g/m2x2dailyx3days Methotrexate (it) 10-12 mg (age adj.) Interim maintenance (w 28-35) Prednisolone (po) 40 mg/m2/d Vincristine (iv) 2 mg/m2 6-MP (po) 75 mg/m2/d Methotrexate (po) 20 mg/m2/w Late Intensification (w 36-42) Consolidation-2 HR (w 44-62) Methotrexate (iv) 8 g/m2 Cytarabine (iv) 2 g/m2x2dailyx3days Methotrexate (it) 10-12 mg (age adj.) Prednisolone (po) 60 mg/m2/d Vincristine (iv) 2 mg/m2 (max 2 mg) 6-MP (po) 75 mg/m2/d Methotrexate (po) 20 mg/m2/w Maintenance (w 64-2 yrs) 6-MP (po) 75 mg/m2/d Methotrexate (po) 20 mg/m2/w Prednisolone (po) 60 mg/m2/dx7 Vincristine (iv) 2 mg/m2 (max 2 mg) Methotrexate (it) 10-12 mg (age adj.) Very High Risk (VHR ) week: 0-42 CNS therapy (w 43-46) Cranial RT 18 Gy 6-MP (po) 50-75 mg/m2/d Methotrexate (it) 12 mg Maintenance-LSA2L2 (w 47-94) 6-Thioguanine (po) 300 mg/m2/d Methotrexate (it) 12 mg Cyclophosphamide (iv) 600 mg/m2 Hydroxy-urea (po) 2400 mg/m2/d Daunorubicin (iv) 30 mg/m2 Methotrexate (po) 10 mg/m2/d Carmustine (iv) 30 mg/m2 Cytarabine (iv) 150 mg/m2/d Vincristine (iv) 2 mg/m2 (max 2 mg) Maintenance (w 96-) 6-MP (po) 75 mg/m2/d Methotrexate (po) 20 mg/m2/w. 20. See Induction: w 0-7 See IR: w 8-14 1, 43 22, 65 1, 43. Total dose: 2 x 12 g/m2. 1-8, 29-35 1,29 1-57 1-50 See IR: w 24-30 1, 99 15, 113 1, 99 43-49, 71-78 43, 71 43-98 43-91. Total dose: 2 x 12 g/m2. 1 until 2 yrs from diagnosis 1 until 2 yrs from diagnosis 1, 57, 113, 169, 225 1, 57, 113, 169, 225 1, 57, 113, 169, 225 Same as HR 1-22 1-29 1,8,15 6 cycles x d 1-56 1-4 1 5 15-18 19 29-32 33 43-46 47. Cy 5-6:Pred (d 15-22) Cy 5-6:Vincristine (iv). 1 until 2 yrs from diagnosis 1 until 2 yrs from diagnosis.

(32) iv=intravenous, im=intramuscular, it=intrathecal, po=orally, 6-MP=6-mercaptopurine. Table 3 gives an overview of the NOPHO ALL-92 protocol, which was used in all children with ALL in the Nordic countries during the study period. A new protocol has now been established, NOPHO-ALL 2000, effective from January 2002 in all Nordic countries.. Treatment failure Achievement of the ultimate goal of curing a child with ALL is dependent on eradication of the malignant cells and on the child´s possibility of staying in complete remission and surviving the treatment. The clinical response to a cytotoxic drug is probably determined by many factors. The following are believed to play a major role (56-58). ♦Cellular drug resistance, primary (intrinsic) or acquired (secondary) ♦Pharmacokinetics of the drug ♦Regrowth capacity of remaining malignant cells Cellular drug resistance Cellular drug resistance is probably a major limitation to the success of chemotherapy in childhood ALL. The mechanisms of leukemic cell resistance can be classified into four major groups: altered plasma membrane transport, decreased cellular drug activation, increased drug inactivation, and altered cellular drug targets. Many anticancer drugs have structural similarities to natural metabolites and share a common carrier system for cellular uptake (59). Melphalan, for example, has been reported to be accumulated inside cells by active membrane transport systems normally used by amino acids, and alterations of the affinity of these carriers for melphalan have been found to cause decreased uptake of the drug,. 21.

(33) leading to resistance (60). The most well studied form of resistance involving altered membrane transport is multidrug resistance (MDR). MDR is a phenomena in which a cellular phenotype which develops resistance in vitro to single cytotoxic drug, such as a vinca alkaloid, an anthracycline or a podophyllotoxin,. also. becomes. resistant. to. other. structurally. and/or. mechanistically dissimilar drugs (61, 62). Many forms of MDR result from expression of ATP-dependent efflux pumps with broad substrate specificity. These pumps belong to a family of ATP-binding cassette (ABC) transporters that share sequence and structural homology, and so far 48 human ABC genes have been identified. Among the most studied and best known ABC transporter are Pglycoprotein (PGP), multidrugresistance associated protein (MRP) and breast cancer related protein (BCRP) (63). Some drugs, e.g. Ara-C, require activation after entering the cell. Enzyme inactivation or a decrease in enzyme activity may lead to resistance. Ara-C is phosphorylated by deoxycytidine kinase (dck). Ara-C resistant cell lines have been reported to show a significantly lower level of the dck enzyme (64). The purine analogue, 6-thioguanine (6-TG) requires conversion to the active nucleotide 6-thioguanylate to cause damage to DNA, and studies have indicated that resistance may be caused by reduction of this conversion ability (59, 65). The most thoroughly studied cellular mechanism for inactivation of cytotoxic drugs is a change in the activity of glutathione S-transferase (GST) and in the level of glutathione (GSH) (59, 66). Alteration of the GSH/GST system has been correlated with development of resistance to a variety of drugs, including alkylating agents, doxorubicin and cisplatin (66, 67). Alteration of the subcellular distribution of the drug by “trapping” in intracellular organelles is another way in which the cell can inactivate the drug. This resistance mechanism has been described for anthracyclines (68), and is probably a component of same MDR phenotypes.. 22.

(34) Resistance to a drug may result from changes in the level of expression or the biochemical properties of a molecule and lead to increased production of a target enzyme (69). This is a type of acquired drug resistance. Increased enzyme expression may also be a result of increased translation or transcription or may be secondary to gene amplification (59). A cellular target for many important cytotoxic drugs is topoisomerase II (Topo II). Topoisomerases are nuclear enzymes that modulate the topological structure of DNA by making transient DNA breaks (70). Topo II inhibitors are thought to act by stabilization of enzyme-DNA binding. Many important anti-leukemic drugs inhibit Topo II, like anthracyclines and epipodophyllotoxins. The resulting cellular phenotype expresses atypical MDR, to distinguish it from classical MDR. It is characterized by cross-resistance among different Topo II inhibitors and also by restained sensitivity to vinca alkaloids (59, 61). Programmed cell death, apoptosis, plays a critical role in chemotherapyinduced cell death. Changes in signal pathways important for the aptotic process may thus affect the sensitivity to cytotoxic drugs. For example increased expression of the bcl2 protein or inactivation of the p53 protein can prevent the induction of apoptosis in the tumor cell (63, 71, 72). In vitro cytotoxicity assays Cell culture assays for measurement of cytotoxic drug resistance in leukemia were used as early as in the beginning of the last century. Pappenheimer added already 1917 trypan blue to fresh thymic lymphocytes that had been exposed to toxic agents (73). Trypan blue stained the dead cells blue, while the living cells remained clear. In 1954, Black and Spear compared the clinical outcome with the in vitro response of patient tumor cells in a succinate dehydrogenase-dependent dye reduction assay system (74). Various improvements in the assay have been made since then, leading to the evolution of today’s chemoresistance assays.. 23.

(35) Today, numerous in vitro assays are used for chemoresistance testing (75, 76). They can be divided into two main groups: cell proliferation assays and total cell kill (TCK) assays. The cell proliferation assays can be categorized into shortterm assays and clonogenic assays. The clonogenic assays were standard in early studies, but as ALL cells have a very low clonogenic capacity these assays are poorly suitable in childhood leukemia research (77-79). TCK type assays are believed to measure cell death in the whole population of tumor cells. Examples of this form of assay are the colorimetric tetrazolium saltbased assays, such as the methyl-thiazol-tetrazolium (MTT) assay, and the differential staining cytotoxicity (DiSC) assay (80). The MTT assay is based on the ability of viable cells to convert a tetrazolium salt into an insoluble formazan precipitate, which is easily quantified by measurement of its absorbency (78). The DiSC assay is based on the ability of viable cells to exclude vital dyes after exposure in vitro for 3-4 days. The results are evaluated by visual inspection of the cell preparation in the microscope. This assay can discriminate between drug effects on malignant and non-malignant cells in a mixed cell population. This is in contrast to the MTT assay, which therefore requires relatively homogeneous leukemic samples. Clonogenic assays and the DiSC assay have shown a good predictive capacity, but are labor-intensive and time-consuming (80-82). The DiSC assay also has the disadvantage of being subjective. The TCK assay used in the present studies was the fluorometric microculture cytotoxicity assay (FMCA). The FMCA was developed in Uppsala in the late 1980s, and is based on the uptake of fluorescein diacetate (FDA) by viable cells and its conversion to fluorescent fluorescein after exposure to cytotoxic drugs in primary cultures (83). The end point for recognizing cell death is loss of cell membrane integrity. The generated fluorescence is linearly related to the number of living cells. The assay cannot differentiate between apoptotic and necrotic cell death.. 24.

(36) Both the MTT assay and FMCA have yielded estimates of drug cytotoxicity that have correlated with those obtained with the DiSC assay (78, 84, 85). In childhood leukemias, the MTT assay has been used in most studies (34). The feasibility and validity of FMCA for in vitro drug resistance testing of tumor cells from cell lines and patients have been documented (86, 87), including the use of samples from patients with ALL (84). Differences between the MTT assay and FMCA The MTT assay and FMCA are both total cell kill assays with great similarities, but they differ in that FMCA is based on detection of membrane disintegration, which is a late and irreversible step in the events leading to cell death (80), while the MTT assay depends on peroxidase activity to reduce MTT to colored formazan crystals. Theoretically the enzyme activity necessary for MTT reduction may be influenced by temporary, and reversible energy depletion (88). The incubation time with the cytotoxic drugs differs between the two assays. In FMCA the malignant cells are exposed to the drugs for 72 hours, whereas the duration of exposure is 96 hours in the MTT assay. Choice of drug concentrations The drug concentration lethal to 50% or 70% of the ALL cells (IC50 or IC70) has usually been determined as the measure of resistance in studies using the MTT assay. Another approach, used in most FMCA studies, is to compare cell survival in different samples at fixed drug concentrations, chosen to give a wide scatter of survival, denoted survival index (SI) or % surviving cells. The selection of concentrations for FMCA is an important issue: for each drug, concentration-response curves spanning a large range of concentrations are generated for a number of samples (89), and the cut-off concentration is then defined as the concentration producing the largest scatter of test results.. 25.

(37) The results of a drug resistance assay can only be interpreted in relation to the results for other samples. The classification of a sample that is resistant or sensitive is always done after comparison with the results for other samples, preferably from patients with the same disease. Studies of in vitro resistance in childhood ALL A number of studies have shown significant correlations between results of in vitro drug resistance testing and the clinical outcome in childhood ALL, most of them retrospective and/or with small numbers of patients and/or only a few drugs tested (84, 90-92). Kaspers et al, in 1997, published data on in vitro resistance to 12 drugs in 152 children with newly diagnosed ALL, studied by the MTT method (34). Hongo et al reported on tests by the same method in samples from 209 children with ALL, using a panel of 16 drugs (35, 93). Both groups reported that a combination of data for prednisolone, asparaginase and vincristine created a drug-resistance profile with prognostic significance for probability of disease free survival (p-DFS). No prospective studies using in vitro cellular testing for choice of treatment in newly diagnosed ALL have yet been reported. This approach is being used, however, in the ongoing study COALL-06-97 (Germany) (37). Pharmacokinetics and clinical response Dose intensity has been shown to be of great importance for the clinical outcome in acute leukemia (57, 94, 95). The treatment intensity is dependent not only on the dose, but also on inter-individual variations in pharmacokinetic parameters such as the absorption, distribution and elimination of the drug. Most antileukemic drugs studied have displayed a large inter-individual variation in systemic exposure. The variation is often of even greater magnitude in children than in adults because of age-related maturation of physiological processes responsible for drug disposition. The systemic clearance of many anticancer. 26.

(38) drugs differs by a factor of 3 to 10 among patients (56). If a drug shows a steep dose-response curve, a small change in AUC may greatly affect the outcome. The variability in drug disposition may have important influence on the efficacy of the drug. In a study by Evans et al, conventional vs. individualized chemotherapy was tested in children with ALL (96). It was shown that adjustment of the dose of methotrexate by considering the patient´s ability to clear the drug improved the outcome in B-lineage ALL. The problem of suboptimal dosing of methotrexate might be overcome by selecting a dose that produces adequate concentrations even in patients with rapid drug clearance. In the Nordic ALL protocol this is achieved by giving very high doses of methotrexate, 5-8 grams/m2 body surface area depending on the risk group. However, this strategy is often impossible to use, since most cytotoxic drugs have a narrow therapeutic span. Two important drugs used in the induction phase of the NOPHO ALL-92 protocol are doxorubicin and vincristine. These two drugs have been used for more than 30 years in the treatment of ALL and many other pediatric tumors, but the pharmacokinetic knowledge is still sparse, especially of vincristine. Anthracyclines Anthracycline drugs are antibiotics that are active against a wide variety of malignant tumors. Daunorubicin was discovered to have anti-leukemic activity as early as in 1966 (97), and several analogues have subsequently been developed, including doxorubicin, idarubicin and epirubicin. Doxorubicin is widely used today in the treatment of malignant disease, including leukemias and many different solid tumors (98, 99). The cytotoxic effect is explained by several mechanisms of action, both inside the cell and at the cell surface. The drug hampers the transcription, replication and repair of DNA. It is believed to intercalate with the DNA and also to interact with DNA-associated enzymes such as DNA helices and DNA Topo II, thereby. 27.

(39) inhibiting replication and transcription (100, 101). Anthracyclines most likely enter the cell by passive diffusion of the electrically neutral molecule. No single mechanism can explain anthracycline resistance. In a study of the resistance mechanism, Den Boer et al of found that lung resistance protein (LRP) and the intracellular level of daunorubicin correlated with daunorubicin resistance in childhood leukemia (102). As with most chemotherapeutic drugs, common side effects of anthracyclines are, bone marrow depression, alopecia, stomatitis, nausea, and vomiting. In addition, and a limitation to its usage, there is a risk of congestive heart failure. This side effect is related to the cumulative dose (103, 104). Cardiotoxicity is more likely to occur in children than in adults, but can be reduced by slowing the infusion, or by concomitant use of cardioprotectants. After intravenous infusion, anthracyclines are rapidly cleared from the plasma and accumulate in the tissue. It has been shown in adults that the pharmacokinetics of doxorubicin are highly variable, with an almost tenfold inter-patient variation of the area under the plasma concentration-time curve (AUC), despite standardization of the dose, based on body surface area (98, 105107). Very few data on the pharmacokinetics of doxorubicin have been reported for infants <1 year of age. On the basis of body surface area, McLeod et al (108) found a trend toward a lower rate of systemic clearance. The reason for the high variability of this drug pharmacokinetics is largely unknown. Previous publications on the pharmacokinetics of doxorubicin in children are few (108-110). In a recent paper, data on doxorubicin and 4’-epidoxorubicin were presented for 31 children with ALL (111). All these reports are from single-center studies with relatively small numbers of patients, and/or with patients treated for a variety of malignancies. No distinct age dependency or gender. differences. have. been. observed.. 28. Dose-normalized. steady-state.

(40) concentrations of doxorubicin tended to be higher in patients with a low body mass index, but the difference was not statistically significant (111) Vincristine Vincristine is an alkaloid derived from the leaves of the periwinkle plant Catharanthus roseus (see painting on front cover). Linnaeus formerly called this plant Vinca rosea. It originates from Madagascar and its medicinal properties have been known since the seventeenth century. Vincristine exerts its effect on microtubules by binding to tubulin or microtubules (112, 113), and it inhibits polymerization of mitotic spindle microtubules. The mechanism of mitotic inhibition is well known. New insights into the cytotoxic effects of vincristine are emerging, including induction of apoptosis (114). Vincristine has been used in clinical practice since the early 1960s (115). Today it is a key drug in the combination chemotherapy for childhood and adult ALL. Vincristine is also part of the front-line treatment for lymphomas, Wilms’ tumor, rhabdomyosarcoma, Ewing’s sarcoma, neuroblastoma and many brain tumors in children. Vincristine is used more frequently and more effectively in pediatric patients than in adults. This may be due to the higher level of sensitivity of pediatric tumors to vincristine. and/or to better tolerance of children to. relatively higher doses of vincristine (116). Vincristine resistance is related to the expression of the MDR phenotype. Resistance after drug exposure can be primary or secondary (117). The side effects of vincristine differ from those of most other antineoplastic agents. Myelosuppression is generally mild and reversible, and the dose-limiting side effect is neurotoxicity, which causes a peripheral, symmetrical, mixed sensory-motor and autonomic polyneuropathy (for reviews see (118-121)). In children the first symptoms are often abdominal pain, constipation, or even paralytic ileus, occurring within days after administration. Depression or loss of. 29.

(41) deep tendon reflexes follows after a week or two. Other neurological side effects are ptosis, paresthesias and muscle cramps, increasing numbness, and weakness (119). Despite extensive clinical application of vincristine over the past thirty to forty years, uncertainty about the optimal dosing still persists. The standard dose in adults is 1.5 mg/m2 (max 2.0 mg) and in children 1.5-2.0 mg/m2 (max 2.0 mg), administered up to once weekly. However, the practice of limiting the single dose to 2.0 mg appears to be based on medical tradition and the fear of neurotoxicity, rather than on reliable pharmacodynamic and pharmacokinetic data. These dosing recommendations have also recently been challenged (122). Up to a decade ago, there were no specific and sensitive analytical methods for measuring vincristine concentrations in biological fluids. Instead, non-specific methods were used, such as radioimmunoassays and measurement of tritiumlabeled vincristine, which were unable to separate the parent drug from active and inactive metabolites (123-125). The introduction in 1985 of a specific high performance liquid chromatographic (HPLC) assay that is sufficiently sensitive for clinical use in children, made appropriate studies of vincristine possible (126). This method is highly specific for vincristine and when more recently it was refined, to a low detection limit of 0.3 ng/ml, it became possible to perform pharmacokinetic studies. It has since been found that both the intra- and inter-patient variability of pharmacokinetic variables are large in pediatric cancer patients, and that the tumor type, concomitant medication and liver function might be of importance (127-131). None of these studies showed any clear correlation between age and pharmacokinetic parameters in the children studied. However, these studies included only few adolescents.. 30.

(42) Regrowth capacity of malignant cells Regrowth of malignant cells as an explanation for treatment failure was studied in AML by Preisler et al who concluded that it was a major cause of treatment failure in AML patients with a poor prognosis (132). Generally speaking, treatment will fail even if the leukemic cells are pharmacodynamically sensitive to the drug and enough systemic exposure is achieved if the cells regrow faster than they can be killed. One strategy to overcome this problem is to reduce leukemic regrowth between courses (63). Treatment failure due to regrowth is probably also relevant in ALL (133). The extent of leukemic cell regrowth between treatment courses is most likely determined by several factors such as the number of cells surviving chemotherapy, percentage of cells in cycle, proliferation potential, cell cycle time, ability to differentiate, and other undefined factors (58). Regrowth has been measured by repeated bone marrow samples and blast cell quantification (63). Hence, there is a need for less laborious laboratory methods to estimate regrowth. In this context it is worth noting that high levels of the oncogene c-myc and low levels of c-fms have been associated with short remission duration (134).. Development of new anticancer drugs In the search for ways to circumvent drug resistance, either acquired or inherited in the cell, development of new drugs may be another line of action. Large scale screening of compounds for cytotoxic activity has been carried out at the National Cancer Institute (NCI) since 1955. Other compounds have been specifically designed to interfere with known biochemical pathways in the cell or to be analogues of drugs with known activity (135). In the development of a new drug, preclinical studies have to be performed to characterize the primary pharmacodynamics of an agent prior to the first clinical trial. In previous studies it was shown by the use of FMCA that the diagnosis-. 31.

(43) specific activity of cytotoxic drugs measured in tumor cells from patients correlated well with the clinical disease-specific activity pattern, pointing to the usefulness of in vitro tests in drug development, especially in the selection of diagnoses for clinical phase II studies (136, 137). In vitro studies can also indicate the therapeutic effect of combined agents, as well as providing information on synergistic or antagonistic interactions (138). CHS 828, a pyridyl cyanoguanidine, was accidentally found to have antitumor properties in the early 1990s. It was in the search for new antihypertensive agents acting on potassium channels that Leo Pharmaceutical Products synthesized a number of cyanoguanidine analogues. When these compounds were run through a routine screening test in tumor bearing rats the anticancer activity was discovered (139). In a study by Hjarnaa et al, the activity pattern obtained in cell lines, of CHS 828 was shown to have low correlation with the activity patterns of known anticancer agents, and no sensitivity to any known mechanism of multidrug resistance was observed (140). The cell lines represented the mechanism of resistance, including those associated with expression of P-glycoprotein, altered Topo II, increased levels of reduced glutathione (GSH) and tubulin defects. The cytotoxicity was in the same range, however as that of standard cytotoxic compounds for the ten cell lines being tested. Activity was observed in the nM and µM range. The mechanism of action of CHS 828 does not appear to fall into one of the common categories and it does not demonstrate cross-resistance to other standard cancer chemotherapeutic drugs (141-143). Extensive research has been conducted and is still in progress to elucidate the exact mechanism and mode of cell death induced by CHS 828 (144, 145). The first clinical trial was initiated in 1998 and the first phase II study is now proceeding in adult patients with chronic lymphoblstic leukemia (CLL).. 32.

(44) Aims of the investigation. The aims of the present investigations were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL).. The more specific aims were ♦. to evaluate the clinical utility of in vitro chemosensitivity testing at onset of ALL by correlating the results for ten tested drugs with the clinical outcome.. ♦. to compare the in vitro chemosensitivity in ALL cells from children with Down’s syndrome (DS) with that in samples from non-DS children.. ♦. to investigate the in vitro sensitivity of leukemia cells from children with ALL and AML to a novel cyanoguanidine CHS 828.. ♦. to study the pharmacokinetics of two cytotoxic drugs used in induction treatment of ALL, namely doxorubicin and vincristine.. 33.

(45) Material and Methods. Patients Study I (paper I) comprised children (aged 1 to 18 years) with non-B ALL diagnosed between 1992 and 2000. Swedish centers for pediatric oncology provided us with samples from 347 patients. After 1995, a number of centers in the other Nordic countries also took part in the study: 86 patients were included from Norway, 35 from Denmark, 32 from Finland and 6 from Iceland. Figure 1 shows the inclusion of patients over time, illustrating how the study started as a single center study (Uppsala) then became a Swedish national study and finally developed into a Nordic study.. 60. Number of patients. 50. 40. Country of origin. 30. Sweden 20. Denmark Norway. 10 Finland 0. Iceland 92. 93. 94. 95. 96. 97. 98. 99. 2000. Year of Diagnosis. Figure 1. Year of diagnosis and number of patients included from each of the Nordic countries in the study of in vitro resistance in ALL (paper I).. 34.

(46) Parallel to this study, the pharmacokinetic investigations of doxorubicin and vincristine were performed. All Swedish centers for pediatric oncology participated in these latter studies, which ran between February 1995 and November 1999 (papers IV and V). In the doxorubicin study (paper IV) 107 children of ages 1.3–17.3 years were investigated at day one of induction therapy. Five infants, 3-9 months old, were also included. The vincristine study (paper V) comprised 98 children aged 1.3–17.3 years. Some of the patients were included in more than one of the studies (Figure 2).. ALL in vitro study (paper I) n=370. Vincristine study (paper V) n=98. 66. 83. 44. Doxorubicin study (paper IV) n=107. 65. Figure 2. Number of patients participating in the studies presented in paper I, IV and V. In both papers I and V were 66 patients, in I and IV were 83, in paper IV and V were 65 patients included. 44 patients took part in all three studies.. During 1995-1999 about 300 Swedish children >1 year of age were diagnosed with ALL, i.e., the patients of studies in paper IV and V represent 1/3 of the whole patient population. Reasons for not including children in the pharmacokinetic studies were mostly practical difficulties, such as lack of extravenous access or lack of staff to handle research samples. Ten children with Down’s syndrome and acute leukemia participated in study II (paper II), 5 with ALL and 5 with AML. The 5 children with ALL were also included in study I (paper I). In the CHS 828 study (paper III), samples from 42 children with ALL and 23 with AML were analyzed between June 1996 and November 2000. The patients. 35.

(47) with newly diagnosed ALL were also included in the in vitro ALL study (paper I). All patients with newly diagnosed ALL included in the studies received treatment according to the NOPHO ALL-92 protocol (10). Clinical follow-up was achieved by annual reports to the Nordic childhood leukemia registry (Childhood Cancer Research Unit, Karolinska Institute, Stockholm, Sweden) from all centers involved, and the last date of follow-up was December 31, 2001.. Samples Studies of in vitro cellular resistance (papers I-III) Bone marrow aspirates and blood samples were collected in heparinized glass test tubes, kept at room temperature and sent by mail or international express delivery companies. As a rule they reached the laboratory for processing within 24-36 h. The majority of the samples were analyzed freshly, but for practical reasons some were cryopreserved in culture medium containing 10% dimethyl sulfoxide (DMSO) and 50% fetal calf serum (FCS) by initial freezing for 24 h at -70oC followed by storage in liquid nitrogen. The cells were later thawed and analyzed. Previous studies showed that cryopreservation did not affect in the vitro sensitivity to standard drugs (84, 146, 147). Leukemic cells were prepared by density-gradient centrifugation with 1.077g/ml Ficoll-Isopaque (Pharmacia, Uppsala, Sweden). The viability was determined by the trypan-blue exclusion test. Median viability was 95% and the FMCA was performed only when viability was >70%. An independent hematologist estimated the proportion of leukemic cells on May-GrüsnwaldGiemsa stained cytocentrifugate preparations, using light microscopy. The median proportion of lymphoblasts after separation was 90% and FMCA was performed only when this proportion was >70%.. 36.

(48) Pharmacokinetic studies (papers IV-V) Patients were studied on the first day of induction treatment. Blood was drawn from a peripheral vein (never through the catheter used for drug administration) and collected in a heparinized glass test tube. The tube containing the sample was immediately put into ice water and centrifuged within 60 min. Plasma was then removed and stored at -70o C until analyzed. In the doxorubicin study (paper IV), plasma samples were drawn 23 h after the start of a 24-h infusion of doxorubicin 40 mg/m2. In the vincristine study (paper V), blood was collected before and 10, 30, 360, and 1380 min after a one-minute vincristine injection. Patient data (body weight, height, actual dose administered), and the exact times of starting and terminating doxorubicin and vincristine administration, and of blood sampling, were noted. The serum concentrations of creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin, which were determined before the administration of the drugs, were also recorded.. Drugs and Reagents Fluorescein diacetate (Sigma, St. Louis, MO) was dissolved in DMSO (Sigma) and kept frozen (-20oC) as a stock solution (10 mg/ml) protected from light. The culture medium RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 50 µg/ml streptomycin and 60 µg/ml penicillin was used throughout. Cytotoxic drugs in clinical use were obtained from commercial sources and tested at the concentrations shown in Table 4. The active metabolite of cyclophosphamide, 4-hydroperoxy-cyclophosphamide, was used. Experimental plates were prepared with 20 µl/well of drug solution at 10 x the desired final concentration with a programmable pipetting robot (Pro/Pette; Perkin Elmer,. 37.

(49) Norwalk, CT). Each drug and concentration was tested in triplicate wells. The plates were stored at -70 oC pending further use. Table 4. Drugs, origin, solvents and concentrations used in the in vitro drug resistance testing.. Drug Dexamethasone (Dexa). Origin MSD. Etoposide (VP-16). Bristol-Myers PBS Squibb Pharmacia PBS. 5 µg/ml. 1 µg/ml. Prednisolone (Pred). Bristol-Myers SW Squibb Organon PBS. Cytarabine (Ara-C). Sigma. PBS. 0.5 µg/ml. Vincristine (Vcr). Lilly. PBS. 0.5 µg/ml. 6-thioguanine (6-TG). Sigma. NaOH/SW. 10 µg/ml. Asparaginase (Asp). Ipsen. SW+PBS. 10 U/ml. PBS. 2 µg/ml. Doxorubicin (Dox) Amsacrine (Amsa). 4-hydroperoxyDuke cyclophosphamide (4-HC) University. Solvent PBS. Concentration 1.4 µg/ml. 0.5 µg/ml. 50 µg/ml. SW=sterile water, PBS=phosphate buffered saline, NaOH=sodium hydroxide. Drugs were used at empirically derived cut-off concentrations, chosen to produce a large scatter of SI values among the samples (84). As the amount of cells varied and sometimes was a limiting factor, all drugs could not be tested in all samples. Experimental drug CHS 828, used in study III (paper III) was obtained from Leo Pharmaceutical Products, Copenhagen, Denmark. It was dissolved in 100% DMSO and was kept at -20oC as a stock solution of 10 mM, and further dilutions were made in 30% DMSO (1 mM CHS 828) and phosphate buffered saline (PBS). CHS 828 was. 38.

(50) tested at six tenfold dilutions starting from 1 µM. For comparison and in combination experiments, four standard drugs from commercial sources were chosen, representing different mechanistic classes: the alkylating agent melphalan, the antimetabolite Ara-C, the anthracycline doxorubicin and the Topo II inhibitor etoposide.. Methods Fluorometric Microculture Cytotoxicity Assay (FMCA) The FMCA is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate (FDA) to fluorescein by cells with intact plasma membranes and has been described in detail previously (83, 86, 136, 148). One hundred thousand leukemic cells in 180 µl culture medium were seeded per well of 96-well microtiter plates prepared in advance with the different drugs to be tested. The culture plates were incubated at 37oC in a humidified atmosphere containing 95% air and 5% CO2 for 72 h continuous drug exposure. The plates were then centrifuged (200 g, 5 min) and the medium was removed by automatic pipetting. After one wash with PBS, 200 µl/well of PBS containing FDA (10 g/ml) was added. Subsequently, the plates were incubated for 1 h at 37oC reading the fluorescence was read by a scanning fluorometer (Fluoroscan 2; Labsystems OY, Helsinki, Finland). Drugs were tested in triplicates. Six wells without drugs served as controls and six wells containing culture medium only served as blanks. Quality criteria for a technically successful assay included a proportion of leukemic cells of >70% in control wells after 72 h incubation, a fluorescence signal in control wells of >5 times the mean blank value, and a mean CV in control wells of <30%. The results are presented as survival index (SI), defined as fluorescence in test wells/fluorescence in control wells (blank values subtracted) x 100 or as percent surviving cells. Thus,. 39.

(51) a low numerical value indicates high sensitivity to the cytotoxic effect of the drug. Measurement of drug concentrations Doxorubicin and doxorubicinol were assayed by an analytical procedure based on reversed-phase liquid chromatography with fluorometric detection, as described in detail previously (149). All the doxorubicin samples (study IV, paper IV) were processed in the Karolinska Pharmacy, Stockholm. The detection limit of doxorubicin and doxorubicinol was 0.2 ng/ml. All plasma concentrations reported are mean values of duplicate analyses. The precision of the analytical procedure (coefficient of variation) was 2.2% (intraday) and 3.4% (inter-day). Vincristine concentrations were measured by high perfomance liquid chromatography (HPLC) with electrochemical detection. The vincristine samples were analyzed in the Department of Pharmacy, University Hospital, Groningen, The Netherlands, for detail concerning the procedure see reference (150). The sensitivity of the HPLC method for vincristine was 0.48 µg/l according to good laboratory practice (GLP) rules, with a coefficient of variation of 6.2% at the lower limit of detection (150). Pharmacokinetic methodology Plasma concentrations of doxorubicin and doxorubicinol were measured 23 hours after the start of a 24-hour constant rate infusion. Since the dose actually administered sometimes differed from the target dose, the observed doxorubicin and doxorubicinol concentrations were normalized for a dose of 40 mg/m2 by the formula: Observed concentration x target dose/actual dose. Plasma clearance (Cl) was calculated according to the formula Cl = D/T/Css, where D/T is the actual dose rate and Css is the observed steady-state concentration of the drug. It has been shown that about 93% of the steady-state concentration of doxorubicin is. 40.

(52) reached after 23 h of infusion (111), and this was compensated for in the calculations of plasma clearance by dividing the observed 23 h doxorubicin concentration by 0.93. The maximum plasma concentration reached at the end of a constant infusion can be used as a substitute for measuring AUC (111, 151). For vincristine a limited sampling strategy and Bayesian analysis were used to fit a two-compartment model to the concentration-time data, using the parameter estimation module of the ADAPT II pharmacokinetic software package (152). Priors for the Bayesian analysis were obtained from previous studies (128, 129). Estimated primary pharmacokinetic parameters included volume of central compartment (Vc), first-order rate constant for overall elimination of drug from central compartment (Ke), and first-order rate constants for drug transport between central and peripheral compartment (Kcp and Kpc). From the primary parameters the following secondary parameters were derived: distribution and elimination half-lives (t½ α and t½ β), total body clearance (Cl), and volume of distribution at steady-state (Vdss). Area under the concentration-time curve (AUC) was calculated using the formula Cl = Dose/AUC.. Statistical methods Differences in distribution of variables were tested with the Mann-Whitney U test, Kruskal-Wallis H, test or the chi-square test. The Spearman correlation coefficient was used to examine relationships between continuous variables. Curves illustrating the probability of disease free survival (p-DFS) were calculated by the Kaplan-Meier method. The log rank test was used to compare survival curves. Statistical comparisons of outcome were conducted by simple and multiple Cox proportional hazard regression analysis. The SPSS 10.0 software package (SPSS Inc. Chicago, IL) was used for the calculations. All analyses were two-tailed and the level of statistical significance was set at p<0.05.. 41.

(53) Results and Discussion. In vitro resistance in ALL (paper I) Cellular resistance was tested successfully by FMCA in 370 out of 506 samples reaching the laboratory. Reasons for failure were: total number of cells too small (n=39), too low percentage (<70%) of lymphoblasts before test (n=8), too low percentage (<70%) of lymphoblasts in control wells after 72 h of incubation (n=17), too low signal in controls compared to blanks, i.e. signal-tonoise <5 (n=48), and a coefficient of variation in controls >30% (n=24). Table 5 shows characteristics of the successfully tested cases, compared to all children treated according the NOPHO ALL-92 protocol in the Nordic countries during the study period. Table 5. Characteristics of 370 children with newly diagnosed ALL, successfully tested for in vitro cellular drug resistance, compared to not tested or not successfully tested patients (n=1219).. No of pat Age, years median (range) 1-9/>=10 years ratio Male/female ratio M/F WBC x 109/l median <10 10-50 >50. Tested. Not tested or All children not successfully tested. 370. 1219. 1589. 4.8(1.1-17.4) 291/79 3.7/1 203/167 1.2/1. 4.5 (1.0-17.4) 1006/213 4.7/1 657/562 1.2/1. 4.6 (1.0-17.4) 1298/291 4.5/1 859/730 1.2/1. (0.5-768) 9.3 (0.3-990) (52%) 632 (52%) (30%) 372 (30%) (18%) 215 (18%). 9.4 (0.3-990) 825 (52%) 484 (30%) 280 (18%). 9.5 193 112 65. 42. p values. 0.063 0.092 0.72 0.37 0.99.

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