DRUG RESISTANCE IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

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					RESISTENSI OBAT PADA ANAK-ANAK DENGAN LEKEMIA LIMFOBLAS AKUT ( DRUG RESISTANCE IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA ) IDG. Ugrasena Divisi Hematologi Lab/SMF Ilmu Kesehatan Anak FK. Unair/RSUD Dr. Soetomo

Abstrak

Keberhasilan pengobatan dengan kemoterapi tergantung pada 2 hal yaitu: sel kanker harus dapat dijangkau oleh konsentrasi obat yang cukup ( faktor farmakokinetik ) dan sel kanker harus cukup sensitif terhadap kemoterapi yang digunakan ( faktor resistensi obat ). Ada beberapa mekanisme resistensi: obat tidak ditranfer kedalam sel melalui membrane sel, obat mengalami detoksifikasi intraseluler, atau obat tidak mencapai DNA. Beberapa

resistensi obat bisa terjadi oleh karena bekerja sebagai pompa toksin sehingga konsentrasi obat intra seluler menurun. Saai ini resistensi sel leukemi terhadapa beberapa sitostatika dapat ditentukan secara in vitro. Derajat resistensi sangat berguna menentukan prognosis. Sebagian besar faktor prognosis yang sudah dikenal selama ini dapat diterangkan lewat pemeriksaan resistensi obat ini ( pemeriksaan MTT ). Sebagai contoh yaitu penderita LLA hiperdiploid yang dikenal prognosanya baik, hal ini disebabkan karena sel ini sensitif dengan obat 6-MP demikian pula obat anti metabolit lainnya. Usia lebih dari 10 tahun atau kurang dari 1 tahun dikenal prognosisnya buruk karena resisten terhadap steroid.

Abstract Success of chemotherapy depends upon two factors: the malignant cells must be reached by sufficient drug concentration ( pharmacokinetic factors ) and the cells must be sufficiently sensitive to drug used ( drug resistance factors ). The mechanisms of drug resistance are manifolds: cells may not transport drugs over the cell membrane, drug may be detoxified intracellularly, or may be segregated in vesicles, or may not reached the nuclear DNA. Some of the drug resistance act as a pump for toxin, thus lowering the concentration of drug inside the cell.

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The resistance of leukemic cells to various cytostatic drugs can be measured in vitro. The degree of resistance is very powerful prognostic factors. Most other prognostic factors are actually explained by drug resistance. For examples, hyperdiploid ALL has very good prognosis because the cells are very sensitive to 6MP and other anti metabolites, and also worse prognosis of children over 10 or under 1.5 year of age result from resistance to steroid.

INTRODUCTION. Acute Lymphoblastic Leukemia ( ALL ) is the most frequently occurring type of cancer in children constituting 25% - 30 % of all childhood malignancies1. The results of chemotherapy in childhood ALL have markedly improved during the past decades2,3. The remission rate of patients treated with chemotherapy is more than 95%, and the long term disease- free survival rate about 75 – 80%. In spite of the excellent results 25 – 30 % of the patients will experience a relapse with poor prognosis. This treatment failure may be explained by unfavourable pharmacokinetics, regrowth potential of the residual leukemic cells and by cellular drug resistance3,4. Although the prognosis of children with newly diagnosed ALL has improved greatly with the use of current combination chemotherapy, in less privileged countries, the results are less good, although progress is being made. In Child health Department Dr. Soetomo Hospital, ALL patients who are being treated by WK (Wijaya Kusuma)-ALL 2000 protocol for ALL , 87 % achieved complete remission, 30 % relapsed during the treatment, 40% were drop out and 28.5 % died. Because treatment with cytotoxic drugs is not always curative in children with ALL and sometimes accompanied by severe side effects, there is still need for improvement of the results of chemotherapy. Since cellular drug resistance is an important determinant of clinical outcome after chemotherapy, drug resistance studies have to be done to further improve the therapy. In our country there is very limited information or study on ALL including drug resistance in relation to the successfulness of treatment.

Prognostic factors The identification of prognostic factors has become an essential element in the design and analysis of current therapeutic protocols in ALL. Prognostic factors used for risk group
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classification are partly based on clinical and cell biological patient features, such as sex, age, leukemic cell burden at diagnosis, immunophenotype, and chromosomal abnormalities1,2,5. The biologic explanation of the prognostic significance of these features is unclear, but is often assumed to be related to cellular drug resistance5. The initial white blood cell count (WBC) is the single most important conventional predictor of clinical outcome for remission induction, duration of remission, and long-term survival. There is a linear relationship between the initial leukocyte counts and outcome in children with ALL. Children with high leukocyte count tend to have a worse prognosis. Although there is no sharp dividing line, patients with an initial leukocyte count more than 50.000 cells / mm3 blood are prognosis
6,7,8,9,21

universally recognized as having

a particularly poor

.

The best continuous complete remission (CCR) was achieved by the 30 % of patients who had an initial WBC of less than 5000 cells/mm3 blood, but the worst survival experience was exhibited by those with initial WBC from 100.000 – 200.000 cells/mm3 blood5. However in another study as reported by Wald et al, the incidence of early death between hyperleukocytic and non hyperleukocytic patient with ALL was not statistically different75. Mediastinal masses have a strong association with the WBC5,8,9, and its presence is not an independent prognostic factor as it is associated with other adverse presenting features including male sex and high leucocytes count76. Males have a less favourable prognosis than females, demonstrated in several childhood ALL studies8,9,10. Age is a well established prognostic factor in childhood ALL8. Children younger than 18 months have the worst prognosis5,11. Leukemia in these children is characterized by a high tumor load, but also by a high incidence of the prognostically unfavorable pro B phenotype11. Patients older than 10 years have a poor prognosis and children with an intermediate age have the best prognosis5,6,12. Immunophenotype is one of the prognostic factors in children with ALL. B-ALL cases had the worst prognosis although this has improved now while patients with B-cell precursor ALL have the most favourable prognosis. Among the patient with B–cell precursor ALL, those with the early pre B–cell phenotype have a more favourable prognosis compared with patients with pro B–cell phenotype, who have a relatively poor prognosis13. T cell ALL is usually associated with male sex , high WBC count, mediastinal mass, and central nervous system (CNS) infiltration, and formerly had a poor outcome in children2,14. Shuster et al15, reported that childhood T-cell ALL with WBC > 50.000/mm3 blood had a poorer outcome
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compared with the group with a WBC < 50.000/mm3. Crist et al, in a larger number of patients found that the high risk features formerly ascribed to pre-B-cell ALL are closely associated with the t (1;19): i.e., increased leukocyte count16. Parts of these findings are still controversial because in one study the immunophenotype is related to other clinical and biological features like WBC and organomegaly. Patients with > 50 chromosomes per leukemic cell account for about one fourth of childhood ALL cases and have proved to have more durable responses to treatment compared with non hyperdiploid cases17,18,19,77. Especially, DNA hyperploidy is associated with favourable prognosis in B- lineage ALL19. The frequency of hyperdiploidy , is highest in cases of CD 10+ early pre-B ALL whereas pseudodiploidy is present in the majority of patients with CD10 – early pre-B ALL and in virtually all cases of B-cell ALL19. Some authors reported that the persistence of circulating leukemic blast cells ( > 1.000/µL blood ) or otherwise measured slow early response after 1 week of single or multiagent remission induction chemotherapy was an important early predictor of worse prognosis20,21,22,23. A number of clinical characteristics and cell biological features as described above has prognostic significance in childhood ALL, and are summarised in the table. These features are especially related to the occurrences of relapses. Relapsed ALL has a poor prognosis, with a cure rate of less than 30%.

Table 1. Factors with prognostic significance in childhood ALL.
_____________________________________________________________________ Category better prognosis poor prognosis _____________________________________________________________________ WBC at diagnosis Age Sex Immunophenotype Ploidy Cytogenetics In vitro drug resistance Circulating blasts at day 8 BM blast at day 8 or 15 Complete remission after induction chemotherapy achieved not achieved < 50.000 cells/mm3 18 Mo-10 years female common, pre-B cell hyperploidy t ( 12;21 ) sensitive <1000/mm3 blood < 25% >50. 000 cells/mm3 <18 Mo or > 10 year male pro-B, T cell non hyperploidy t ( 4;11), t (9;22 ) resistance >1000/mm3 blood > 25%

____________________________________________________________________
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As mentioned previously, the prognostic significance of these factors may partly be caused by cellular drug resistance, which can be measured in vitro. There are many different assays to asses the chemosensitivity of leukemia cells. Clonogenic assays in acute myeloblastic leukemia (AML) patients have successfully been used to predict initial response to chemotherapy24,25,26. A good correlation between in vivo and in vitro results has been demonstrated in a limited number of patients, using the differential staining cytotoxicity (DiSC) assay and it was possible to distinguish between responders and non-responders to initial treatment in ALL27. The significant correlations between in vitro resistance of the leukemic cells measured with the MTT ( 3-4[ 4,5-dimethylthiazol-2-yl ]-2,5-diphenyl tetrazolium bromide) assay and the clinical response showed that these cells play an important role in the clinical situation28,29,30,31,48,51. The MTT assay has also been used to detect differences between relapsed ALL cells ( significantly more resistant to several drugs ) and ALL cells obtained at initial diagnosis30,32,33. ALL subgroups as defined by age and immunophenotype and DNA ploidy revealed specific in vitro drug resistance profiles34,35. As mentioned above clinical pharmacokinetic factors are also related to treatment failure, for example poor compliance, serum protein binding of drug, drug clearance and antibodies to asparaginase4. Theoretically, the use of pharmacokinetic principles to individualize drug dosage so as to achieve targeted blood concentrations should minimize both the likelihood of undertreatment and probability of drug toxicities. In 1984, Evans et al studied the relapse rate of 108 children with ALL who received maintenance therapy with intermediate-dose methotrexate (MTX) 1000 mg/m2 or standard therapy with cranial radiation and oral MTX/6-mercaptopurine (6-MP)36. They found the clearance of MTX to be an independent variable for predicting a relapse that occurred in 22 of the children. Pinkel et al in 1971, demonstrated a relationship between prescribed dosage and efficacy of ALL maintenance therapy, with children given half dosages of 6-MP, vincristine (VCR), cyclophosphamide, and MTX having significantly worse clinical responses37. One of the other causes of treatment failure is the regrowth potential of minimal residual leukemic cells. If the courses of treatment do not kill all of the malignant cells present, then the behaviour of the cells which survived treatment will play a role in determining the actual effect of the treatment38. A number of studies in childhood ALL has now shown that detection of minimal residual disease (MRD) in bone marrow during or at the end of induction, or during the first few months of treatment, provides information of strong

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prognostic value39,40,41,42,49. Control of the immune system may play a role over these residual leukemic cells. The persistence of small numbers of leukemic cells well into the clinical course is an important feature of childhood ALL, but not a focus in this study.

Cellular drug resistance. There are several well established indicators of prognosis including sex, age at initial presentation, presenting peripheral blood white cell count and immunophenotype. Since recently the resistance of leukemic cells to various cytostatic drugs can be measured in vitro and the degree of resistance is a very powerful prognostic factor. Most other prognostic factors are actually explained by drug resistance28,29,30,31,33,35,43,44,45,46,47,48,50. To the best of our knowledge, no information is available on drug resistance in childhood leukemia in Indonesia; therefore we will study that in this thesis.

Drug resistance assay. Since thirty to fourty years various attempts have been made to develop in

vitro chemosensitivity assays with respect to a relevance for clinical practice. Clonogenic assays have long been considered to be the golden standard for in-vitro chemosensitivity testing. They, however have a number of drawbacks. Firstly, the number of patient samples of which the leukemic cells will be clonogenic in vitro is limited, especially in ALL samples. Secondly, the drug effect is measured on a small proportion of cells, i.e. those cells that can be induced to proliferation in vitro, and not on cells that are non dividing or resting at that time. Practical disadvantages are that these assays are very time consuming and laborious. These drawbacks therefore make clonogenic assays less suitable for its use in ALL patients and are the reason that in vitro drug resistance studies in ALL were not performed until 10-15 years ago50,51. Only with the recent introduction of short – term, non clonogenic assays, an increasing number of authors has been studying cellular drug resistance in childhood leukemia51. Examples of these assays are the colorimetric tetrazolium based assays such as the MTT and INT assay4,52, DiSC assay53, and the fluorometric microculture cytotoxicity assay (FMCA)54. The DiSC assay relies on the intactness of the cell membrane in living cells as opposed to dead cells after several days of incubation with drugs. Relatively low numbers of cells are needed to test a range of drugs in different concentrations. A main advantage of this assay is that it can discriminate between malignant and non-malignant cells, in contrast to the MTT assay and FMCA. However, the DiSC assay has the disadvantages of being
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subjective, laborious, and time consuming, which makes it less suited for large-scale patient studies51. These short–term assays, especially the MTT assay and FMCA, are rapid and objective because they need only 2 – 4 days of culture and are semi-automated. MTT assay and FMCA need relatively homogenous leukemic cell
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populations., which is almost

always the case in newly diagnosed childhood ALL . One should use the MTT assay only for samples with at least 70% malignant cells determined cytomorphologically at the start of the assay. In 1983 Mossman described the MTT assay which relies on the demonstration of living cells by their ability to convert a soluble tetrazolium salt into an insoluble formazan. The formazan precipitate is purple and can be dissolved and the extinction read on a 96-well microplate reader (ELISA reader)51. The MTT assay is a rapid and objective cell culture drug resistance assay, and appeared to be an independent and strong predictor of both short-tem response to therapy and long-term survival. The MTT assay is very suitable for assessing chemosensitivity in acute leukemia31,44. This assay will be used to study leukemic sample in this thesis. All of these assays have the disadvantage that MTX cytotoxicity can not be determined55,56, No dose dependent cytotoxic effect of MTX upon ALL cells derived from patients, has been described in the literature using short-term total cell kill assay. This can be explained by the fact that ALL cells are difficult to culture. In four days culture the mean control cell survival of ALL cells without adding any drug is about 65 %55. This means that a relatively large amount of ALL cells is spontaneously dying. Nucleosides and bases such a thymidine and hypoxanthine and folate, that are released by these cells probably salvage the remaining cells from MTX cytotoxicity56.

Cellular drug resistance in relation to clinical and cell biological features Clinical and cell biological features are well known as conventional prognostic factors in childhood ALL, but how this is caused is not well understood. Many experts in this field have studied the correlation between these factors and in vitro drug resistance determined by the MTT assay. Age is well established prognostic factor in childhood ALL. A recent report shows that infant ALL cells are in vitro highly resistant to glucocorticoid (GC) and L-asparaginase (L-asp). This is in agreement with the poor outcome of infants with ALL and with the fact that in vitro resistance to these drugs was associated with poor outcome in ALL30,31,48. Pieters et al in 1993, reported that ALL cells from children <18 month were more resistant to prednisolone (PRD), and daunorubicin (DNR) and that ALL cells from children >
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10 years were more resistant to PRD than those in the intermediate age group (18 month - 10 years)50. The same author in 1998 found that infants < 1,5 years were significantly more resistance to PRD and L-asp but significantly more sensitive to cytarabine (ara-C) compared to the intermediate age group34. In contrast Hongo et al, did not find a relation between age and cellular drug resistance to the four drug : dexamethasone (DXM), PRD, L-asp and VCR30. There was generally no significant difference between female and male in drug resistance35,48. However samples of female patients were significant resistant to ara-C and mercaptopurine (6-MP) than the male samples35. WBC at initial diagnosis was no significant relation with drug resistance30,35. However patients with a WBC of < 10 x 109 l-1 were more resistant to ara-C than patients with a WBC 10-50 x 109 l-1 (35). There were no significant differences in drug resistance between FAB classification L1 cases and FAB classification L2 cases, nor within the total childhood ALL group, nor within the group of c-ALL and pre B –ALL patients35. In general, pro B and T ALL samples were more drug resistant than the group of c ALL and pre B ALL samples35. Rots et al, reported that T-ALL samples were 3.4 fold more resistant to MTX compared with c/pre-B ALL samples56. Pro B ALL samples were significantly more resistant to L-asp, and 6thioguanine (6-TG) but T ALL samples were significantly more resistant to PRD, DXM, VCR, L-asp, DNR than the group of cALL and pre B ALL samples35. Pieters et al, reported that which anthracyclinespro B ALL cells were significantly more resistant to GC, L-asp, 6 TG, 6 MP compared to c ALL /pre B-ALL but more sensitive to ara-C34. In contrast Hongo et al, found no relation between immunophenotype and DXM, PRD, L-asp, VCR (DPAV) sensitivity30. DNA hyperploidy is strongly and independently associated with a good prognosis in childhood ALL. The relation between DNA hyperploidy and drug resistance as reported by Kaspers et al, showed that hyperploid ALL cells were more sensitive to mercaptopurine, thioguanine, cytarabine and to L-asp than were non hyperdiploid ALL cells, but these two ploidy group did not differ significantly in resistance to PRD, DXM, VCR, DNR35. The t (12;21) translocation resulting in TEL/AML1 gene fusion was reported by Ramakers-Van Woerden et al, showed that to be associated with a high in vitro sensitivity, to L-asp but not to other drugs57.

Cellular drug resistance in relation to disease status and clinical outcome.

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In vitro drug resistance in relation to disease it self and clinical outcome has been studied by several authors. Using the in situ thymidilate synthase inhibition (TSI) assay, from newly diagnosed precursor B-ALL patients58. Previously

Rots et al, reported that relapsed precursor B-ALL cells were threefold more resistant to MTX than cells obtained

Klumper et al, reported that compared with cells obtained at initial diagnosis, relapsed ALL cells were more resistant to GC, L-asp, anthracyclines and to thiopurines, whereas no resistance was observed for vinca alkaloid, cytarabine, epipodophyllotoxins33. The MTT assay provides drug resistance profiles which accurately predict the clinical outcome after chemotherapy in childhood ALL31,78. The first study of drug resistance that related initial chemosensitivity to long –term clinical outcome reported by Pieters at al, showed children with poor response to 1 week monotherapy with PRD have poor clinical outcome than those with good response to PRD48. CCR was significantly lower in patients with resistant cells than in those with sensitive cells for thioguanine, DNR and PRD. Regarding the correlation between PRD in vitro and in vivo, Kaspers et al reported that the clinically poor PRD responders to a 1 week PRD monotherapy were nearly 100-fold more resistance to PRD in vitro than clinically good responders and that increased in vitro resistance to PRD was associated with a worse long-term clinical outcome45. In 1999 Hongo et al, reported that the three drug ( PRD, L-asp, VCR ) resistant group correlated with both short-term and long-term treatment failure43. Zwaan et al, reported that AML cells were significantly more resistant than ALL cells to the following drug: GC, VCR, L-asp and anthracycline79.

Mechanisms of action and resistance Cellular drug resistance is generally recognized as an important determinant of the clinical outcome after chemotherapy. Even if optimal tumor cell exposure is achieved, a number of cellular factors may be responsible for drug resistance, which is also focus of this study. During recent years several mechanisms have been described, which will be discussed below. In general decreased transport of drug into the cell, defective intracellular

metabolism of the drug to its active compound, increased drug inactivation, enhanced cellular repair mechanisms, altered target molecules, and multidrug resistance (MDR) by increased drug efflux represent know mechanisms of chemo resistance80. In addition, resistance caused by altered cell death regulators involved in drug induced apoptosis play an important role2.
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Because many drugs are used in the treatment of childhood leukemia and many factors may be responsible for resistance to each drug, it is unlikely that one single mechanism is responsible for clinical resistance to the complete treatment. The advantage of cell culture drug resistance assays as described above is that they measure the endpoint of different mechanisms of resistance, i.e, cell kill. However, to understand and modulate or circumvent resistance, studies on resistance mechanisms in patient cells are obligatory81.

Glucocorticoids The use of glucocorticoids ( GC ) in childhood leukemia dates from the late 1940`s, early 1950`s. Initially cortisone and adrenocorticotropic hormone were used, later PRD, and occasionally DXM. It became clear that this type of with response rates higher than any other single drug. The mechanism of action of GC is that GC diffuses through the cell membrane, bind to their intracellular receptor to form a complex that is activated and then translocates into the nucleus. Here it binds to DNA, finally leading to DNA fragmentation and DNA repair associated with NAD and ATP depletion resulting in cell death. The events leading to apoptosis after binding of the GC-receptor complex to DNA are essentially unknown44. However, the activity depends on the type of leukemia, ALL being much more sensitive than ANLL, and the disease status, GC being more effective in untreated patient than in patients who present with relapsed leukemia44. Despite the effectiveness of GC in ALL, these drugs are not curative when used as single agent. Since resistance to GC occurs in clinical practice, both at initial diagnosis and relapse, it is important to study resistance to this type of drugs. Several explanations can be given why the prognosis is worse in case of GC resistance: 1, lack of antileukemic effect of potentially effective GC, 2. when GC are ineffective, no favourable drug interactions are likely to occur either, 3. associated with a more general drug resistance44. Most studies focussed on the glucocorticoid receptor (GR) as a mechanism of resistance. Werner et al, described a polymorphism resulting in a superactive form of GR, corresponding to a mutation: C638G82. Patients with a low number of GR appeared resistant to a short-term course of GC and GR number greater than 8,000 sites/leukemic cell is a favourable prognostic marker46,47. Hala et al reported about a leukemic cell line in which a glucocorticoid receptor gene defect (L753V) is associated with resistance83, while Hurley et al reported glucocorticoid resistance in families with familial glucocorticoid resistance due to a point mutation in the steroid binding domain of the glucocorticoid receptor84.
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drugs had marked cytotoxic activity,

L – Asparaginase ( L-Asp ) L – asparaginase is also always being used in ALL treatment protocols. L-

asp catalyzes the hydrolysis of L-asparagine to aspartic acid. Its cytotoxic effect is related to L- asparagine depletion of cells that require exogenous L-asparagine for their protein synthesis. It is assumed that elevated L-asparagine synthesis mediated by asparagine synthetase is related to L-asp resistance4. Dubbers at al reported a significantly lower asparagine synthetase activity in AML M5 and precursor B- ALL compared with AML non M5 and higher asparagine synthetase activity in T-ALL compared with blast of the B lineage85. This may at least partly explain the relatively increased sensitivity to L-

asparaginase in these AML and precursor B-cell ALL subgroups.

Methotrexate ( MTX ) MTX is one of the mostly frequently used cytostatic drugs, and has several unique features ; it has an antidote (folinic acid), and it is possible to determine MTX and MTX metabolites concentration. The mechanism of action of MTX: MTX is transported across the membrane mainly by the reduced folate carrier (RFC). Intracellularly, MTX will be polyglutamylated by folylpolyglutamate synthetase (FPGS) to MTX-polyglutamates (MTX –PGs). These MTXPGs can subsequently be broken down by folylpolyglutamate hydrolase (FPGH). MTX will inhibit DHFR (dihydrofolate reductase ), whereas MTX- PGs inhibit DHFR, TS (thymidilate synthase) and enzymes involved in the purine de novo synthesis I (PDNS)58,59. Defective transport as a mechanism of acquired MTX resistance has been studied in a small number of ALL cases. Two out of four children with relapsed ALL showed evidence of impaired uptake of MTX60. Whitehead et al in their study correlated the accumulation of MTX and formation of MTX-PGs with clinical outcome in childhood ALL61. Out of 35 children with non–T, non-B-cell ALL, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year EFS than those whose lymphoblasts did not. The same author also reported that high levels of MTXPGs were found in the hyperdiploid ALL samples62. Defects at the DHFR level have been reported by Matherly et al, in the patients with WBC less than 50 x 10 9/l blood. High level of DHFR was associated with poor outcome63. Ravindranath et al, in a preliminary study results suggested that the relationship between MTX sensitivity and MTHFR genotype64. Reduced activity of the MTHFR by the
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C677T variant was associated with increased risk of higher MTX toxicity among marrow transplantation patients65.

Thiopurines 6-MP is a common antimetabolite used in the oral maintenance treatment of ALL since its introduction in 194759. 6-TG is an analogue of 6-MP and the other well known purine analogue. The mechanism of action: The thiopurine drugs have no direct anti-leukemic activity; they are prodrugs which undergo extensive intestinal and hepatic metabolism after oral dosing. The initial transformations occur along three competing routes of mercaptopurine metabolism catalysed by thiopurine methyltransferase (TPMT), xanthine oxidase (XO) and hypoxanthine guanine phosphoribosyltransferase (HGPRT). 6-MP and 6-TG are analogues of respectively hypoxanthine and guanine. Both thiopurines are converted to their active nucleotide derivatives 6-thioIMP and 6-thioGMP respectively by HGPRT. 6-Thio-IMP is subsequently converted to 6-thioGMP by HGPRT. Incorporation into DNA and RNA of this nucleotide is a mechanism of toxicity of thiopurine. Beside this, 6-thioGMP and 6-thioIMP inhibit purine the novo synthesis by inhibiting phosphoribosyl pyrophosphate (PRPP) amidotransferase. They also affect the purine salvage pathway by inhibiting several enzymes of this pathway, such as TPMT. Nucleotidases catalyze the dephosphorylation of purine nucleotides like GMP, IMP and AMP to their corresponding nucleosides guanosine, inosine and adenosine respectively. Ecto-5’ nucleotidase (ecto-5’NT) is localized on the cell membrane with the enzymatic activity facing the external medium. Its function involves extracellular dephosphorylation of nucleotides to which cells are impermeable, into readily transported nucleosides4. The moleculer causes of resistance to 6-MP may include alteration in any factor of the enzyme system, which may be acquired or controlled by common genetic polymorphism in the case of TPMT. TPMT activity in leukemic blasts at diagnosis was significantly correlated with TPMT activity in erythrocytes before therapy. Thus, erythrocytes may serve as surrogate of leukemic cells for studies on mercaptopurine metabolism and TPMT activity in childhood ALL66. Coulthard et al, described the relationship between genotype and TPMT activity in the target of drug action, the leukemic cell. They demonstrated that TPMT activity in normal homozygotes was significantly higher than that in heterozygotes. The latter patients had a significantly increased risk of toxicity when treated with 6-MP67.

Anthracyclines
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The mechanisms of action of anthracyclines are not very well described. They interact with DNA and topoisomerase II which leads to DNA strand breaks but also a direct cytotoxic effect of anthracyclines by damaging the cell membrane has been suggested4. Multi drug resistance (MDR) phenotype is the most and best described mechanism of resistance of anthracyclines. The drug efflux pump P-glycoprotein (P-gp) in the cell membrane decreases the intracellular levels of this drug , but also other pumps play a role4.

Vinca alkaloids VCR is the most common vinca alkaloid used in ALL. The known working mechanism of vinca alkaloids is that they prevent spindle formation and inhibit ribosomal RNA and protein synthesis. In addition there are other mechanisms of vinca alkaloid resistance such as alteration in cell membrane and MDR. The drug efflux pump P-gp in the cell membrane decreases the intracellular levels of this drug4.

Cytosine arabinoside (Ara-C) Ara-C treatment is not applied in WK-ALL 2000 protocol, but is being used in some high risk patients in other protocols. This cytotoxic drug is transported across the cell membrane by facilitated diffusion, phosphorylated to its active metabolite ara CTP by deoxycytidine kinase (dCK) and subsequently incorporated into DNA as araCMP by DNA polymerase 4. The clinical relevance of possible mechanisms of resistance such as decreased membrane transport, decreased phosphorylation by low dCK activity, increased breakdown of araCTP by high cytidine deaminase and altered DNA polymerase, is largely unknown in childhood ALL4.

Alkylating agents Alkylating drugs are used principally for the treatment of lymphoma and pediatric solid tumors. The mechanism of resistance to alkylating agents in childhood ALL are unknown4. Glutathione (GSH) may be accelerated by glutathione S-transferase (GSTs). Significant correlation was found also between GSH levels and in vitro sensitivity to DNR, PRD and melphalan but not to mitoxantrone, VP16 and 6TG68. DNA repair is another mechanism of resistance. Recently much effort is focused on identifying novel drug targets which leads to antiCD20 and STI571. Anti-CD 20 is known as safe and shows significant clinical activity in patients with bulky relapse or refractory low grade B-cell non Hodgkin,s lymphoma69. STI571
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(formerly known as CGP57148B) is an active and relatively specific inhibitor of Bcr-Abl kinase activity and has remarkable activity as a single agent treatment for CML and Philadelphia chromosome positive ALL70,71. The possible mechanisms of resistance to the drugs discussed above are given in table 24. Table 24: Drug resistance mechanisms in childhood acute lymphoblastic leukemia
Class of drug Glucocorticoids ( GC ) Suggested mechanisms Number of GC receptors (GCR) Affinity of receptor Function of receptor Nuclear translocation of the GCR complex. DNA binding of the GC R complex GCR polymorphism (?) Asparagine synthetase Membrane transport MTX polyglutamylation,and folylpolyglutamate synthetase (FPGS) / folylpolyglutamate hydrolase (FPGH) Active efflux Intracellular normal folate pools Dihydrofolate reductase, Thymidylate synthase ( TS) Methylenetetrahydrofolate reductase( MTHFR ) (?) Nucleoside concentration,ecto-5’ nucleotidase Cyto-5’ nucleotidase and phosphatases Phosphoribosyl pyyrophosphate (PRPP) and PRPP Amidotransferase Hypoxanthine-guanine phosphoribosyl transferase Thiopurine methyltransferase (TPMT) Ara CTP formation Ara C transport Ara-C and Ara-CTP deamination DNA incorporation MDR-1/P-glycoprotein Multidrug resistance related protein ( MRP ) Lung resistance protein (LRP) Topoisomerase II BCRP (breast cancer resistance protein ) Glutathione Glutathione and glutathione S-transferases DNA repair

L-Asparaginase Methotrexate ( MTX )

Thiopurines

Cytosine arabinoside (ara-C)

Anthracylines, Vinca-alkaloids, And Epipodophyllotoxins

Alkylating agents

Modulation of resistance

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Since drug resistance has a major impact on the success of chemotherapy, it is of clinical importance to identify possibilities to modulate or circumvent each type of drug resistance. Recently, some laboratory studies on modulators have been done such as N(phosphon)-acetyl-L-aspartate (PALA) modulation of ara-C resistance72, verapamil and cyclosporin in AML relapse73, meta-iodobenzylguanidine (MIBG) and 5-aza-2-deoxycytidine (5-AZA) for GC resistance74. In general, drug resistance modulation has not yet proven to be of major clinical significance.

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