Recent advances in the treatment and understanding of childhood acute lymphoblastic leukaemia

CANCER TREATMENT REVIEWS 2003; 29: 31–44 doi:10.1016/S0305-7372(02)00106-8

ANTI-TUMOUR TREATMENT

Recent advances in the treatment and understanding of childhood acute lymphoblastic leukaemia Jeffrey E. Rubnitz and Ching-Hon Pui Department of Haematology-Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA, Department of Paediatrics, University of Tennessee Health Science Center, College of Medicine, Memphis, TN, USA Clinical trials have advanced the cure rate of childhood acute lymphoblastic leukaemia to near 80%. Treatment response, as measured by minimal residual disease, has allowed us to refine risk classification schemes and better tailor the intensity of therapy for each patient. More complete molecular analysis of leukaemia cells, pharmacodynamic and pharmacogenetic studies, and the development of targeted therapy should ultimately lead to further improvements in treatment. Pharmacogenetic studies should allow treatment refinements that will decrease the risk of complications while maintaining high cure rates. In addition, gene expression profiling may improve the genetic classification of leukaemia and identify clinically important subgroups. It may also lead to the identification of new targets for novel antileukaemic agents. c 2003 Elsevier Science Ltd. All rights reserved. s Key words: Leukaemia; childhood; minimal residual disease; cytogenetics; pharmacogenetics; therapy.

INTRODUCTION Recent clinical trials have achieved 5-year event-free (EFS) survival rates greater than 70% for patients with childhood acute lymphoblastic leukaemia (ALL) (1–4). Our current efforts to improve outcome include the development of more precise risk classification strategies based on the level of minimal residual disease (MRD), the optimization of therapy through pharmacodynamic and pharmacogenomic studies, and the development of more specific therapies. Here, we summarize some of the recent advances in the treatment and understanding of childhood ALL.

Correspondence to: Jeffrey E. Rubnitz, MD, PhD, Department of Haematology-Oncology, St. Jude Children’s Research Hospital, Mail Stop 260 332 N. Lauderdale Street, Memphis, TN 38105-2794, USA. Tel.: +1-901-495-2388; Fax: +1-901-521-9005; E-mail: jeffrey. [email protected]

GENETIC ALTERATIONS IN LEUKAEMIC CELLS Cytogenetic and molecular analyses of leukaemic blasts have enhanced our understanding of the pathogenesis of ALL and have identified therapeutically important subgroups of disease (5,6). To date, specific genetic abnormalities with clinical relevance have been identified in approximately 75% of childhood ALL cases (Table 1). Gene expression profiling will probably identify additional prognostically important genetic subgroups in the near future (7). Recently, microarray analysis of T-cell ALL identified four major genetic subgroups with prognostic significance: the MLL-ENL fusion gene and expression of HOX11 were associated with a favorable outcome, whereas activation of TAL1 or LYL1 conferred a poor prognosis (8). Additionally, HOX11L2 activation also appears to be associated with a dismal outcome in T-cell ALL (8,9). Although the predictive value of genetic abnormalities is superior to that of clinical features, the

c 2003 ELSEVIER SCIENCE LTD. ALL RIGHTS RESERVED. 0305-7372/03/$ - see front matter s

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J.E. RUBNITZ AND C.-H. PUI

TA B L E 1 Outcome and frequency of genetic subgroups in childhood acute lymphoblastic leukemia Subgroup

Frequency (%)

5-year event-free survival estimate (%)

B cell (rearranged MYC gene) B precursor Hyperdiploid > 50 TEL-AML1 E2A-PBX1 BCR-ABL MLL-AF4 Hypodiploid < 45 MLL rearranged T cell MLL-ENL HOX11 TAL1 LYL1

2–3

75–85

25 22 5 3 2 1 5

80–90 85–90 75–85 20–40 20–35 25–40 30–50

1 3 6–7 1

85–95 80–90 30–40 30–40

prognostic impact of these abnormalities is dependent on the efficacy of the treatment given. In addition, heterogeneity exists even within genetic subgroups, further limiting their usefulness in predicting outcome. For example, although patients with Philadelphia (Ph) chromosome-positive ALL generally have a poor prognosis, about one third can be cured by chemotherapy alone (10). Among patients with Phþ ALL, those who have a low leukocyte count at initial examination and are between 1 and 9 years of age have a favorable outcome (11). Similarly, the t(4;11) confers a poor prognosis overall, but there is heterogeneity within this genetic subgroup as well (12). Approximately 50% of patients who have t(4;11)-positive disease and are older than 1 year at the time of diagnosis are cured by modern therapy; in contrast, less than 20% of infants with the identical translocation are cured (12). Among patients with hypodiploidy (fewer than 45 chromosomes), the outcome worsens as the chromosome number decreases: patients with 24–28 chromosomes have the worst prognosis (13). Other genetic features, such as hyperdiploidy and the TEL-AML1 fusion gene, confer a good prognosis: EFS estimates exceed 80% (14,15). Within the hyperdiploid group, patients with trisomies 4, 10, and 17 have a superior outcome; this finding again demonstrates the heterogeneity of ALL (14,16,17). In contrast, prognostic indicators have not yet been identified in cases of TEL-AML1-positive ALL. However, the components of treatment probably affect the outcome of TEL-AML1-positive ALL; low relapse rates were observed in some protocols that featured intensified L -asparaginase therapy (18–20) but not in others (21–23). Leukaemic blasts that express TEL-AML1 are more sensitive to L -asparaginase

in vitro (24), and treatment regimens that include prolonged exposure to this agent are extremely effective at treating this type of leukaemia (3). When TEL-AML1-positive ALL relapses, the disease is often still sensitive to chemotherapy (21,23,25). Although patients had a favorable outcome even after relapse (5-year EFS and survival estimates, 63% and 82%, respectively), a matched-pair analysis demonstrated that TEL-AML1 expression was not an independent predictor of outcome after adjustments were made for time to relapse and site of relapse (21). In contrast to leukaemic cells from older children, blasts from infants with ALL are resistant to prednisolone and L -asparaginase in vitro, but are more sensitive to cytarabine (26). On the basis of these findings, high-dose cytarabine has been incorporated into the treatment regimen of a current international trial for infant ALL. Similarly, high-dose methotrexate is an important component of the treatment of hyperdiploid ALL, as leukaemic blasts from these cases are more sensitive to methotrexate in vitro (27) and accumulate higher levels of methotrexate polyglutamates (the active metabolites of methotrexate) (28). Patients with T-cell ALL may benefit from very high-dose methotrexate (14), a result consistent with our finding that T-lineage blasts accumulate methotrexate polyglutamates less avidly than do B-lineage blasts and therefore higher serum concentrations of methotrexate are needed for adequate responses in T-cell ALL (29). In addition to adding prognostic information, the analysis of genetic alterations in leukaemic cells has provided insight into the origins of childhood ALL. Genetic studies of identical twins with leukaemia (30,31) and the detection of leukaemia-specific fusion genes (32) or immunoglobulin gene rearrangements (33) in neonatal blood spots have definitely established the prenatal origin of some cases of leukaemia. With regard to the development of TEL-AML1positive ALL, the Greaves laboratory demonstrated that a pair of monozygotic twins who developed TELAML1-positive ALL shared a unique TEL-AML1 genomic breakpoint. The presence of the unique breakpoint in each child suggested that the leukaemic clone originated in one fetus and was transferred to the other fetus transplacentally (34). Because leukaemia did not develop until one was 3 years and 6 months old and the other was 4 years and 10 months old, the investigators hypothesized that secondary mutations were necessary for the full leukaemic phenotype. This hypothesis was supported by the results of a subsequent study that demonstrated the presence of TEL-AML1 fusion sequences in blood spots of 8 of 11 neonates in whom ALL developed later in childhood; such a finding again suggested that a preleukaemic clone containing the fusion

ADVANCES IN TREATMENT OF ALL

develops in utero (32). Analysis of a second set of twins concordant for TEL-AML1-positive leukaemia but discordant for age at the time of diagnosis revealed identical genomic translocations at diagnosis (31). Furthermore, twin B harbored low levels of the TEL-AML1 gene rearrangement when the leukaemia in twin A was diagnosed (9 years before that of twin B); this finding demonstrates that the putative second event required for leukaemic transformation may occur after a variable and protracted latency. Subsequent analysis of a set of triplets demonstrated that all three children harbored identical genomic TEL-AML1 sequences (35). Interestingly, the two monozygotic twins experienced the onset of ALL at the same time (21 months of age), whereas the third sibling at this time had very low levels of the TELAML1 fusion gene in his bone marrow without an increase in leukaemic blasts. At the time of diagnosis, the leukaemic blasts from monozygotic twins harbored different deletions of the TEL allele that had not been translocated. These patients apparently had undergone distinct second events after birth, further supporting the multihit model of leukemogenesis. Recently, Greaves demonstrated that the TEL-AML1 fusion is present in cord blood of about 1% of randomly selected newborns, a frequency about 100-fold higher than that of TEL-AML1-positive ALL (36). This finding also suggests that preleukaemic clones are generated at high frequencies in utero, but that additional environmentally induced or spontaneous genetic alterations lead to the full leukaemic phenotype in a subset of children.

PHARMACODYNAMICS AND PHARMACOGENOMICS OF ALL Host factors such as pharmacodynamics and pharmacogenomics have a significant effect on treatment outcome in ALL (37–39). There is wide interpatient variability in the pharmacokinetics of common antileukaemic drugs used in the treatment of ALL. Therefore, in some patients, relapse may be attributed to inadequate drug exposure rather than drug resistance of leukaemic cells. Low systemic exposure to methotrexate and low dose-intensity of mercaptopurine have each been associated with inferior outcome (40,41). We have also demonstrated that individualized dosing of methotrexate based on pharmacokinetic parameters leads to an improved outcome for children with B-cell precursor ALL (40). We are therefore applying this treatment strategy by targeting systemic methotrexate exposure during consolidation therapy on our current ALL protocol. Because cytochrome P450 (CYP) enzymes are responsible for the oxidative metabolism of many

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chemotherapeutic agents, administration of anticonvulsants that induce CYP enzymes can increase the systemic clearance of these chemotherapeutic agents. Patients who received phenobarbital, phenytoin, or carbamazepine had a significantly worse outcome than patients who did not receive anticonvulsants (42). For patients who require anticonvulsants, we now use agents, such as gabapentin, that are less likely to induce drug-metabolizing enzymes. In contrast, azole antifungals (e.g., fluconazole and itraconazole) and the macrolide group of antibiotics (e.g., erythromycin, rifampin, and azithromycin) can inhibit CYP enzymes and thereby decrease clearance and increase the exposure to and the toxicity of certain antileukaemic agents (vincristine and anthracyclines) (43). Polymorphisms of genes encoding proteins involved in drug disposition (drug-metabolizing enzymes, drug transporters, and drug receptors) affect the metabolism and therefore the efficacy and toxicity of antileukaemic agents (37,38). Pharmacogenomics – the study of such genes – uses conventional genetic studies as well as newly developed microarray techniques to identify and characterize these polymorphisms. Pharmacogenomic research may ultimately allow us to select drug and dosage based on each patientÕs ability to metabolize, eliminate, and respond to specific drugs. Some important polymorphisms that are relevant to ALL therapy are shown in Table 2. Thiopurine methyltransferase (TPMT) is an enzyme that converts mercaptopurine, thioguanine, and azathioprine into inactive methylated metabolites; this conversion prevents these drugs from becoming thioguanine nucleotides, which are incorporated into DNA and thus induce an antileukaemic effect. TPMT activity is inherited as an autosomal co-dominant trait. Approximately 90% of the population are homozygous for the wild-type allele and have full enzyme activity, about 10% are heterozygous for the polymorphism and have intermediate levels of enzyme activity, while one in 300 persons carries two mutant TPMT alleles and does not express functional TPMT (44–48). Cells of patients with heterozygous TPMT alleles and intermediate levels of enzyme activity accumulate more thioguanine nucleotides after mercaptopurine or thioguanine therapy than do cells of persons homozygous for the wild-type TPMT alleles. Therefore, patients with intermediate levels of TPMT activity require modest reductions in dosage to prevent excessive myelosuppression (49,50). Moreover, patients with a TMPT deficiency due to homozygous mutant alleles accumulate very high levels of thioguanine nucleotides, and these patients experience life-threatening hematopoietic toxicity if they are treated with standard doses of mercaptopurine, azathioprine, or thioguanine; therefore, the dose must be

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J.E. RUBNITZ AND C.-H. PUI

TA B L E 2 Genetic polymorphisms of enzymes that affect treatment response Gene product

Polymorphism

Drugs affected by polymorphism

Effect of polymorphism

Thiopurine methyltransferase (TPMT) Glutathione S-transferase (GST)

Single-nucleotide polymorphisms that lead to unstable protein Completion deletions or point mutations

Mercaptopurine, azathioprine, thioguanine Alkylating agents, glucocorticoids, epipodophyllotoxins, anthracyclines

Cytochrome P450 3A4 (CYP3A4)

Single-nucleotide polymorphisms that lead to nonfunctional enzyme Point mutations that lead to protein instability Single-nucleotide polymorphisms that lead to nonfunctional enzyme

Vincristine, anthracyclines, epipodophyllotoxins, glucocorticoids Methotrexate

Myelosuppression, increased risk of therapy-related cancer Increased sensitivity to antileukemic drugs, increased risk of primary or therapy-related leukemia Decreased risk of therapy-related leukemia

Methylenetetrahydrofolate reductase (MTHFR) NAD(P)H:quinone oxidoreductase (NQO1)

reduced to 10–15% of conventional doses. Patients who have ALL and at least one mutant TPMT allele tend to respond better to mercaptopurine therapy and have better outcomes than do those who have two wild-type alleles (41). However, patients with at least one mutant TPMT allele are at an increased risk of epipodophyllotoxin-related acute myeloid leukaemia (AML) (51) or irradiation-induced brain tumors (52) in the context of antimetabolite therapy. In fact, AML has been identified in these patients even when their treatment consisted primarily of antimetabolites (53). We now prospectively identify patients with TPMT deficiency and reduce their dosage of mercaptopurine in an effort to reduce toxicity and improve clinical outcome. Glutathione S-transferases (GSTs) detoxify a wide range of xenobiotics, including cancer chemotherapeutic agents, environmental carcinogens, and reactive oxygen products. GSTs are highly polymorphic: approximately 50% of persons have a homozygous deletion of GSTM1, and 25% have a homozygous deletion of GSTT1 (54). In addition, GSTP1 is subject to polymorphisms: persons with GSTP1 Val105 =Val105 have reduced GST activity (55,56). Deficiency of GST has been associated with lower relapse rates in some studies, possibly because of the reduction in inactivation of cytotoxic chemotherapy (56–59). In contrast, GST genotype did not affect outcome in a recent ChildrenÕs Cancer Group analysis (60). GST deficiency has also been associated with increased toxicity of some anticancer agents, and increased expression of GST has been associated with in vitro drug resistance of cancer cell lines (61). In patients who had AML and received intensive chemotherapy, the GSTT1-null genotype was associated with greater toxicity and with death during remission (62,63). Whether this relationship extends to patients who receive intensive chemotherapy for high-risk ALL or

Alkylating agents, mitomycin C

Increased risk of mucositis, decreased risk of primary ALL Increased risk of infant and childhood ALL, adult AML and therapy-related leukemia; worse outcome of ALL

undergo transplantation for very high-risk ALL remains to be studied. GST polymorphisms have also been implicated in the development of de novo ALL (58,64,65) and therapy-related AML (55,66). Cytochrome P450 (CYP) enzymes, which are localized primarily in the liver and gastrointestinal tract, are responsible for the oxidative metabolism of endogenous steroids, hormones, and drugs (38). These enzymes are involved in activation (e.g., epipodophyllotoxins) or inactivation (e.g., vinca alkaloids) of anticancer drugs or in forming metabolites that are toxic to host tissues (67). The CYP3A family, whose two main forms are CYP3A4 and CYP3A5, is responsible for about half of the drug metabolism carried out by CYP enzymes (61,68). Therefore, patients who inherit functional CYP3A4 and CYP3A5 phenotypes catabolize many antileukaemic agents faster than other patients do (61,68). As mentioned above, the induction of CYP3A by phenytoin or phenobarbital may explain the worse clinical outcome of patients who received these agents for seizure control (42), whereas azole antifungal agents and the macrolide group of antibiotics inhibit CYPs and cause increased toxicity (43). Regarding polymorphisms in the CYP genes, patients carrying the CYP3A4*1B variant allele are at a decreased risk of therapy-related AML (69). In addition, a recent study showed that a variant of CYP1A1 is associated with a worse outcome in childhood ALL, especially in patients with MLH1 Ile219 , a variant of a DNA repair enzyme (70). The CYP2E1*5 (71) and the CYP1A1*2A (64) alleles are associated with a greater risk of de novo ALL. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, thereby maintaining normal concentrations of reduced folates and homocysteine. Two polymorphisms associated with

ADVANCES IN TREATMENT OF ALL

reduced MTHFR activity have been described: substitution of thymine for cytosine at nucleotide 677 (677C > T; C ! T; alanine ! valine) (72,73) and substitution of cytosine for adenine at nucleotide 1298 (1298A > C; A ! C; glutamine ! alanine) (74). Approximately 10% of the population are homozygous for the 677T variant, which encodes an enzyme with approximately 30% of the wild-type activity, and 40% have a heterozygous genotype with enzyme activity that is about 60% of wild-type. Because the active polyglutamate metabolites of methotrexate can inhibit MTHFR, patients with reduced MTHFR activity, such as that caused by the 677C > T genotype, are at an increased risk of methotrexate-related toxicity, including oral mucositis (75,76). This increased risk of toxicity after low-dose methotrexate therapy has only been reported for patients with the MTHFR 677C > T allele; this risk after high-dose methotrexate therapy and leucovorin rescue has not been reported (76). We believe that leucovorin rescue after high-dose methotrexate may attenuate the increased risk of toxicity by providing an exogenous source of reduced folates that compensates for the low folate concentrations in these patients. Recent findings have suggested that folate plays a role in the development of childhood ALL (77) and that the MTHFR polymorphism protects against the development of ALL (78–80). In one study, MTHFR polymorphism was associated with genetically defined subgroups of childhood ALL: a low number of carriers of the 677C > T allele were present among patients with MLL-rearranged leukaemia, and a lower number of patients with the 1298A > C allele were observed in the hyperdiploid ALL subgroup (80). This seemingly paradoxical protective effect may be caused by the increased fidelity of DNA synthesis afforded by the greater availability of the 5,10-methylenetetrahydrofolate (80). NAD(P)H:quinone oxidoreductase (NQO1) is an enzyme that converts toxic benzoquinones to hydroxyl metabolites. Two polymorphisms have been identified: NQO1*2, which results in the absence of enzyme function, and NQO1*3, which causes diminished enzyme function (71). The function of NQO1 suggests that patients with decreased or no enzyme activity are more susceptible to toxic and carcinogenic effects of benzene and certain chemotherapeutic drugs. As predicted, a higher prevalence of the inactivating polymorphisms has been associated with infant or childhood ALL (71,81), adult AML (82), and therapy-related leukaemia (83,84). In one study of childhood ALL, patients with the NQO1*2 variant had a worse treatment outcome. The authors speculated that impaired protection of cells from free radicals contributes to recurrent leukaemia in patients with this variant (70).

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Polymorphisms of drug targets or drug transporters may also play a role in response to chemotherapy and susceptibility to leukaemia. In this regard, a recent study showed that a triple tandem polymorphism in the promoter of thymidylate synthase, an important target of methotrexate, was associated with a worse outcome in children with ALL (85). Polymorphisms of thymidylate synthase and another folate-metabolizing enzyme (serine hydroxymethyltransferase) are associated with a lower risk of adult ALL (86). Membrane transporters are involved in drug absorption, distribution into tissues, and efflux from cells. The ATP-binding cassette (ABC) family of membrane transporters comprising P-glycoprotein (PGP) and the multidrug resistanceassociated proteins (MRPs) has been recently reviewed (87). Although overexpression of ABC transporters has been associated with resistance to a broad spectrum of antileukaemia agents in cell lines, the clinical relevance of such overexpression in patients with ALL is unknown (61). Similarly, genetic polymorphisms occur in the human ABC transporters (61,87), but the clinical relevance of the polymorphisms is also unclear.

MINIMAL RESIDUAL DISEASE IN ALL Most current trials for childhood ALL attempt to tailor the intensity of therapy to the risk of relapse (6). Risk classification schemes are based on clinical features (e.g., age and leukocyte count at diagnosis), biological features (e.g., phenotype, karyotype, and molecular abnormalities), and response to therapy. Early response to therapy, which is influenced by the sensitivity of the leukaemic cells to chemotherapy as well as host pharmacodynamic and pharmacogenomic characteristics, is probably the best predictor of outcome. In fact, simple morphologic examination of the peripheral blood after 1 week of therapy is a relatively good indicator of ultimate outcome (88,89). Even within high-risk subgroups such as Phþ ALL and infant ALL, measurement of circulating blasts after 1 week of prednisone therapy is prognostically important (90,91). Similarly, morphologic examination of the bone marrow after 1 to 3 weeks of induction therapy is an independent predictor of outcome (92–94). Recently, we demonstrated that the presence of any blasts in the bone marrow after 3 weeks of induction therapy portends a particularly dismal outcome (95). In contrast to morphologic examination, which tends to be subjective and imprecise, minimal residual disease (MRD) assays provide objective and sensitive measurements of low levels of leukaemic cells (96–98). MRD may be assessed by DNA-based

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polymerase chain reaction (PCR) analysis of clonal antigen receptor gene rearrangements, RNA-based PCR analysis of leukaemia-specific gene fusions, or flow cytometric detection of aberrant immunophenotypes (96–98). Through a series of studies using immunologic detection of leukaemic cells, we have clearly demonstrated the clinical importance of MRD measurement in the treatment of ALL (99– 102). Patients who attained MRD negativity (defined as less than 1 leukaemic cell among 10,000 mononuclear bone marrow cells) had a significantly lower relapse rate than patients who did not achieve this status (99). Sequential monitoring of MRD further improved the predictive value: persistence of MRD at week 14 of continuation therapy was associated with a 70% cumulative risk of relapse (100). We also demonstrated that approximately one half of patients achieved MRD negativity at day 19 of induction and had an exceptionally good prognosis, with a cumulative risk of relapse of only 6% (101). More recently, we determined that peripheral blood, rather than bone marrow, may be used to monitor MRD in patients with T-cell ALL (102). In addition, application of both flow cytometry and PCR testing has allowed us to successfully study 100% of patients with newly diagnosed disease (103). Although a complete review of all MRD studies in ALL is beyond the scope of this article, it should be mentioned that many other investigators have also demonstrated the prognostic importance of MRD in the context of various chemotherapeutic regimens (98). One recent study suggested that the absolute number of leukaemic cells per milliliter of bone marrow may be even more accurate at predicting outcome than relative measurements are (104). We are now using MRD quantitation to tailor the intensity of therapy we deliver to patients treated for ALL on our current clinical trial. We hope that early intensification of therapy will improve the outcome of patients for whom MRD measurements indicate a slow response early in treatment.

RECENT ADVANCES IN THE TREATMENT OF ALL Treatment groups Characterized by MLL gene rearrangements, unique gene expression profiles, and a poor prognosis, infant ALL is usually considered a separate subgroup of childhood ALL (105,106). Leukaemic blasts from infants with ALL are more resistant in vitro to prednisolone and L -asparaginase but are more sensitive to cytarabine than blasts from older children;

J.E. RUBNITZ AND C.-H. PUI

this finding suggests that infants may benefit from treatment with this agent (26). The use of cytarabine in infant ALL is also supported by the excellent results of a Dana–Farber Cancer Institute (DFCI) trial that featured high-dose cytarabine as postremission intensification therapy (107). However, the results of a recent trial in the United Kingdom (Infant 92) were not as encouraging: the 4-year EFS estimate was only 33% (108). Infants with MLL rearrangements who were treated on the Japan Infant Leukaemia Study MLL96 had a remarkably similar outcome, i.e., a 3year EFS estimate of 34% (109). Currently, two large prospective studies of infant ALL are testing the efficacy of additional intensification of high-dose cytarabine and high-dose methotrexate therapy. Although the high relapse rates associated with infant ALL imply that further intensification is needed, several studies have suggested that allogeneic stem cell transplantation does not improve the outcome of these patients (12,108). In fact, among patients with the t(4;11), any type of transplantation was associated with worse disease-free and overall survivals (12). ALL patients older than 1 year of age are generally divided into three categories based on the patientsÕ risk of relapse, although some investigators have defined four risk groups (6,14). Patients who have an excellent prognosis and are sometimes considered to have low-risk disease are those with B-lineage ALL, between 1 and 9 years of age, and have initial leukocyte counts less than 50  109 =L, and either the TEL-AML1 fusion or trisomies 4, 10, and 17. Patients with standard-risk disease have B-lineage a favorable age and leukocyte count, but lack TEL-AML1 or the trisomies. Other patients with B-lineage ALL and those with T-cell ALL (except those whose leukaemic cells overexpress HOX11) are at high risk of relapse and are therefore treated with more intensive chemotherapy regimens. Finally, the very high-risk group consists of a small subset of patients, primarily those whose disease fails to enter remission after standard induction therapy and those with Phþ ALL; these patients are candidates for allogeneic stem cell transplantation (SCT) during first remission (11). Although other features such as male sex (110,111) and black or Hispanic race (112) are also associated with an inferior outcome, these characteristics are generally not included in risk classification schemes; however, some investigators recommend extending continuation treatment for boys.

Remission induction therapy Some remission induction regimens include only three drugs for low-risk patients – a glucocorticoid,

ADVANCES IN TREATMENT OF ALL

vincristine, and L -asparaginase – and reserve the addition of an anthracycline for patients with standard and high-risk disease. Other regimens, however, use four to seven drugs for all patients, in an attempt to induce a faster and deeper reduction in leukaemic cell burden, thereby preventing the emergence of drug resistance. These intensive remission induction regimens appear to have improved the outcome of patients older than 10 years and those with high-risk ALL (17,113). However, intensive induction therapy may not be necessary for patients with low or standard-risk disease, as long as they receive intensification therapy after their disease enters remission (17). In addition, L -asparaginase may not be a necessary component of induction therapy; several trials that included this agent only in the postremission setting achieved excellent remission induction rates and high probability of long-term survival (114,115). The absence of L asparaginase was also associated with fewer thrombotic complications during remission induction therapy. In contrast, glucocorticoids are an essential constituent of induction therapy, although the optimal steroid is unknown. Dexamethasone may provide better leukaemic control and CNS penetration than prednisone (17,116), but in one study dexamethasone was associated with unacceptable toxicity when used in induction therapy (117).

Intensification therapy Clinical trials performed by the ChildrenÕs Cancer Group (CCG) and the Berlin–Frankfurt–Muenster (BFM) study group demonstrated that delayed intensification (also called reinduction) therapy improved the outcome of patients with standard or high-risk ALL (17,113). Additional intensification, designated augmented BFM or double-delayed intensification by the CCG, improved outcome even further (118,119). While this intensification therapy improved the outcome of children younger than 10 years with high-risk ALL and a slow early-response to therapy, it was associated with a high incidence of avascular necrosis of the bone (118). Recently, the results of the AIEOP ALL95 study demonstrated that the addition of two courses of Protocol II improved the outcome of patients who had poor responses to prednisone (120). In contrast, additional blocks of intensification failed to alter EFS estimates on a recent trial in the United Kingdom, possibly because of concomitant decreases in other elements of therapy and in a lack of sustained, or metronomic, therapy (121). Whereas delayed intensification therapy is generally given about 3 months after induction therapy

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is completed and includes the same or similar drugs as those used in induction therapy, consolidation therapy includes high-dose methotrexate and mercaptopurine given at the time remission is achieved (5,6). Although the optimal dose of methotrexate is still a subject of investigation, 2.5 g=m2 is probably adequate for patients with B-lineage ALL, and 5 g=m2 for T-cell ALL (6,14,113). In addition, we previously showed that dosing based on plasma concentrations of methotrexate rather than simply on body surface area is beneficial for B-lineage cases (40). A third form of intensification therapy consists of the prolonged use of intensive L -asparaginase therapy to continuously deplete serum of asparagine. A Paediatric Oncology (POG) Group study demonstrated that this strategy improved the outcome of patients with T-cell ALL; however, the regimen used in that study also resulted in a higher incidence of treatment-related AML due to the concomitant use of etoposide (122). The prolonged use of L -asparaginase and doxorubicin in DFCI protocols has contributed to excellent overall outcome, especially for patients with TEL-AML1-positive B-lineage ALL and for patients with T-cell ALL (3,115,123). In the most recent DFCI trial, patients who received at least 26 weekly doses of L -asparaginase had a better outcome than those who did not (3). However, the DFCI trial was also associated with subclinical cardiomyopathy, which was not prevented by the use of continuous infusion, rather than bolus, administration of doxorubicin (124). Finally, allogeneic SCT represents the most intensive form of intensification. Patients with Phþ ALL are clearly candidates for allogeneic SCT during first remission; an international collaborative study demonstrated the outcome of this group of patients who received transplants of stem cells from an HLA-matched related donor is superior that of the same group who received only intensive chemotherapy (11). In contrast, allogeneic SCT does not appear to improve the outcome of patients with t(4;11)-positive ALL (12) or of infants with ALL (108).

Continuation therapy Children with B-precursor or T-cell ALL require continuous postremission therapy for optimal success (6,17,113,115). Although effective for other malignancies, high-dose pulse therapy with intermittent rest periods is associated with an inferior outcome in ALL (6,113). In addition, continuation therapy should be prolonged; attempts to shorten therapy to 12 or 18 months have resulted in inferior overall outcomes

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(113,125). Although a subset of patients may be cured with a shorter duration of therapy, most clinical trials currently prescribe at least 2–2.5 years of therapy for all patients. Furthermore, because the outcome for boys is generally inferior to that of girls (110,111), several studies, including the current St. Jude trial, are examining the benefit of extending continuation therapy to 3 years for boys. Weekly low-dose methotrexate and daily oral mercaptopurine make up the backbone of most continuation regimens for ALL. Although improved outcome has been associated with increasing mercaptopurine dosages to the limits of individual tolerance (as indicated by neutrophil counts) (126), one must not to be too aggressive. The use of mercaptopurine at dosages that cause neutropenia has been associated with increased rates of infection, an inability to deliver all planned therapy, and a higher relapse rate (41,127). As discussed above, the dosage of mercaptopurine may need to be decreased in patients with TPMT deficiency to decrease acute toxicity and the risk of secondary cancers (45,50– 53,128). We now prospectively decrease the mercaptopurine dose slightly in patients with TPMT deficiency to determine whether this approach will improve outcome. The addition of intermittent pulses of steroids and vincristine to the antimetabolite backbone improves outcome and is included in most modern continuation regimens (17,129). Because of better antileukaemic effect, dexamethasone has replaced prednisone as the steroid of choice, but dexamethasone is associated with increased risks of avascular necrosis, osteoporosis, and fractures (130–132).

Therapy for central nervous system leukaemia The treatment of subclinical or overt central nervous system (CNS) leukaemia has made a tremendous impact on the overall cure rates for childhood ALL (133). Certain patients, including those with high leukocytes counts at diagnosis, T-cell disease, or leukaemic blasts in the cerebrospinal fluid (CSF), are at higher risk of CNS relapse and therefore require more intensive therapy directed at the CNS (134). CNS-directed therapy includes cranial irradiation, intrathecal chemotherapy, and systemic chemotherapy with dexamethasone and high-dose methotrexate (1,17,135). Although cranial irradiation is the most effective modality, it is associated with significant morbidity, including neurotoxic effects, endocrinopathy, and second malignant neoplasms (5,6,52,136,137). Therefore, intrathecal therapy has largely replaced cranial irradiation for all patients except those at very high risk of CNS relapse (2,138).

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In fact, three studies omitted cranial irradiation in all patients regardless of their risk classification and still achieved CNS relapse rates of less than 10% (4,139,140). For patients at high risk of CNS relapse, the BFM study group demonstrated that a radiation dose of 12 Gy, when given with effective intrathecal and systemic chemotherapy, provided adequate CNS control (113). Recently, we have demonstrated that for patients in whom growth hormone deficiency occurs as a result of CNS-directed therapy, growth hormone replacement appears to be safe and effective (141). An area of active discussion remains the optimal treatment of patients with low numbers of identifiable leukaemic blasts in the CSF (i.e., CNS2 status, defined as leukaemic blasts present on cytospin, but fewer than 5 leukocytes per microliter of CSF) (142). Previously, we demonstrated that CNS2 status conferred an adverse prognosis among patients treated on St. Jude Total Therapy Studies XI and XII (143), a finding confirmed by the POG (144). However, three other studies did not show an association between CNS2 status and outcome (145–147). This apparent discrepancy may be explained by differences in treatment regimens, including early CNS-directed therapy on the CCG and BFM studies (145,147) that was more intensive than that on Studies XI and XII (143). We intensified intrathecal therapy for CNS2 patients during remission induction and postremission therapy on our subsequent trials Studies XIIIA (1) and XIIIB and have reduced the cumulative incidence of any CNS relapse in CNS2 patients to less than 5% (unpublished data). To this end, recent CCG studies of patients of all risk groups demonstrated that CNS2 patients in the cohort with presenting leukocyte count > 50  109 =L or age > 10 years had an increased risk for both haematological and CNS relapse, as compared to the CNS1 patients (148). Hence, CNS2 patients should at least receive more intensive CNS-directed therapy. A distinct category of CNS status at diagnosis is traumatic lumbar puncture with identifiable blasts. We previously demonstrated an inferior outcome for this group of patients (134), a finding recently confirmed by the BFM study group (147). Possible explanations for this observation include the iatrogenic introduction of blasts into the CSF and decreased efficacy of subsequent intrathecal treatment after a traumatic lumbar puncture (134,142,147). Although intensive intrathecal therapy can abolish the poor prognostic impact of a traumatic lumbar puncture with blasts, it can also adversely affect neuropsychologic and spinal cord function (149–151). To reduce the frequency of traumatic lumbar puncture, we now routinely perform the diagnostic procedure under deep sedation or general anesthesia, and intrathecal chemotherapy is always administered immediately

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after the collection of CSF. In addition, the most experienced clinicians perform diagnostic lumbar punctures at our institution; this decision is based on our observation that a less-experienced clinician is a risk factor for traumatic lumbar puncture (152). Because a low platelet count (<100  109 =L) is also a risk factor, we now transfuse platelets into all thrombocytopenic patients before they undergo initial puncture. Together, these preventive measures have lowered the frequency of traumatic lumbar puncture with or without blasts at diagnosis to 4% each in our current trial. We previously reported data indicating that lumbar puncture is generally safe in patients with thrombocytopenia (153). Therefore, we still contend that platelet transfusion is not necessary with subsequent lumbar punctures when patients lack circulating blasts, especially during remission induction therapy that includes prednisone, vincristine and L asparaginase, which generally induce a hypercoagulable state (154).

FUTURE DIRECTIONS Current studies of ABC transporters and other potential mechanisms of drug resistance will hopefully lead to therapeutic methods to overcome leukaemic cell resistance to conventional chemotherapy (61,87,155–157). More promising, however, is the development of novel therapies, particularly those targeted to specific abnormalities in the leukaemic cell. Imatinib mesylate (Gleevec), a selective inhibitor of the BCR-ABL tyrosine kinase, induces durable responses in patients with chronic-phase chronic myelogenous leukaemia (CML) (158,159). The inhibitor also has activity in accelerated-phase or blastcrisis CML, although the results are not as long-lived (160–162). Similarly, a recent phase II study demonstrated that imatinib induced complete bone marrow responses in about one third of patients with relapsed or refractory Phþ ALL, but resistant disease developed rapidly (163). Other kinase inhibitors under development include selective inhibitors of FLT3, which is mutated or overexpressed in subsets of ALL and AML (164–166). Gene expression profiling, in which the expression of thousands of genes is analyzed, has the potential to more accurately predict relapse and the development of secondary cancers (7,106). This method may therefore allow the construction of more precise risk classification schemes and the tailoring of the intensity of therapy to a greater extent than is currently possible. More important, gene expression profiling may identify new targets against which novel and specific therapies may be developed.

39

ACKNOWLEDGEMENTS We thank Julia Cay Jones for expert editorial review. This work was supported in part by the Cancer Center Support Grant CA21765 from the National Cancer Institute, a Center of Excellence grant from the State of Tennessee, and by the American Lebanese Syrian Associated Charities (ALSAC). C.-H. Pui is the American Cancer Society – F.M. Kirby Clinical Research Professor.

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