Hong Kong Journal of Paediatrics [HK J Paediatr (New Series) 2003;8:170-183]

Children's Centre of Cancer and Blood Diseases, Department of Paediatrics and Adolescent Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam Road, Hong Kong, China

GCF Chan ( 陳志峰 )MD

St. Jude Children's Research Hospital and the University of Tennessee Health Science Center, Memphis, Tennessee, USA

CH Pui ( 裴正康 )MD

Correspondence to: Dr GCF Chan

Received October 2, 2002

Supported in part by grant CA-21765 from the National Institute of Health, by a Center of Excellence grant from the State of Tennessee, and by the American Lebanese Syrian Associated Charities (ALSAC). CH Pui is an American Cancer Society-FM Kirby Clinical Research Professor.

Acute lymphoblastic leukaemia (ALL) is the commonest form of childhood malignancy with an estimated annual incidence of 40 to 50 new cases (<15 years) in Hong Kong. The cure rate for ALL in children has improved drastically over the past 2 decades and is approaching 70% currently. Well-designed collaborative clinical trials had contributed greatly to this success in treatment outcome. The advances in new technology especially in the field of molecular biology also help to revolutionise the process of risk assessment, treatment stratification and disease monitoring. In addition to the advances in treatment, progresses had also been made in the understanding of leukaemogenesis and its associated risk factors, individual susceptibility and prognosis assessment. But while there are more and more childhood ALL children being cured, long-term therapy-related complications starts to emerge and becomes a new challenge. This review article will mainly focus on the recent advances in the areas of leukaemogenesis, prognostic assessment, current treatment design and late effect of ALL management.

Background

Acute lymphoblastic leukaemia (ALL) is the most common form of childhood malignancy and its annual incidence varies with different ethnic groups and geographic regions. In Hong Kong, the annual incidence is around 4.1 per million children as compared to the 6.1 among American White and 5.1 among the American Black (HKPHOSG 2002 & SEER 1988 data). During the past decade, the cure rate for childhood ALL in most developed countries reached 63-83% (Table 1). This success can be principally attributed to the effectiveness of risk-directed therapy developed through well-designed clinical trials. Chinese children who live in more affluent areas of the world have benefited from these achievements and experience a similar cure rate. However, because of a lack of access to treatment, inadequate treatment, and poor supportive care, many Chinese children with ALL still suffer a poor outcome. A simplified, tailor-made treatment approach may be more practical for Chinese patients who live in less developed regions.

Because of the remarkable advances made in the field of molecular biology during recent years, we have a better understanding of the molecular abnormalities that underlie leukaemogenesis and drug resistance. This information has led to the development of new therapeutic strategies, including immunotherapy and therapies that are molecularly targeted toward genetic lesions. However, because those new treatments are expected to be costly initially, it will be many years before children from underdeveloped or developing countries will benefit from them.

Another key advance in the treatment of leukaemia is the emergence of the field of pharmacogenetics. Genetic polymorphisms of certain drug-metabolising enzymes, transporters, receptors, or targets have been linked with host susceptibility to the development of de novo leukaemia or therapy-related second cancers. Furthermore, recognition of inherited differences in patients' ability to metabolise antileukaemic agents has provided rational selection criteria for optimal drug dosages and scheduling. Distinct ethnic or racial differences in the pharmacogenetic characteristics of Chinese children probably exist; however, at this time, the related data remains quite sparse. Collaborative studies that include multiple Chinese paediatric oncology centers and those abroad are currently generating more useful information in this area.

Finally, the intensity of therapy can be adjusted on the basis of treatment response. Treatment response, which is assessed by measurement of subclinical leukaemia, or minimal residual disease (MRD), has emerged as a powerful and independent prognostic indicator (Figure 1). MRD is an indicator of the sensitivity or resistance of leukaemic cells to drugs and the pharmacokinetic and pharma-cogenetic properties of the host; measurement of MRD will probably become the gold standard for assessing patients' risk of relapse.

Treatment Outcome of Childhood ALL

Contemporary risk-directed therapy has resulted in 5-year event-free survival rates that have ranged between 63% and 83% in children treated for ALL in developed countries (Table 1).^1-12 Although some clinical trials resulted in a relatively low event-free survival rate, retrieval therapy boosted the overall survival to approximately 80%. A comparable treatment outcome has been reported in Chinese children treated for ALL in Taiwan and Hong Kong in recent years;^13,14 however, in some regions of China and Southeast Asia, a substantial number of children with ALL still do not receive adequate treatment or supportive care. This may be due to either financial or facility constraints. A simplified, less expensive chemotherapy regimen may be useful in this setting. In addition, a collaborative, central facility may help to establish proper diagnosis and risk stratification approaches.

Leukaemogenesis in Children

Numerous investigations have examined the demographic and environmental risk factors of the development of childhood leukaemia. The factors associated with an increased risk are male sex, aged 2 to 5 years, high socioeconomic status, white race, in utero

X-ray exposure, and the presence of some congenital syndromes such as Down syndrome or neurofibromatosis (Table 2).¹⁵ Other possible risk factors include increased birth weight, maternal history of fetal loss, paternal smoking before or during pregnancy, parental occupational exposure to carcinogens, postnatal infections, and diet.

This hypothesis may explain the higher risk of ALL in children from higher socioeconomic status or developed countries. It states that delayed exposure of these children to infectious agents contributes to the etiology of childhood ALL. Descriptive epidemiologic and case-control studies conducted in developed Western countries¹⁶ and Hong Kong¹⁷ have provided substantial indirect support for this theory. In the Hong Kong study, roseola, fever and rash, or both experienced during the first year of life reduced the risk of ALL (odds ratio, 0.33; 95% confidence interval, 0.16 to 0.68), whereas tonsillitis during the 3 to 12-month period before reference date (date of diagnosis for cases, corresponding date for controls) increased the risk of ALL (odds ratio, 2.56; 95% confidence interval, 1.22 to 5.38). Some other indirect measures of exposure to infection at crucial times were associated with predicted patterns of risk, but day-care attendance failed to show associations.

Although traditional epidemiologic studies have yielded a considerable amount of information about leukaemogenesis in children, the advances made in molecular biology provide a more powerful tool for further study. One current primary focus of these studies is infant ALL, which frequently involves the MLL gene located on the chromosome band 11q23.¹⁸ MLL gene (also known the mixed lineage leukaemia gene or myeloid lymphoid leukaemia gene) serves as a homeotic gene. Translocations of MLL with other genes can induce regulator of leukaemia possibly via loss of wild-type MLL function together with gain of additional signals from sequences provided by the partner chromosomes. Many of the MLL translocation partners (>30) encode proteins involved in modulating gene expression. For example, the AF4 protein targeted by the t(4;11) appears to function directly as transcription factor. Approximately 75% of infants with ALL have MLL gene rearrangements. MLL rearrangements are also common in therapy-related acute myeloid or lymphoid leukaemia in all age groups. Therapy-related acute leukaemia arises shortly after treatment with topoisomerase II inhibitors such as epipodophyllotoxins [i.e. etoposide (VP16)].¹⁹ DNA topoisomerases are nuclear enzymes that can repair the structure of DNA by both breaking and rejoining action. Topoisomerase I acts on single stranded DNA and topoisomerase II produces double stranded DNA breakage-rejoining. A topoisomerase II inhibitor acts by forming a stable tertiary DNA-topo-II-drug complex and interferes with DNA replication, repair and transcription. The similarity in the genetic abnormalities associated with infant leukaemia and topoisomerase II inhibitor-related leukaemia suggests that transplacental fetal exposure to topoisomerase II inhibitors might induce leukaemogenesis in infants. Chemicals such as flavonoids (in food and drink), quinolone antibiotics, benzene metabolites, catechins, and estrogens can inhibit topoisomerase II in vivo and in vitro; thus, these chemicals may be potential mutagens.²⁰ Because dietary and environmental exposures provide much lower functional doses of topoisomerase II inhibitors than anticancer chemotherapy, infants with leukaemia or their mothers may have low activities of enzymes that detoxify carcinogens. Quinones induce topoisomerase II-mediated DNA cleavage,²¹ and low activity of NAD(P)H: quinone oxidoreductase, an enzyme that converts benzoquinones to less toxic hydroxy metabolites, has been associated with infant leukaemia with MLL-AF4 fusions.²²

Genetic polymorphisms of other enzymes capable of detoxifying carcinogens may also affect the risk of de novo leukaemia. For example, the deficiency of glutathione S-transferases (GST-M1 and GST-T1), which detoxify electrophilic metabolites by catalyzing their conjugation to glutathione, has been associated with infant leukaemias without MLL rearrangements¹⁸ and with ALL in black children.²³ Another recent study related GST-M1-null and cytochrome P-450 1A1*2A genotypes to an increased risk for childhood ALL; children carrying both genotypes are at a particularly high risk.²⁴

Prenatal Origin of Genetic Abnormalities Associated with Childhood Leukaemia

Genetic studies of identical twins with concordant leukaemia^25-27 and studies that backtracked leukaemia-specific translocated fusion-gene sequences (e.g., MLL-AF4, TEL-AML1, AML1-ETO) to neonatal blood Guthrie spots^28-30 established the prenatal origin of leukaemias in many cases. Indeed, detection of clonotypic immunoglobulin heavy-chain gene or T-cell receptor gene rearrangements at birth^31,32 suggests that most, if not all, cases of childhood ALL are fetal in origin. However, these genetic aberrations may have a different effect on the onset of leukaemia. In the t(4;11) with MLL-AF4, the high concordant rate in identical twins (25% to 100%), and the brief latency period (weeks to months) suggests that this fusion is sufficient to induce leukaemogenesis.³³ In contrast, childhood t(12;21) ALL with TEL-AML fusion, which is present in 20% to 25% of 1- to 10-year-old diagnosed with childhood ALL, and childhood T-cell ALL have a lower concordance rate in identical twins (~5%), a longer and variable postnatal latency period, diverse clinical presentations, and variable outcome of therapy among identical twins. This finding suggests that in these cases, an additional postnatal molecular event(s) is necessary for full leukaemic transformation.^25,27,33 In fact, additional genetic abnormalities were frequently found in patients who have ALL with the TEL-AML chimeric transcript.³⁴

In a recent report of triplets, concordant leukaemia with identical TEL-AML1 fusions was diagnosed in the monozygotic twins at 3 years of age; the remaining dizygotic co-twin was free of leukaemia and the genomic sequence.³⁵ In addition to the fusion transcript, the identical twins had another independent deletion of the normal TEL allele, a finding that suggests that a postnatal event also occurred. Interestingly, the TEL-AML1 fusion is detected in 1% of normal neonatal blood, a rate that is 100 times that of the expected incidence and further supports the theory that a secondary transforming event(s) is necessary to induce leukaemia.

Prognostic Assessment and Disease Monitoring

Although different prognostic factors have been proposed by various study groups, there is no argument that treatment regimen is the single most important prognostic factor known currently. Many clinical and biologic variables lost predictive strength in contemporary treatment programs. Age at the time of diagnosis of ALL and leukocyte count have consistent prognostic significance in B-lineage but not T-lineage ALL.^9,10,12 Yet, these criteria are inadequate even for B-lineage leukaemia, because as many as one third of patients with standard risk (aged 1 to 9 years with a leukocyte count <50x10⁹/L), may relapse.

In the U.S. collaborative group studies, African American and Hispanic children had a significantly worse outcome than did Caucasian children, even after adjusting for other prognostic features.^36,37 However, the risk factor associated with race may be abolished by a more effective treatment approach.³⁸ The treatment outcome of Chinese children treated for ALL in Hong Kong also appears to be dependent on the treatment protocols adopted. Replacing the UKALL regimens, which were commonly used in the 1980s, with the Berlin-Frankfurt-Münster protocols in the 1990s significantly improved the overall result (Li CK, et al. HKPHOSG Annual Scientific Workshop Report 2002).

Assigning risk on the basis of the primary genetic abnormalities of leukaemic cells is insufficient because of the clinical heterogeneity within the various genetic subgroups. For example, as many as 20% of children with favourable genetic features (i.e., TEL-AML1 fusion and hyperdiploidy [>50 chromosomes]) eventually relapse, and approximately one third of those with high-risk abnormalities (i.e., the Philadelphia Chromosome with BCR-ABL fusion and the t(4;11) with MLL-AF4 fusion) are cured with chemotherapy alone.³⁹

Pharmacodynamic and pharmacogenetic characteristics are important determinants of treatment outcome.⁴⁰ There is a wide variability in the rate of systemic clearance of antileukaemic agents and in the absorption of orally administered chemotherapy. Low systemic exposure to methotrexate and low dose intensity of 6-mercaptopurine have each been associated with inferior treatment outcome.^41-43 These findings indicate that treatment is unsuccessful in some patients, because they received inadequate doses of drugs and not because their leukaemia was drug-resistant.

Concomitant administration of cytochrome P450 enzyme-inducing anticonvulsants (phenytoin, phenobarbital, and carbamazepine) can significantly increase the rate of systemic clearance of several antileukaemic agents and decrease the efficacy of chemotherapy.⁴⁴ The probability of event-free survival is higher in patients who have a homozygous or heterozygous deficiency in thiopurine methyltransferase (TPMT), the enzyme that catalyzes the S-methylation (inactivation) of mercaptopurine, than it is in those who have normal TPMT activity; this difference may be caused by higher exposure to active metabolites of 6-mercaptopurine (i.e., thioguanine nucleotides).⁴³ The GSTM1-null, GSTT1-null, and the GSTP1 Val₁₀₅/Val₁₀₅ genotypes have also been associated with a lower risk of relapse, perhaps because patients with these genotypes experience reduced detoxification of cytotoxic chemotherapy.⁴⁵

As mentioned above, the most important prognostic indicator is the response to treatment, because it reflects the intrinsic drug sensitivity or resistance of leukaemic cells. Sensitivity and resistance are determined by expression of ATP-binding cassette transporters, various aberrant intracellular processes that prevent apoptosis,^46-50 and the pharmacodynamic and pharmacogenetic properties of the patient.⁴⁰ Since the early 1980s, the extent of clearance of leukaemic cells from the blood or bone marrow during the early phase of therapy has been an independent prognostic factor that is recognized by investigators of the Children's Cancer Group and the Berlin-Frankfurt-Münster consortium.⁵¹ These investigators assessed the response to treatment by morphologic examination of the bone marrow or peripheral blood. Morphologically identifiable, persistent disease (as little as 1-4% blast cells) on day 15 of remission-induction therapy was associated with a poor prognosis, and that detected on days 22 to 25 was associated with a particularly dismal outcome. The prognostic effect of persistent morphologic disease is independent of other known risk factors, including treatment, age at the time of diagnosis, white blood cell count, DNA index, cell lineage, central nervous system status, and National Cancer Institute-Rome criteria, which is based on age and white blood cell count. Although morphologic methods can be readily applied at any center, these methods are subjective and lack precision. Approximately 20% of patients with a good response will eventually experience a relapse of disease, and a third of the patients with a poor response may become long-term survivors when treated with intensive chemotherapy alone.⁵²

Several methods are currently available to measure MRD during or after initial remission-induction chemotherapy. By determining aberrant surface antigens expression (immunophenotypes) using 2 or 3 fluorescent label antibodies simultaneously, one can distinguish blast cells from normal cells by flow cytometer at cellular level.^53,54 A more sensitive but tedious method is to use semi-quantitative polymerase chain reaction (PCR) analysis of leukaemic specific clonal antigen-receptor gene rearrangements.^55-58 When applied together, these methods enable us to monitor MRD in virtually all cases of ALL. Patients who experience remission of their disease, as determined by immunologic or molecular measures (i.e., leukaemic involvement of <0.01% of nucleated bone marrow cells at the end of remission-induction therapy), are predicted to have a better clinical outcome than patients whose remission is defined solely by morphologic criteria. In studies to date, patients with MRD at a level of 1% or more at the end of remission-induction therapy have fared almost as poorly as those who experience induction failure (i.e., those with 5% or more blast cells in the bone marrow).

Sequential monitoring of MRD can further improve the clinical usefulness of risk assessment. Approximately 50% of children have fewer than 0.01% blast cells after only 2 weeks of remission-induction therapy, and these patients have a particularly favorable clinical outcome (Figure 1). The finding of comparable levels of leukaemic blast cells in bone marrow and blood of patients with T-cell ALL suggests that blood samples can be used for clinical monitoring in patients with this subtype of leukaemia.

One prerequisite for the clinical application of MRD studies is the ability to study all patients. Recent advances in real-time quantitative PCR (RT-PCR) have facilitated MRD studies and allowed successful study in up to 90% of cases.⁵⁹ In addition, comparative analyses of gene expression in normal B-cell progenitors and B-lineage leukaemic cells have identified new leukaemia-associated markers (e.g., CD58), thereby also boosting the number of cases that can be studied by flow cytometry to 90%.⁶⁰ Tandem application of flow cytometry and PCR testing resulted in a successful study in 100% of cases of ALL at St. Jude Children's Research Hospital (St. Jude).⁶¹ This measure has, therefore, been incorporated in the risk classification schema at St. Jude (Table 3).

Evolution of Childhood ALL Treatment

The improved cure rate of ALL can be attributed mainly to the development of more effective combination chemotherapy regimens. Virtually all of the chemo-therapeutic agents currently used to treat ALL were discovered between the early 1950s and the late 1970s. Hence, the recent improvement in treatment must be attributed to the optimal and more rational use of the existing agents and not to the discovery of new agents. In both children and adults with mature B-cell ALL (FAB-L3), a short-term (2 to 8 months), intensive chemotherapy regimen primarily based on cyclophosphamide, methotrexate, cytarabine, and intrathecal therapy resulted in a cure rate of 75-85%.^62,63 The recent development of rasburicase, a recombinant urate oxidase that is a highly effective uricolytic agent, may further improve this cure rate by reducing early morbidity and mortality caused by tumour lysis syndrome and acute renal failure.⁶⁴

Most study groups treat infants as a unique subgroup; infants are generally treated with multiple drugs at high doses, and no cranial irradiation is administered. The prognosis for infants with ALL, especially those with 11q23/MLL rearrangements, remains poor. Despite current treatment regimens, the probability of event-free survival in these patients is 20-35%.¹⁸ In several recent clinical trials, however, high-dose cytarabine, high-dose metho-trexate, and intensive consolidation-reinduction therapy appeared to result in improved clinical outcome.^65,66 Intensive systemic and intrathecal treatments without cranial irradiation appeared to provide adequate CNS protection, even in infants with CNS leukaemia at the time of diagnosis.⁶⁷

The basic approach to treating ALL in children and adults consists of a relatively brief remission-induction phase, followed by intensification (consolidation) therapy, and then prolonged continuation treatment. All patients require treatment for subclinical CNS leukaemia; this treatment should be initiated early in the form of intrathecal therapy.

The first goal of therapy is to induce complete remission and restore normal haematopoiesis. Induction regimens invariably include a glucocorticoid (prednisone, prednisolone, or dexamethasone), vincristine, and at least one other agent (asparaginase or anthracycline). Because of the improvements in supportive care and chemotherapy, the rate of complete remission of ALL now ranges from 96% to 99%.^1-12 Intensification of remission-induction therapy may not provide additional benefit to patients with standard-risk leukaemia, provided that those patients receive post-induction intensification therapy.^3,4 Highly intensive remission-induction therapy may even lead to inferior overall outcome due to increased early morbidity and mortality. The use of asparaginase and dexamethasone (instead of prednisone) during remission induction has recently been challenged;¹³ these agents have demonstrated marginal clinical benefit and higher therapy-related morbidity and mortality. However, more intensive remission induction is necessary for patients with high-risk or very-high risk ALL, especially those with Philadelphia chromosome-positive ALL or T-cell ALL with a poor early response.

After restoration of normal haematopoiesis, patients in remission should receive intensification (consolidation) therapy; however, a consensus on the standard regimens or the optimal duration of this treatment has not yet been reached. Delayed intensification (or reinduction) therapy, which was pioneered by the investigators of the Berlin-Frankfurt-Münster consortium, is the most widely adopted regimen. This regimen, which is administered 3 months after the patient enters remission, is basically a repeat of the initial remission-induction therapy and is most beneficial for patients with standard-risk ALL.^2,3 Investigators in the Children's Cancer Group have shown that double-delayed intensification (i.e. that administered 6 months after remission) improved the outcome of patients with high-risk or very high-risk leukaemia and slow early treatment response.³

The use of different intensification regimens in various clinical trials has led to the identification of effective treatment components for certain subtypes of leukaemia. For example, the improved outcome in patients with T-cell ALL in the clinical trials of the Dana-Farber Cancer Institute Consortium⁶ and Children's Cancer Group³ has been credited to the intensive use of asparaginase, a finding that has been confirmed in a randomised study done by the Pediatric Oncology Group.⁶⁸ Very high doses of metho-trexate (5 gm/m²) appeared to improve outcome in patients with T-cell ALL.² An in vitro study has also shown that

T-lineage blast cells accumulate methotrexate polyglutamates, the active metabolites of methotrexate, less avidly than do B-lineage blast cells; thus, a higher serum concentration of methotrexate is needed for an adequate response.⁶⁹ High-dose methotrexate also benefits patients with B-lineage ALL, but a lower dose (2.5 g/m²) of the agent should be adequate for most cases.⁷⁰

Among the various types of postremission intensification regimens, continuation treatment is the most successful.⁷¹ High-dose pulse therapy with prolonged rest periods necessitated by myelosuppression can result in an inferior outcome,⁷² perhaps because rest periods provide time for slowly proliferating tumour endothelial cells to repair and recover.⁷³ Continuous or frequent administration of cytotoxic drugs improves outcome by abrogating this process, a finding that is consistent with the concept of metronomic dosing.

Children with ALL, except those with mature B-cell ALL, require prolonged continuation treatment for reasons that are poorly understood. The attempt to intensify early therapy and shorten the total duration of treatment to 1 year resulted in inferior overall event-free survival.⁷⁴ Interestingly, the abbreviated therapy appeared to be adequate for a subset of patients with very high-risk ALL who responded well to prednisolone treatment. Because those patients who can be cured with abbreviated therapy cannot be identified with certainty, the general rule is to provide therapy for 2 to 2.5 years. Some investigators prefer to arbitrarily extend treatment of boys to 3 years, because boys generally experience a poorer outcome.^75-77 However, the efficacy of this approach remains to be proven.

A combination of daily 6-mercaptopurine and weekly methotrexate administration constitutes the standard regimen of continuation treatment. Individualising the doses of these agents to the patient's limit of tolerance (as indicated by low neutrophil count) has been associated with an improved clinical outcome.⁷⁸ However, overzealous use of 6-mercaptopurine is counterproductive, because this agent induces neutropenia, which necessitates the frequent interruption of chemotherapy and reduces overall dose intensity.⁷⁹

In rare cases (1 in 300), patients are homozygous deficient for TPMT and experience extreme sensitivity to 6-mercaptopurine. Recent study showed that the 10% of patients, who are heterozygous for this deficiency and thus have intermediate levels of enzyme activity, might also require a moderate reduction in the dose of 6-mercaptopurine to avert side effects.⁷⁹ The identification of the genetic basis of this autosomal codominant trait has made the molecular diagnosis of these cases possible.⁸⁰ Studies can now be performed in patients who have poor tolerance to the combination of methotrexate and 6-mercaptopurine to identify the drug responsible for the increased myelosuppression and to selectively reduce its dosage. While undergoing antimetabolite-based therapy, patients with defective TPMT are at risk of radiation-induced brain tumour⁸¹ and epipodophyllotoxin- or alkylating agent-induced acute myeloid leukaemia.⁸² Hence, identification of these cases has important therapeutic implications.

Clinical observations have revealed that most Chinese patients with ALL cannot tolerate standard-dose mercaptopurine, despite the fact that the incidence of heterozygous and homozygous TPMT-deficiencies in these patients is comparable to that seen in Caucasian patients.⁸³ This finding suggests that other factors (e.g., the difference in the amount of dietary folate intake) are involved. Nonetheless, multivitamins and folate supplements should not be given during antileukaemia therapy, because these substances can reduce the efficacy of chemotherapy.

Adding intermittent pulses of vincristine and a glucocorticoid to the antimetabolite continuation regimen improves results,⁸⁴ and this approach has been widely adopted. Many clinical trials have substituted dexamethasone for prednisone during continuation therapy, because the clinical efficacy of dexamethasone is superior. However, dexamethasone also appears to increase the frequency of avascular necrosis of bone and bone morbidity (e.g. osteoporosis);^85,86 therefore, additional studies are needed to determine the optimal dosage and duration of dexamethasone therapy during this phase of treatment.

Patients with high-risk genetic features, large leukaemic-cell burden, T-cell leukaemia, or leukaemic cells in the cerebrospinal fluid (even iatrogenic introduction during a traumatic lumbar puncture) are at increased risk of CNS relapse and require more intensive CNS-directed therapy.⁸⁷ Although high-dose methotrexate is useful for preventing haematologic or testicular relapse, this treatment has only marginal, if any, effect on the control of CNS leukaemia. In contrast, dexamethasone improves CNS control.^3,5 Whether triple intrathecal therapy (methotrexate, hydrocortisone, and cytarabine) is more efficacious than intrathecal methotrexate alone remains unknown.

Cranial irradiation is, perhaps, the most effective CNS-directed therapy, but this treatment is associated with substantial side effects such as neurotoxicity and second cancers, especially brain tumours. Currently, intensive intrathecal and systemic chemotherapy has replaced cranial irradiation in 90% or more patients. This approach, in combination with cranial irradiation for selected very high-risk cases, has lowered the rate of CNS relapse to less than 5%.^4,88,89 The dose of radiation can be lowered to 12 Gy without increasing the risk of CNS relapse, if effective systemic intensive chemotherapy is used.⁹⁰ In a recent retrospective study of T-cell ALL with either a high leukocyte count (>50x10⁹/L) or CNS leukaemia at the time of diagnosis, CNS irradiation reduced the rate of CNS relapse but failed to improve event-free survival. Hence, whether CNS irradiation can improve haematologic control remains controversial.⁹¹

Three studies that omitted cranial irradiation for all patients resulted in CNS relapse in approximately 3-4% of the patients; patients with CD10^- or T-cell ALL and those with CNS leukaemia at the time of diagnosis had higher risk of CNS relapse. However, the overall rate of event-free survival in these studies was only 60-70%. More effective systemic therapy and intensification of intrathecal therapy for patients with high-risk or very high-risk leukaemia may further reduce the risk of CNS relapse. Ongoing trials at St. Jude are testing whether cranial irradiation can be omitted regardless of the patient's risk features and reserved only for retrieval therapy in those who experience CNS relapse.

Many advances have been made in transplantation; these include the prevention of graft-versus-host disease, expansion of the pool of suitable unrelated or related donors, acceleration of engraftment, enhancement of the graft-versus-leukaemia effort, and supportive care. Because of the high response rate to chemotherapy and the questionable efficacy of bone marrow transplantation (BMT) in patients with t(4;11), only Philadelphia chromosome-positive ALL and early haematologic relapse are clear indications for transplantation. Indications for transplantation should be subjected to constant review. Preliminary findings from the Berlin-Frankfurt-Münster consortium suggest that patients with T-cell ALL with a slow early response also benefit from this procedure (M. Schrappe, personal communication).

Late Effects of Treatment for ALL

Most current protocols avoid the use of regimens that can induce second cancer and emphasise the use of glucocorticoids, antimetabolites, and asparaginase as the main agents of treatment. However, the increasing use of glucocorticoids during reinduction and continuation therapy has been associated with a marked increase in the occurrence of osteonecrosis. This complication is more common in older children (>=10 years) and those of female sex and white race (as compared with black race).^86,92 The increased risk in girls may be related to early pubertal development, because maturing bones with epiphyseal closure and reduced intramedullary blood flow are more susceptible to osteonecrosis. The factor(s) that contributes to the racial difference in the incidence of osteonecrosis is unknown. The difference induced by equivalent doses of dexamethasone, prednisone, and prednisolone is also unknown. Recently, several study groups started to decrease the duration of dexamethasone therapy, because the preliminary results of the Children's Cancer Group study indicated that intermittent use of dexamethasone reduces the risk of osteonecrosis (J Nachman, personal communication). Prospective monitoring and early intervention could prevent this debilitating complication, and early osteonecrotic changes may be reversible with proper conservative management. (Pui CH, unpublished observation).

Symptomatic avascular necrosis of the hip joints was not reported in the Hong Kong childhood ALL cohorts in either the HKALL-93 protocol, which was part of UKALL-XI and included prednisone administration, or the HKALL-97 protocol, which was part of BFM-95 and included dexamethasone treatment. The recent findings cannot rule out the possibility that early asymptomatic changes occurred in Chinese patients, because this phenomenon has been described in some studies that used magnetic resonance imaging to screen for the complication.

Another late effect found in the bone is decreased bone mineral density, which has been attributed to cranial irradiation and intensive systemic chemotherapy, especially regimens that include high-dose antimetabolites or glucocorticoids.^93,94 Male sex and white race are significant predictors of low bone mineral density.⁹³ However, the incidence of low bone mineral density in Chinese children is not available. Studies are ongoing to determine whether genetic polymorphisms of the vitamin D receptor influence the severity of low bone mineral density. Although treatment of decreased bone mineral density should reduce the risk of osteoporosis and fractures later in life, intervention studies are needed to determine the optimal therapy (e.g., nutritional counseling, exercise, vitamin D, phosphate or calcium supplementation, and bisphosphonates) to prevent this late effect.

Thrombotic complications occur in approximately 2.4-11.5% of the patients who receive a glucocorticoid, vincristine, and asparaginase as remission induction or reinduction therapy.^95,96 Cerebral venous thrombosis accounts for half of the thrombotic complications. This high frequency can be attributed to the combined use of asparaginase and a glucocorticoid, heightened awareness of the possibility of this complication, frequent placement of central lines, and improved diagnostic imaging methods.

German investigators found that 27 of their 32 patients with thrombotic complications had one or more hereditary prothrombotic defects.⁹⁶ In fact, half of the patients with a prothrombotic defect experienced thrombosis. This finding may pave the way for effective prophylaxis; for example, during glucocorticoid-vincristine-asparaginase treatment, low-molecular weight heparin could be given to patients with hereditary prothrombotic defects. However, preliminary result of a St Jude study did not appear to support the finding (Pui CH, unpublished observation).

The incidence of thrombosis appeared to be lower in Chinese children. Only two of the 145 children on the HKALL-93 (modified UKALL-XI) protocol experienced cerebral venous thrombosis, and neither patient had the hereditary prothrombotic defect described in the literature. There may be genetic differences in the incidence of these defects among ethnic groups; the prothrombotic factor V Leidens mutation, which occurs in factor V Leiden allele, is present in about 5% of the Caucasian individuals (Europeans, Jews, Israeli Arabs, and Indians) and is virtually absent in Africans, Asians including Chinese.⁹⁷

CNS-directed therapy, even that which does not include cranial irradiation, has been associated with adverse cognitive and academic late effects, particularly in girls.⁹⁸ In one study, visual and verbal short-term memory deficits were observed in children who had received approximately 20 triple intrathecal treatments (methotrexate, hydrocortisone, and cytarabine) as the sole CNS-directed therapy;⁹⁹ methotrexate was not administered intravenously. Clearly, neuropsychological function should be assessed in survivors of childhood ALL. Identification of specific therapy-induced cognitive impairments will facilitate the development of appropriate remediation programs. Currently in Hong Kong, a cross sectional functional (by IQ testing and 128 leads EEG) and structural (by MRI and DTI) assessment of the cognitive function among a cohort of childhood leukaemia and brain tumour survivors have been undergoing. By comparing different cohorts of patients who underwent high or low dose cranial irradiation, intrathecal therapy, systemic chemotherapy and a control group, we hope to find out the impact and risk factors of different treatment modalities on the cognitive function of children.

In a retrospective review of 9720 children who received the Children's Cancer Study Group ALL protocols from 1970 to 1988, 43 second cancers were identified. Among these, brain tumours (mainly in the forms of high grade glioma and meningioma) accounted for the majority (56%, 24/43). Acute myeloid leukaemia was the next commonest form of cancer (23%, 10/43).¹⁰⁰ Others included a variety of solid tumours such as thyroid or parotid carcinoma. The age adjusted relative risk of cancer for childhood ALL survivors were 7-fold higher than the normal childhood population. Brain tumours were mainly found in young patients (<5 years) who received cranial irradiation¹⁰⁰ and also those with genetic defect in thiopurine catabolism (i.e. deficiency of thiopurine methyltransferase).¹⁰¹ It can occur many years later and the prognosis was poor except for those with resectable meningioma. The risk of secondary AML appears to be closely associated with the use of epipodophyllotoxin such as VP16 as described in the previous section.¹⁹ It has a relatively brief latency period and associated with 11q23 rearrangement in contrary to the myelodysplastic leukaemia induced by alkylating agents (i.e. cyclophosphamide). The judicious use of cranial irradiation and chemotherapy in current protocols may help to reduce this alarming complication.

The study of genetic polymorphisms in drug-metabolising enzymes, drug transporters, and targets of drug action has attracted intense interest over the past few years. This information can be used to individualise drug dosages (especially those with a low therapeutic index) and drug combinations (to enhance antileukaemic effects and reduce late sequelae). Efforts are also being made to identify new antileukaemic drugs and approaches to therapy.

Identification of specific oncoproteins and the elucidation of the molecular processes that regulate leukaemic-cell survival and apoptosis could also improve treatment.¹⁰² One of the classic examples is imatinib mesylate (Gleevec), which selectively inhibits BCR-ABL tyrosine kinase. Inhibition of this kinase leads to growth inhibition and apoptosis of leukaemic cells that contain the BCR-ABL fusion product. While this agent induced an overall response rate of 82% and a complete response rate of 55% in patients with BCR-ABL⁺ ALL or chronic myeloid leukaemia in lymphoid blast cell crisis,¹⁰³ these responses had been transient. Additional studies are needed to determine if this agent can improve outcome in patients with newly diagnosed BCR-ABL⁺ ALL.

Recently, molecular manipulation of interleukin-4 was shown to abrogate its proinflammatory activity. This finding has provided a novel and therapeutically promising cytokine that induces apoptosis of leukaemic cells in vitro but does not affect the growth of normal haematopoietic cells.¹⁰⁴ Whether this cytokine is clinically useful will require additional studies.

As new information continues to emerge from the Human Genome Project, DNA microarray studies, advanced bioinformatics analyses, high-throughput DNA-screening systems, and proteomics, one can expect accelerated advances in research. DNA microarray technology already has considerable value in molecular diagnosis and identification of new leukaemia-associated markers for disease monitoring.¹⁰⁵ Ultimately, such advances will be used as guides for optimising and individualising therapy.

References

1. Conter V, Arico M, Valsecchi MG, et al. Long-term results of the Italian Association of Pediatric Hematology and Oncology (AIEOP) acute lymphoblastic leukemia studies, 1982-1995. Leukemia 2000;14:2196-204.

2. Schrappe M, Reiter A, Zimmermann M, et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Münster. Leukemia 2000;14:2205-22.

3. Gaynon PS, Trigg ME, Heerema NA, et al. Children's Cancer Group trails in childhood acute lymphoblastic leukemia: 1983-1995. Leukemia 2000;14:2223-33.

4. Harms DO, Janka-Schaub GE. Co-operative study of the childhood acute lymphoblastic leukemia (COALL): long-term follow-up trials 82, 85, 89, and 92. Leukemia 2000;14:2234-9.

5. Kamps WA, Bokkerink JP, Hakvoort-Cammel FG, et al. BFM-oriented treatment for children with acute lymphoblastic leukemia without cranial irradiation and treatment reduction for standard risk patients: results of DCLSG protocol ALL-8 (1991-1996). Leukemia 2002;16:1099-111.

6. Silverman LB, Declerck L, Gelber RD, et al. Results of Dana-Farber Cancer Institute Consortium protocols for children with newly diagnosed acute lymphoblastic leukemia (1981-1995). Leukemia 2000;14:2247-56.

7. Vilmer E, Suciu S, Ferster A, et al. Long-term results of three randomized trials (58831, 58832, 58881) in childhood acute lymphoblastic leukemia: a CLCG-EORTC report. Children Leukemia Cooperative Group. Leukemia 2000;14:2257-66.

8. Gustafsson G, Schmiegelow K, Forestier E, et al. Improving outcome through two decades in childhood ALL in the Nordic countries: the impact of high-dose methotrexate in the reduction of CNS irradiation. Nordic Society of Paediatric Haematology and Oncology (NOPHO). Leukemia 2000;14:2267-75.

9. Maloney KW, Shuster JJ, Murphy S, Pullen J, Camitta BA. Long-term results of treatment studies for childhood acute lymphoblastic leukemia: Pediatric Oncology Group studies from 1986-1994. Leukemia 2000;14:2276-85.

10. Pui CH, Boyett JM, Rivera GK, et al. Long-term results of Total Therapy studies 11,12 and 13A for childhood acute lymphoblastic leukemia at St Jude Children's Research Hospital. Leukemia 2000;14:2286-94.

11. Tsuchida M, Ikuta K, Hanada R, et al. Long-term follow-up of childhood acute lymphoblastic leukemia in Tokyo Children's Cancer Study Group 1981-1995.Leukemia 2000;14:2295-306.

12. Eden OB, Harrison G, Richards S, et al. Long-term follow-up of the United Kingdom Medical Research Council protocols for childhood acute lymphoblastic leukemia, 1980-1997. Medical Research Council Childhood Leukemia Working Party. Leukemia 2000;14:2307-20.

13. Liang DC, Hung IJ, Yang CP, et al. Unexpected mortality from the use of E. coli L-asparaginase during remission induction therapy for childhood acute lymphoblastic leukemia: a report from the Taiwan Pediatric Oncology Group. Leukemia 1999;13:155-60.

14. Li CK, Chik KW, Chan GCF, et al. Treatment of acute lymphoblastic leukaemia in Hong Kong children: HKALL-93 study. Heamatol Oncol 2003;21:1-9.

15. Bhatia S, Ross JA, Greaves MF, et al. Epidemiology and etiology. In: Pui CH, editors. Childhood Leukemias. Cambridge, UK: Cambridge University Press, 1999:38-49.

16. Birch JM, Alexander FE, Blair V, Eden OB, Taylor GM, McNally RJ. Space-time clustering patterns in childhood leukaemia support a role for infection. Br J Cancer 2000;82:1571-6.

17. Chan LC, Lam TH, Li CK, Pui CH. Is the timing of exposure to infection a major determinant of acute lymphoblastic leukaemia in Hong Kong? Paediatr Perinat Epidemiol 2002;16:154-65.

18. Biondi A, Cimino G, Pieters R, et al.Biological and therapeutic aspects of infant leukemia. Blood 2000;96:24-33.

19. Pui CH, Relling MV.Topoisomerase II inhibitor-related acute myeloid leukaemia. Br J Haematol 2000;109:13-23.

20. Hutt AM, Kalf GF. Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene. Environ Health Perspect 1996;104 Suppl 6:1265-9.

21. Frydman B, Marton LJ, Sun JS, et al. Induction of DNA topoisomerase II-mediated DNA cleavage by beta-lapachone and related naphthoquinones. Cancer Res 1997;57:620-7.

22. Wiemels JL, Pagnamenta A, Taylor GM, Eden OB, Alexander FE, Greaves MF. A lack of a functional NAD(P)H:quinone oxidoreductase allele is selectively associated with pediatric leukemias that have MLL fusions. United Kingdom Childhood Cancer Study Investigators. Cancer Res 1999;59:4095-9.

23. Chen CL, Liu Q, Pui CH, et al. Higher frequency of glutathione S-transferase deletions in black children with acute lymphoblastic leukemia. Blood 1997;89:1701-7.

24. Krajinovic M, Labuda D, Richer C, Karimi S, Sinnett D. Susceptibility to childhood acute lymphoblastic leukemia: influence of CYP1A1, CYP2D6, GSTM1, and GSTT1 genetic polymorphisms.Blood 1999;93:1496-501.

25. Ford AM, Pombo-de-Oliveira MS, McCarthy KP, et al. Monoclonal origin of concordant T-cell malignancy in identical twins.Blood 1997;89:281-5.

26. Ford AM, Bennett CA, Price CM, Bruin MC, Van Wering ER, Greaves M. Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia. Proc Natl Acad Sci U S A 1998; 95:4584-8.

27. Wiemels JL, Ford AM, Van Wering ER, Postma A, Greaves M. Protracted and variable latency of acute lymphoblastic leukemia after TEL-AML1 gene fusion in utero. Blood 1999;94:1057-62.

28. Gale KB, Ford AM, Repp R, et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A 1997;94:13950-4.

29. Wiemels JL, Cazzaniga G, Daniotti M, et al. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet 1999;354: 1499-503.

30. Wiemels JL, Xiao Z, Buffler PA, et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002;99:3801-5.

31. Fasching K, Panzer S, Haas OA, et al.Presence of clone-specific antigen receptor gene rearrangements at birth indicates an in utero origin of diverse types of early childhood acute lymphoblastic leukemia. Blood 2000;95:2722-4.

32. Yagi T, Hibi S, Tabata Y, et al.Detection of clonotypic IGH and TCR rearrangements in the neonatal blood spots of infants and children with B-cell precursor acute lymphoblastic leukemia. Blood 2000;96:264-8.

33. Greaves M. Molecular genetics, natural history and the demise of childhood leukaemia. Eur J Cancer 1999;35:173-85.

34. Ma SK, Wan TS, Cheuk AT, et al. Characterization of additional genetic events in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion: a molecular cytogenetics study. Leukemia 2001;15:1442-7

35. Maia AT, Ford AM, Jalali GR, et al. Molecular tracking of leukemogenesis in a triplet pregnancy. Blood 2001:98:478-82.

36. Bhatia S, Sather H, Zhang J, et al. Ethnicity and survival following childhood acute lymphoblastic leukemia (ALL): follow-up of the Children's Cancer Group (CCG) Cohort. Proc Am Soc Clin Oncol 1999;18:568a.

37. Pollock BH, DeBaun MR, Camitta BM, et al. Racial differences in the survival of childhood B-precursor acute lymphoblastic leukemia: a Pediatric Oncology Group Study. J Clin Oncol 2000;18:813-23.

38. Pui CH, Boyett JM, Hancock ML, Pratt CB, Meyer WH, Crist WM. Outcome of treatment for childhood cancer in black as compared with white children. The St Jude Children's Research Hospital experience, 1962 through 1992.JAMA 1995;273:633-7.

39. Pui CH, Evans WE. Acute lymphoblastic leukemia. N Eng J Med 1998;339:605-15.

40. Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999; 286:487-91.

41. Schmiegelow K, Schroder H, Gustafsson G, et al.Risk of relapse in childhood acute lymphoblastic leukemia is related to RBC methotrexate and mercaptopurine metabolites during maintenance chemotherapy. Nordic Society for Paediatric Haematology and Oncology. J Clin Oncol 1995;13:345-51.

42. Evans WE, Relling MV, Rodman JH, Crom WR, Boyett JM, Pui CH. Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia.N Eng J Med 1998;338:499-505.

43. Relling MV, HancockML, Boyett JM, Pui CH, Evans WE. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. Blood 1999;93:2817-23.

44. Relling MV, Pui CH, Sandlund JT,et al. Adverse effect of anticonvulsants on efficacy of chemotherapy for acute lymphoblastic leukemia. Lancet 2000;356:285-90.

45. Stanulla M, Schrappe M, Brechlin AM, Zimmermann M, Welte K. Polymorphisms within glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) and risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia: a case-control study. Blood 2000;95:1222-8.

46. Den Boer ML, Pieters R, Kazemier KM, Janka-Schaub GE, Henze G, Veerman AJ.Relationship between the intracellular daunorubicin concentration, expression of major vault protein/lung resistance protein and resistance to anthracyclines in childhood acute lymphoblastic leukemia. Leukemia 1999;13:2023-30.

47. Dhooge C, De Moerloose B, Laureys G, et al.P-glycoprotien is an independent prognostic factor predicting relapse in childhood acute lymphoblastic leukaemia: results of a 6-year prospective study.Br J Haematol 1999;105:676-83.

48. Zhu YM, Foroni L, McQuaker IG, Papaioannou M, Haynes A, Russell HH.Mechanisms of relapse in acute leukaemia: involvement of p53 mutated subclones in disease progression in acute lymphoblastic leukaemia.Brit J Cancer 1999;79(7-8):1151-7.

49. Carter TL, Watt PM, Kumar R, et al.Hemizygous p16(INK4A) deletion in pediatric acute lymphoblastic leukemia predicts independent risk of relapse.Blood 2001;97:572-4.

50. Prokop A, Wieder T, Sturm I, et al.Relapse in childhood acute lymphoblastic leukemia is associated with a decrease of the Bax/Bcl-2 ratio and loss of spontaneous caspase-3 processing in vivo.Leukemia 2000;14:1606-13.

51. Gaynon PS, DesaiAA, BostromBC, et al. Early response to therapy and outcome in childhood acute lymphoblastic leukemia: a review.Cancer 1997;80:1717-26.

52. Pui CH. Risk assessments in acute lymphoblastic leukemia: beyond leukemia cell characteristics. J. Pediatr Hematol/Oncol2001;23:405-8.

53. Gaynon PS, DesaiAA, BostromBC, et al. Early response to therapy and outcome in childhood acute lymphoblastic leukemia: a review.Cancer 1997;80:1717-26.

54. Coustan-Smith E, Behm FG, Sanchez J, et al.Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 1998;351:550-4.

55. Cave H, van der Werff ten BoschJ, Suciu S, et al.Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia.European Organization for Research and Treatment of Cancer ?Childhood Leukemia Cooperative Group. N Eng JMed 1998;339:591-8.

56. van DongenJJ, Seriu T, Panzer-Grumayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood.Lancet 1998:352:1731-8.

57. Pui CH, Campana D. New definition of remission of childhood acute lymphoblastic leukemia.Leukemia 2000;14:783-5.

58. Coustan-Smith E, Sancho J, Hancock ML, et al.Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia.Blood2000;96:2691-6.

59. VerhagenOJ,Willemse MJ, Breunis WB, et al.Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia.Leukemia2000;14:1426-35.

60. Chen JS, Coustan-Smith E, Suzuki T, et al.Identification of novel markers for monitoring minimal disease in acute lymphoblastic leukemia.Blood 2001;97:2115-20.

61. Neale GA, Coustan-Smith E, Pan Q, et al. Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 1999;13: 1221-6.

63. Reiter A, Schrappe M, Tiemann M, et al. Improved treatment results in childhood B-cell neoplasms with tailored intensification of therapy: a report of the Berlin-Frankfurt-Münster Group Trial NHL-BFM 90.Blood 1999;94:3294-306.

64. Pui CH, Mahmoud HH, Wiley JM, et al. Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients with leukemia or lymphoma. J Clin Oncol 2001;19:697-704.

65. Silverman LB, McLean TW, Gelber RD,et al.Intensified therapy for infants with acute lymphoblastic leukemia:results from the Dana-Farber Cancer Institute Consortium.Cancer 1997;80:2285-95.

66. Dordelmann M, Reiter A, BorkhardtA, et al.Prednisone response is the strongest predictor of treatment outcome in infant acute lymphoblastic leukemia. Blood 1999;94:1209-17.

67. Reaman GH, Sposto R, Sensel MG, et al. Treatment outcome and prognostic factors for infants with acute lymphoblastic leukemia treated on two consecutive trials of Children's Cancer Group.J Clin Oncol 1999;17:445-55.

68. Amylon MD, Shuster J, Pullen J, et al.Intensive high-dose asparaginase consolidation improves survival for pediatric patients with T cell acute lymphoblastic leukemia and advanced stage lymphoblastic lymphoma:a Pediatric Oncology Group study.Leukemia 1999;13:335-42.

69. Belkov VM, Krynetski EY, Schuetz JD, et al.Reduced folate carrier expression in acute lymphoblastic leukemia: a mechanism for ploidy but not lineage differences in methotrexate accumulation.Blood 1999;93:1643-50.

70. Relling MV, HancockML, Boyett JM, Pui CH, Evans WE. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. Blood 1999;93:2817-23.

71. Pui CH, Campana D, Evans WE. Childhood acute lymphoblastic leukaemia-current status and future perspectives. Lancet Oncol 2001:2:597-607.

72. Schrappe M, Reiter A, Zimmermann M, et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Münster. Leukemia 2000;14:2205-22.

73. Hanahan D, Bergers G, Bergsland E. Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest 2000;105:1045-7.

74. Toyoda Y, Manabe A, Tsuchida M, et al. Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood.J Clin Oncol 2000;18:1508-16.

75. Chessells JM, Richards SM, Bailey CC, Lilleyman JS, Eden OB.Gender and treatment outcome in childhood lymphoblastic leukemia: report from the MRC UKALL trials.Br J Haematol 1995;89:364-72.

76. Shuster JJ, Wacker P, Pullen J, et al.Prognostic significance of sex in childhood B-precursor acute lymphoblastic leukemia: a Pediatric Oncology Group Study. J Clin Oncol 1998;16:2854-63.

77. Pui CH, Boyett JM, Relling MV, et al. Sex differences in prognosis for children with acute lymphoblastic leukemia. J Clin Oncol 1999;17:818-24.

78. Chessells JM, Harrison G, Lilleyman JS, Bailey CL, Richards SM.Continuing (maintenance) therapy in lymphoblastic leukaemia:lessons from MRC UKALL X.Medical Research Council Working Party in Childhood Leukaemia. Br J Haematol 1997;98:945-51.

79. Relling MV, Hancock ML, Boyett JM, Pui CH, Evans WE.Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia.Blood 1999;93:2817-23.

80. Yates CR, Krynetski EY, Loennechen T, et al.Molecular diagnosis of thiopurine S-methyltransferase deficiency:genetic basis for azathiopurine and mercaptopurine intolerance.Ann Intern Med 1997;126:608-14.

81. Relling MV, Rubnitz JE, Rivera GK, et al.High incidence of secondary brain tumours after radiotherapy and antimetabolites.Lancet 1999;354:34-9.

82. Relling MV, Yanishevski Y, Nemec J,et al.Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia.Leukemia 1998;12:346-52.

83. Kham SK, Tan PL, Tay AH, Heng CK, Yeoh AE, Quah TC. Thiopurine methyltransferase polymorphisms in a multiracial asian population and children with acute lymphoblastic leukemia. J Pediatr Hematol Oncol 2002;24:353-9

84. Duration and intensity of maintenance chemotherapy in acute lymphoblastic leukaemia:overview of 42 trials involving 12 000 randomised children.Childhood ALL Collaborative Group.Lancet 1996;347:1783-8.

85. Murphy RG, Greenberg ML. Osteonecrosis in pediatric patients with acute lymphoblastic leukemia. Cancer 1990;65:1717-21.

86. Ribeiro RC, Fletcher BD, Kennedy W, et al. Magnetic resonance imaging detection of avascular necrosis of the bone in children receiving intensive prednisone therapy for acute lymphoblastic leukemia or non-Hodgkin lymphoma. Leukemia 2001;15:891-7.

87. Gajjar A, Harrison PL, Sandlund JT, et al.Traumatic lumbar puncture at diagnosis adversely affects outcome in childhood acute lymphoblastic leukemia.Blood 2000;96:3381-4.

88. Silverman LB, Gelber RD, Kimball Dalton V, Palton VK. Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91-01.Blood 2001;97:1211-8.

89. Nachman JB, Sather HN, Sensel MG, et al.Augmented post-induction therapy for children with high-risk acute lymphoblastic leukemia and a slow response to initial therapy.N Engl J Med 1998;338:1663-71.

90. Schrappe M, Reiter A, Ludwig WD, et al.Improved outcome in childhood acute lymphoblastic leukemia despite reduced use of anthracyclines and cranial radiotherapy:results of trial ALL-BFM 90.German-Austrian-Swiss ALL-BFM Study Group. Blood 2000;95:3310-22.

91. Laver JH, Barredo JC, Amylon M, et al.Effects of cranial radiation in children with high risk T cell acute lymphoblastic leukemia: a Pediatric Oncology Group report.Leukemia 2000; 14: 369-73.

92. Mattano LA Jr, Sather HN, Trigg ME, Nachman JB.Osteonecrosis as a complication of treating acute lymphoblastic leukemia in children:a report from the Children's Cancer Group. J Clin Oncol 2000;18:3262-72.

93. Kaste SC, Jones-Wallace D, Rose SR, et al.Bone mineral decrements in survivors of childhood acute lymphoblastic leukemia:frequency of occurrence and risk factors for their development.Leukemia 2001;15:728-34.

94. Pfeilschifter J, Diel IJ.Osteoporosis due to cancer treatment:pathogensis and management.J Clin Oncol 2000;18:1570-93.

95. Sutor AH, Mall V, Thomas KB. Bleeding and thrombosis in children with acute lymphoblastic leukaemia, treated according to the ALL-BFM-90 protocol. Klin Padiatr 1999;211:201-4.

96. Nowak-Gottl U, Wermes C, Junker R, et al.Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carring the MTHFR TT 677 genotype, the prothrombin G20210A variant , and further prothrombotic risk factors.Blood 1999;93:1595-9.

97. De Stefano V, Chiusolo P, Paciaroni K, Leone G. Epidemiology of factor V Leiden: clinical implications. Semin Thromb Hemost 1998;24:367-79.

98. Lesnik PG, Ciesielski KT, Hart BL, Benzel EC, Sanders JA. Evidence for cerebellar-frontal subsystem changes in children treated with intrathecal chemotherapy for leukemia: enhanced data analysis using an effect size model. Arch Neurol 1998;55:1561-8.

99. Hill DE, Ciesielski KT, Sethre-Hofstad L, Duncan MH, Lorenzi M. Visual and verbal short-term memory deficits in childhood leukemia survivors after intrathecal chemotherapy. J Pediatr Psychol 1997;22:861-70.

100. Neglia JP, Meadows AT, Robison LL, et al. Second neoplasms after acute lymphoblastic leukemia in childhood.N Engl J Med 1991;325:1330-6.

101. Relling MV, Rubnitz JE, Rivera GK, et al. High incidence of secondary brain tumours after radiotherapy and antimetabolites. Lancet 1999;354:34-9.

102. Pui CH, Sallan S, Relling MV, Masera G, Evans WE.Meeting report: International Childhood Acute Lymphoblastic Leukemia Workshop: Sausalito, CA, 30 November - 1 December 2000. Leukemia 2001;15:707-15.

103. Talpaz M, Sawyers CL, Kantarjain H, et al.Activity of an ABL specific tyrosine kinase inhibitor in patients with BCR-ABL positive acute leukemias, including chronic myelogenous leukemia in blast crisis.Proc Am Soc Clin Oncol 2000;19:4a.

104. Srivannaboon K, Shanafelt AB, Todisco E, et al. Interleukin-4 variant (BAY 36-1677) selectively induces apoptosis in acute lymphoblastic leukemia cells.Blood 2001;97:752-8.

105. Yeoh EJ, Ross ME, Shurtleff SA, et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002;1:133-43.