How can we improve on the already impressive results in pediatric ALL?

Despite the success of the treatment of childhood acute lymphoblastic leukemia, at the turn of the millennium, still ∼20% of children relapse and 15% succumb to the disease.¹  It is clear from the early days of treatment that a significant proportion of children require less intensive therapy for cure but even with increasing intensity of treatment, there remains a group of children with very resistant disease. Risk groups defined by both clinical and laboratory features, including cytogenetics, have been introduced to guide intensity of treatment but this has been insufficient to discriminate reliably between groups in predicting risk of relapse.¹  For those in morphologic remission, determining response at the molecular level using either PCR or flow cytometry to identify and quantify leukemic clones [minimal residual disease (MRD) detection] and adding this information to the stratification has improved the predictive power of the groupings. Results from the United Kingdom ALL 2003 (UKALL 2003) trial showed successful reduction of therapy in the clinical low-risk and intermediate-risk groups who achieved undetectable MRD at day 29 or week 11, maintaining a 5 year event-free survival (EFS) of >94%.²  Similarly, in the same study, those who were in the clinical low-risk and intermediate-risk groups who had MRD detectable at ≥0.01% at day 29, were randomized to standard versus augmented therapy. Those with increased treatment intensity had an improved 5 year EFS (89.6% vs 82.2%, P = 0.04), but no significant overall survival advantage as yet (92.9% vs 88.9%, P = 0.16). Adverse events were more frequent in the augmented treatment group.³  Despite improved risk stratification, unanswered questions remain such as why there is a poorer outcome in older children and young adults and why those with no identifiable high-risk features can have a poor outcome. With such excellent disease free survival rates, treatment related mortality (TRM) becomes a significant cause of death (3.2% in the UKALL 2003 study) compared with disease-related mortality yet still there is a risk of very early relapse and resistant disease in a similar proportion. There is a clear need therefore to continue to refine definition of risk groups but also to develop therapies and design treatment protocols that will maximize efficacy and minimize toxicity in a patient focused manner.

Sensitive and specific ways of identifying patients at diagnosis who are at high risk of relapse are being developed so that therapy can be optimized without delay. In exploratory studies, by examining the DNA of leukemic cells using comprehensive genomic analysis in patients known to have had a poor outcome, genetic aberrations have been identified. This approach has had the combined benefit of improving the understanding of leukemogenesis, identifying genetic abnormalities conferring a poor prognosis and allowing development of therapies specifically targeted at the genetic abnormality.⁴ 

In treating any leukemia, the benefit–risk balance will vary between individual patients and will depend on both the susceptibility of the leukemia to the treatment and also the susceptibility of the patient to the toxic effects of the treatment. There is interplay between the biological characteristics of the leukemia and host and the responsiveness of each to the therapeutic and adverse effects of treatment. Genomic profiling may help elucidate some of these complex interactions.⁵  Where the leukemia is sensitive, then it is possible that therapy can be tailored according to the patient's pharmacogenomics profile. However, where the genomic aberration in the leukemic cell line predicts a leukemia at high-risk of relapse or poor response to treatment, the increased toxicity of therapy may well be justified and trump the use of pharmacogenomics to modify therapy. However, designing targeted therapies may allow intensification of treatment with acceptable toxicities and some of those therapies may also find a place in the treatment of more responsive leukemias.⁶ 

Just as stratification of patients into standard, intermediate, and high-risk groups using clinical criteria alone was insufficient to predict relapse, so the identification of high- and low-risk genetic signatures using low-resolution molecular techniques do not identify all patients at risk of relapse or poor outcome. Many children who relapse lack a known high-risk chromosomal rearrangement and many of the chimeric fusions identified do not induce leukemia in nonclinical models and indeed may be present many years before leukemia develops. Detailed genomic analysis has begun to answer some of these conundrums and in doing so has identified possible new molecular targets for therapy in those groups at high risk of failure with conventional chemotherapy. It is possible that such therapy can be extended to other groups with lower risk disease as there is evidence that combined therapy may improve outcome by reducing toxicities and also by sensitizing the leukemia cells to conventional therapy so that lower doses can be used.

One successful example of using complementary genomic analysis techniques is that of the study undertaken in the well characterized population treated on the Children's Oncology Group's COG P9906 protocol. A subgroup of 207 patients with high-risk BCR-ABL1–negative B-ALL was studied and a key finding in 29 patients using gene expression profiling was overexpression of CRLF2, a gene encoding cytokine receptor like factor 2,⁷ which is a receptor for thymic stromal lymphopoietin (TSLP). Genomic sequencing revealed rearrangements of this gene in every case. Twenty-seven patients had 1 of 2 rearrangements: 62% with the immunoglobulin heavy chain gene (IGH@) on chromosome 14 and 34% with the P2RY8 (P2Y purinoceptor 8 gene) on the pseudo-autosomal region of chromosome Xp/Yp; 1 had both rearrangements and 1 had a novel rearrangement. Alteration of this gene led to overexpression of the receptor on the cell surface of leukemia cells, which could be detected by flow cytometry.⁷  All had a very poor outcome compared with those without CRLF2 rearrangements (35.3% vs 71.3% 4 year relapse-free survival; P < 0.001).⁷  TSLP activates STAT3, STAT5 and JAK2 pathways, which control cell proliferation and development of the hematopoietic system, giving biological plausibility to its potential role in leukemogenesis. Further study in patients with CRLF2 rearrangements revealed association with activating JAK1/2 mutations in >50% but at a different locus from that seen in myeloproliferative disease. Interestingly, in patients with Down Syndrome ALL, where an increased incidence of relapse and TRM are seen, investigation shows that CRLF2 rearrangements are found in ∼50%, which is 10-fold higher than the rate in unselected ALL cases.^4,8 

In the same patient population using similar techniques, mutations or deletions of IKZF1 were identified in approximately one-third of patients with BCR-ABL1–negative high-risk B-ALL. Alterations of this gene, encoding the early lymphoid transcription factor (IKAROS), had previously been found in BCR-ABL1–positive cases of B-ALL, patients already therefore identifiable as high risk. The gene expression profiles of IKZF1–altered BCR-ABL1–positive and high-risk BCR-ABL1–negative ALL were the same, giving rise to the term BCR-ABL1–like or Philadelphia-like ALL (Ph-like ALL). These leukemias were associated with a poor outcome independent of other risk factors.

The findings of a poorer outcome in pediatric patients with CRLF2 overexpression were replicated in 555 unselected patients treated on the German ALL BFM-2000 protocol. CRLF2 overexpression was found in 8.8% of patients, 43% having the P2RY8-CRLF2 fusion gene, 8% the IGH@- CRLF2 fusion gene, and 18% additional CRLF2 copies. The highest levels of overexpression were seen with the fusion genes rather than with increased copy number and JAK2 mutations where they occurred were again not in this latter group. Patients classified as NCI non-high-risk with the fusion gene had a high rate of relapse (71% ± 19% at 6 years).⁸ 

More recently, a study of 167 unselected patients with pre B-ALL in a Japanese population showed CRLF2 overexpression in 18% (30/167). Only 10% (3/30) had the P2RY8-CRLF2 fusion gene, whereas 60% (18/30) had gain of function. CRLF2 rearrangement was not associated with a poor prognosis (5 year EFS 70.7% with vs 75.4% without; P = 0.68) but in those with IKZF1 deletions in addition, 5 year EFS was significantly reduced (44.4% vs 83.1%; P = 0.02). Using multivariate analysis, only the IKZF1 deletion was a significant predictor of outcome in this population.⁹ 

Subsequent molecular study of Ph-like ALL in an American population revealed that it was genetically complex but with identifiable kinase-activating alterations in >90% of cases.¹⁰  In Roberts' study of 1725 children, adolescents and young adults aged from 1 to 39 years, Ph-Like ALL was found with increasing frequency in the older age groups, from 10% in children with standard risk ALL to 27% in young adults.¹⁰  Rearrangements of CRLF2 were found in 47% of those studied, with associated JAK mutations, most commonly JAK2, in 55%. Another subgroup identified was characterized by fusions with the ABL-class kinase genes. Although many different genetic alterations were identified, there was activation of only a limited number of signaling pathways, most notably JAK2, particularly in young adults. Preclinical studies using cell culture techniques have indicated sensitivity of pre-B cells expressing the dominant negative Ikaros isoform and a variety of fusions to tyrosine kinase inhibitors with a PAX5-JAK2 fusion sensitive to ruxolitninb and ABL-kinase fusions sensitive to dasatinib. Dasatinib also showed activity in a xenograft mouse model of engrafted human ETV6-ABL1 ALL cells. Both dastinib and ruxolitinib have been licensed for other indications and where they have been used in patients with ALL and the relevant fusion, rapid and durable responses have been seen. Trials incorporating screening for Ph-like ALL with genomic analysis are planned to test the utility of chemotherapy and kinase inhibitors in this high-risk population.

Genomic analysis in T-cell ALL has not been as successful in identifying genetic alterations associated with outcome. More recently however, a subtype of T-cell leukemia, early T-cell precursor (ETP) leukemia, with an apparent extremely poor prognosis has been identified.¹¹  It has a characteristic immunophenotype and gene expression profile, including overexpression of MEF2C (myocyte enhancer factor 2C). Homminga et al have shown that overexpression is associated with rearrangements of MEF2C or transcription factors or cofactors targeting the gene.¹²  Further investigation of rearrangements of MEF2C as the underlying genetic lesion for this subtype is in progress.¹³  More recently, inhibition of the JAK/STAT pathway using ruxolitinib has shown efficacy in murine xenograft models of ETP ALL,¹⁴  and encouragingly, results from a study of T-cell leukemia by the Children's Oncology Group (COG) have shown that intensification of therapy stratified according to end-induction MRD overcomes the high-risk profile of ETP-ALL.¹⁵ 

Some of these genetic findings may begin to explain the poorer outcome in adolescents and young adults in whom an increase in T-cell and ETP ALL, a decrease in good prognosis genetic alterations and an increase in BCR-ABL1–positive and Ph-like ALL are found. However, intensification of treatment according to end-induction MRD and use of therapeutic regimens, which include TKIs and JAK/STAT inhibitors, may improve response.

In childhood lymphoblastic leukemia, detailed genomic analysis has mostly focused on the leukemic cells but with the increasing success of treatment for most, reduction of toxicity both acute and long-term is an important goal. Reduction of toxicity, which may also improve adherence to regimens, may be achieved by using targeted therapies, both chemical and biological, but currently the mainstay of therapy remains cytotoxic treatment. Patients show different susceptibilities to the toxicity of the chemotherapy used and some of those differences can be explained by germline DNA variants. The most studied to date is probably the variation of the TPMT gene and sensitivity to purine analogues. Thiopurine methyltransferase metabolizes 6 mercaptopurine (6MP) to thioguanine nucleotide metabolites (TGN), which have both cytotoxic and immunosuppressive properties. 6MP forms additional intermediate metabolites, one of which competes for TPMT to form further metabolites at the expense of forming TGN. Heterozygotes account for 10% of the population and have lower TPMT activity and higher levels of TGN than wild-type and a lower relapse risk. Homozygotes have a frequency of 1 in 300. In the UK ALL97 study, those with the commonest allele variant TPMT*1/*3A had a 5 year EFS of 88% compared with 80% for wild-type (P = 0.05).¹⁶  However, the range of doses for heterozygotes was wide and similar to those of wild-type and does not support prescribing according to genotype but to continue titrating against toxicity particularly as thiopurine cytopenias were not detrimental to outcome.¹⁶  Further investigation of 6MP intolerance by Yang et al using a genome-wide association (GWA) study revealed an association with polymorphic variation of the NUDT15 gene, which mediates the hydrolysis of some nucleoside diphosphate derivatives and is a key modulator of thiopurine cytotoxicity. TPMT and NUDT15 polymorphisms appear to be independent variables and although TPMT has the strongest association, much of the SE Asian sensitivity to 6MP is because of NUDT15.¹⁷  Genotyping of these variants may have a role in individualized dosing in susceptible populations.

The role of TPMT status has also been examined in relation to both myelo- and hepatotoxicity in a regimen where 6MP is combined with high dose methotrexate (HD-MTX). The2 drugs act synergistically and it is possible that heterozygotes will experience more toxicity than those with wild-type resulting in more profound myelosuppression and treatment interruption. In the study by Levinsen et al, the patients were divided into intermediate- and high-TPMT activity groups¹⁸ ; phenotyping can be discordant with genotyping and thus genotype cannot be reliably inferred.¹⁹  However, TPMT activity had no impact on HD-MTX related myelotoxicity, hepatotoxicity, or treatment when patients received the same dose, and had the same blood count and alanine transferase levels at the start of HD-MTX. 6MP dose was the most important predictor and not TPMT activity during high-intensity treatment, and thus again status should not guide dosage.¹⁸ 

More recent reports have shown that some patients have an increased susceptibility to vincristine toxicity. Diouf et al studied 222 children treated for ALL on protocols delivering between 36 and 39 doses of vincristine and using GWA studies showed that a mutation in the promoter region of the CEP72 gene, which encodes a centrosomal protein involved in microtubule formation, had a significant association with vincristine toxicity. Sixteen percent of patients were homozygous for the risk allele, which binds a repressor transcription factor, and were at increased risk and severity of vincristine-related neuropathy with earlier onset compared to those with heterozygous or wild-type genotypes. Cellular studies showed that reducing CEP72 expression in human neurons and leukemia cells increased their sensitivity to vincristine. It is possible therefore that reduction in vincristine dosage in susceptible patients may improve the toxicity profile without compromising efficacy.²⁰ 

Sensitivity to ara-C (cytarabine) has been studied using 8 single-nucleotide polymorphisms (SNPs) in 5 genes key in its cellular transport.²¹  Two SNPs in the DCK gene, which encodes deoxycytidine kinase, were associated with hematologic toxicity. Ara-C is actively transported into cells and then metabolized by deoxycytidine kinase to ara-CMP, the rate-limiting step, with subsequent metabolism to the active ara-CTP. DCK rs12648166 G and rs4694362 T alleles were associated with a higher risk of grade 3/4 leucopenia (but not thrombocytopenia) with homozygotes for the risk alleles giving odds ratios of 2.25 (P = 0.005) and 2.24 (P = 0.0053), respectively compared with wild-type. The frequency of both the G and T risk alleles is 0.60 and they are in strong linkage disequilibrium. Individualized chemotherapy based on genetic profiling may help optimize ara-C dosing and reduce toxicity; the SNPs had no significant influence on survival. GWA studies investigating the genetics of chemotherapeutic susceptibility of ara-C in lymphoblastoid cell lines have not identified ara-C metabolizing enzymes. This study suggests that variation in DCK is more responsible for the side effects of the treatment. However, DCK may be important in ara-C resistance where expression is reduced by 60% in ara-C resistant cells.²¹ 

Although the most common malignancy in children, childhood acute lymphoblastic leukemia is still a rare disease with ∼400 cases (<14years) per annum in the UK and 3600 (<20 years) in the US. Because outcome is in general excellent, trials to evaluate novel therapies in children and young adults with refractory, resistant, or relapsed disease are particularly challenging as the numbers become very small, especially if there is division of these patients into further subgroups according to particular characteristics that may influence success of therapy. Thought needs to be given to prioritizing the most promising agents where trials may compete for patients, particularly if the agent is in the same therapeutic class as others being studied. Cooperation between researchers, industry, the regulatory authorities and other key players is vital to maximize resources.^22,23  However, there are opportunity costs in pursuing only 1 treatment option, particularly if results are not positive and this is felt particularly in rare diseases.²⁴  Development of novel agents has been based on the biology of the leukemia and leukemogenic events, and with the increasing knowledge that genomic analysis has provided, translational research is essential and all trials must aim to collect and process samples in a coordinated way to investigate biological data including perimortem material where possible. It is already known that leukemic blasts at diagnosis show clonal heterogeneity and detailed sequential genomic analysis has shown the disappearance, emergence, and evolution of clones as the leukemia is exposed to treatment.^4,25  Relapsed disease shows biological evolution in the majority of cases and the appearance of clones that are only detectable from the diagnostic sample using molecular methods. Characterization of the molecular response of relapsed disease to novel therapies may be informative for the best use of such therapies or to examine the mode of resistance where such therapies fail.²⁶ 

Trial design must be optimized where the numbers of patients available for recruitment are small and model based designs make better use of all recorded data. Use of a Bayesian design for example where prior knowledge of the drug and trial data are used together to continually update information about the distribution of the observed outcome of interest may be more appropriate than the more traditional 3 + 3 design. The 3 + 3 design, or modifications of it, do not capture late-occurring toxicities or lower-graded, cumulative, or additive toxicities that do not meet dose-limiting toxicity (DLT) criteria. Such observations may yield important safety or activity data and reduce uncertainty about the treatment effect and inform future trial designs.²⁵  The 3 + 3 trial design was used in 96.7% phase I cancer trials in 2007-2008 and only 1.6% followed a Bayesian design²⁷ ; such a design has yet to be used in pediatric leukemia trials.²⁸  Bayesian modeling results in a superior ability to identify the dose with the desired toxicity rate and allocation of a greater proportion of patients to doses at, or close to, that dose which is clearly important in small populations.²⁷ 

Cytotoxic chemotherapy characteristically has a narrow therapeutic range with dose-limiting toxicity. The assumption is that increased toxicity rises in parallel with increased efficacy thereby the highest-tolerated dose will equate with the most efficacious dose. This is not necessarily so for targeted therapies as target effect and toxicity may not be linear.²⁷  Toxicity in nonmalignant tissues may not be a valid surrogate and targeted therapy may have a plateau effect.²⁸  There may also be off-target effects again unrelated to maximum dose. These effects may be unexpected and more varied than with conventional chemotherapy and more difficult to predict from preclinical models because of their biological nature, precise target, or immunogenic properties in species other than man. Phase I trials allow assessment of short-term toxicities and may miss cumulative toxicities in targeted therapies, which may be given continuously for a longer period. Mandatory follow-up is often limited to 3-5 years yet it is possible that effects will be seen after this, such as the growth retardation observed when imatinib was used long-term in prepubertal children,²⁹  found later to be because of disruption of GH/IGF-1 axis.³⁰  There is no mechanism for assessment of tissue maturation and physical growth in early phase trials although there may be helpful data in preclinical juvenile models. Time-To-Event Continual Reassessment Method (TITE CRM) may allow assessment of toxicities over several cycles of treatment and is being introduced into phase I pediatric oncology trials.³¹  In addition to the recording of adverse events, different end points other than toxicity biomarkers, such as on-target and off-target binding or drug concentration in the bone marrow may be more informative, particularly if the precise mode of action is not known. There is a need to understand the underlying biology and pharmacodynamic interactions because cell death may not be an appropriate end point, for instance if the therapy is designed to induce cell maturation; time to progression or remission may be more appropriate end points.²⁸ 

Combination therapy may be difficult to test although probably necessary in a relapsed or resistant population. Phase II window studies in treatment naïve patients where the new agent is used before conventional therapy starts can partly overcome this problem, as can phase II multi-arm studies. Phase III studies with incorporation of the novel drug with conventional therapy are challenging. If there is a very high cure rate as in ALL, the goal may be equivalence or reduced toxicity but in the relapsed or resistant setting, evidence of some activity with acceptable toxicity may be the aim. To improve the safety of introducing new therapies into the pediatric population, safety data from adult use where the drug may be the same but the target defines a different disease, such as the TKIs in chronic myeloid leukemia.

Last, the regulatory bodies have schemes to encourage development of pediatric medicines and promising medicines in rare diseases. The European Medicines Agency (EMA) may designate a new therapy as a promising innovative medicine allowing application to the Early Access to Medicines Scheme route to licensing. This scheme aims to provide earlier availability of promising new unlicensed medicines to patients who have a high, unmet clinical need. It is primarily aimed at medicines that have completed phase III trials, but may be applied to completed phase II trials in exceptional circumstances.

Cytotoxic chemotherapy remains the mainstay of treatment for childhood ALL. Such therapy however is limited by its toxicity, both short-term and long-term, and also by lack of efficacy in a significant proportion of patients because of inherent or acquired resistance. Detailed genomic analysis of leukemia cells, particularly in subgroups known to respond poorly to chemotherapy, has identified recurrent chromosomal rearrangements and submicroscopic genetic alterations that may be amenable to targeted therapy including small molecule inhibitors, immunotherapies, or small interfering RNAs (siRNAs). The assumption is that targeted therapies will be safe, effective, and potentially curative and because of their specificity will have reduced toxicity and no off-target effects. It may also be possible to identify mechanisms that have rendered the leukemia resistant to therapy, and either design therapies that will overcome these alterations or use engineered siRNAs to silence the signaling pathway at the RNA level. Some targeted therapies will be made using autologous cells and developed as advanced therapeutic medicinal products, such as chimeric antigen receptor (CAR) T cells. These therapies will be made on an individual basis and with current manufacturing technologies will not be widely applicable, but for those with multiple-relapsing or resistant disease may achieve significant response, and enable proof of principle and safety to be established.

The tyrosine kinase inhibitors (TKIs) imatinib and dasatinib have greatly improved the response and outcome in patients with BCR-ABL1–positive ALL,³²  and as discussed above, trials are being designed to introduce them, along with other TKIs such as ruxolitinib, depending on the fusion identified, in patients with Ph-like ALL. Although targeted, imatinib and related TKIs have off-target effects, because they are not specific for the BCR-ABL fusion protein and thus may affect other c-ABL kinases. Mutations in BCR-ABL1 prevent first- and second-generation TKIs from binding leading to resistance, which has limited their use in some patients with chronic myeloid leukemia. Ponatinib, a multi-targeted TKI, was designed to bind across the mutated sites and overcome such resistance but has more off-target effects, including significant cardiovascular toxicity due to inhibition of survival pathways shared by both cancer and cardiac cells.^33,34 

Another small molecule inhibitor, EPZ 5675, is showing targeted activity in the treatment of MLL-rearranged acute leukemia. Due to genetic alterations in MLL, DOT1, which encodes a histone methyl transferase, is misregulated resulting in the increased expression of the genes HOXA9 and MEIS1 that promote leukemogenesis. The histone methyl transferase inhibitor, EPZ 5675, inhibits DOT1L and has shown selective killing of several cell lines bearing MLL-fusion proteins and tumor regression in a rat xenograft model. It is currently undergoing trials in the treatment of pediatric patients with MLL-rearranged acute leukemia including ALL.³⁵ 

The immune system is highly specific and adaptive and with manipulation has been used to treat malignant disease including leukemia. Various classes of agent are available including monoclonal antibodies and CAR T-cells.³⁶  The principle underlying CAR T-cell therapy is the generation of large numbers of autologous peripheral T cells, which recognize a surface antigen expressed on malignant cells. This is achieved by ex vivo transduction of the patient's peripheral blood T cells with a gene encoding a CAR. For B-ALL, CD19 has been selected as the target antigen as it is not found in other organs or hematopoietic stem cells. A dose-escalation trial using CD19-CAR T cells in 21 young children and adults with resistant pre B-ALL showed potent antileukemic activity with cytokine-release syndrome (CRS) as a notable but manageable toxicity.³⁷  Promising results have been reported in 30 patients with relapsed or resistant disease, including 25 children of whom 18 were post-allogeneic transplant, with 90% achieving complete remission, some lasting up to 24 months, and a 6 month EFS of 67%. CRS was seen in all patients and severity was related to burden of disease rather than dose of cells.²⁶ 

Small interfering RNAs (siRNAs) are double-stranded RNAs between 21 and 25 oligonucleotides in length and are designed to specifically degrade the target mRNA and thus inhibit protein expression. They have been used in several settings including gene knockdown models and are showing potential as therapeutic agents to “silence” or modulate genes, such as the multidrug resistance gene in leukemia cells restoring or increasing drug sensitivity.³⁸  Increasingly, as molecular alterations in acute leukemia are being identified and linked to aberrant function through the action of fusion proteins, so the possibility of gene silencing with siRNAs becomes a therapeutic option. This approach has been used with targets, such as BCR1-ABL and MLL gene rearrangements. One of the challenges with using siRNAs as therapeutic agents is safe and effective delivery to the target cell, and the development of nanotechnology in parallel will be key to the success of this developing therapeutic area.^39-41 

Harnessing the power of the new methods of genomic analysis to understand leukemogenesis, stratify patients, predict risk of relapse, and identify targets for novel therapy has increased the opportunity for tailoring intensity of treatment and targeting therapy according to new criteria and genetic markers. Many challenges remain but continued communication and development of collaborative links between scientists, academics, clinical investigators, industry, and regulatory bodies should help speed the path for basic research to translate into improved patient outcomes in pediatric ALL.

Angela Thomas, Royal Hospital for Sick Children, Edinburgh EH9 1LF, UK; Phone: +44 131 536 0000; Fax: +44 131 536 0430; e-mail: angela.thomas2@nhs.net.