Role of DNA mismatch repair genetic polymorphisms in the risk of childhood acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer. Genetic variants in the coding regions of the mismatch repair genes MLH1 (Ile-219Val) and MSH3 (Arg-940Glu and Thr-1036Ala) could contribute to an individual's susceptibility as modifiers in leukaemogenesis. To investigate this possibility, we conducted a case-control study on 287 children with ALL and 320 healthy controls both of French-Canadian origin. MLH1 and MSH3 variants, when taken independently, seemed to play little or no role in the aetiology of childhood ALL. However, when the MLH1 genotypes were combined with genotypes previously shown to influence ALL susceptibility, we found that the MLH1 variant Val-219 further increases the risk of GSTM1 null and CYP1A1*2A genotypes [combined odds ratio (OR) = 6.0, P = 0.002] as well as that of CYP2E1*5 (OR = 15.8, P = 0.001). No association was found with MSH3 variants. This study suggests an association of leukaemogenesis in children with both xenobiotic metabolism and DNA repair, and thus points to the effect of environmental exposure.     

An infectious aetiology for childhood acute leukaemia: a review of the evidence

There are three current hypotheses concerning infectious mechanisms in the aetiology of childhood leukaemia: exposure in utero or around the time of birth, delayed exposure beyond the first year of life to common infections and unusual population mixing. No specific virus has been definitively linked with childhood leukaemia and there is no evidence to date of viral genomic inclusions within leukaemic cells. The case-control and cohort studies have revealed equivocal results. Maternal infection during pregnancy has been linked with increased risk whilst breast feeding and day care attendance in the first year of life appear to be protective. There is inconclusive evidence from studies on early childhood infectious exposures, vaccination and social mixing. Some supportive evidence for an infectious aetiology is provided by the findings of space-time clustering and seasonal variation. Spatial clustering suggests that higher incidence is confined to specific areas with increased levels of population mixing, particularly in previously isolated populations. Ecological studies have also shown excess incidence with higher population mixing. The marked childhood peak in resource-rich countries and an increased incidence of the childhood peak in acute lymphoblastic leukaemia (ALL) (occurring at ages 2-6 years predominantly with precursor B-cell ALL) is supportive of the concept that reduced early infection may play a role. Genetically determined individual response to infection may be critical in the proliferation of preleukaemic clones as evidenced by the human leucocyte antigen class II polymorphic variant association with precursor B-cell and T-cell ALL.     

Philadelphia positive acute lymphoblastic leukaemia of childhood

On current chemotherapeutic regimens, children with Philadelphia positive acute lymphoblastic leukaemia show a heterogeneous response to treatment. A few respond quickly to treatment and achieve long-term remission. Some fail to achieve remission after induction and the majority respond slowly to treatment. Relapse on treatment is common and remission is sustained in a proportion of cases only after allogeneic stem cell transplantation (allo-SCT). The use of imatinib along with conventional cytoreductive therapy, prior to allo-SCT appears to be the most promising strategy. The future lies in the molecular evaluation of response to treatment and combination targeted chemotherapy.     

Somatic PTPN11 mutations in childhood acute myeloid leukaemia

Somatic mutations in PTPN11, the gene encoding the transducer SHP-2, have emerged as a novel class of lesions that upregulate RAS signalling and contribute to leukaemogenesis. In a recent study of 69 children and adolescents with de novo acute myeloid leukaemia (AML), we documented a non-random distribution of PTPN11 mutations among French-American-British (FAB) subtypes. Lesions were restricted to FAB-M5 cases, where they were relatively common (four of 12 cases). Here, we report on the results of a molecular screening performed on 181 additional unselected patients, enrolled in participating institutions of the Associazione Italiana Ematologia Oncologia Pediatrica-AML Study Group, to provide a more accurate picture of the prevalence, spectrum and distribution of PTPN11 mutations in childhood AML and to investigate their clinical relevance. We concluded that PTPN11 defects do not represent a frequent event in this heterogeneous group of malignancies (4.4%), although they recur in a considerable percentage of patients with FAB-M5 (18%). PTPN11 lesions rarely occur in other subtypes. Within the FAB-M5 group no clear association of PTPN11 mutations with any clinical variable was evident. Nearly two third of the patients with this subtype were found to harbour an activating mutation in PTPN11, NRAS, KRAS2 or FLT3.     

Predicting relapse risk in childhood acute lymphoblastic leukaemia

Intensive multi‐agent chemotherapy regimens and the introduction of risk‐stratified therapy have substantially improved cure rates for children with acute lymphoblastic leukaemia (ALL). Current risk allocation schemas are imperfect, as some children are classified as lower‐risk and treated with less intensive therapy relapse, while others deemed higher‐risk are probably over‐treated. Most cooperative groups previously used morphological clearance of blasts in blood and marrow during the initial phases of chemotherapy as a primary factor for risk group allocation; however, this has largely been replaced by the detection of minimal residual disease (MRD). Other than age and white blood cell count (WBC) at presentation, many clinical variables previously used for risk group allocation are no longer prognostic, as MRD and the presence of sentinel genetic lesions are more reliable at predicting outcome. Currently, a number of sentinel genetic lesions are used by most cooperative groups for risk stratification; however, in the near future patients will probably be risk‐stratified using genomic signatures and clustering algorithms, rather than individual genetic alterations. This review will describe the clinical, biological, and response‐based features known to predict relapse risk in childhood ALL, including those currently used and those likely to be used in the near future to risk‐stratify therapy.     

Intraocular relapse of childhood acute lymphoblastic leukaemia

Relapse of childhood acute lymphoblastic leukaemia (ALL) involving the eye is a rare but challenging problem. Twenty cases occurred in patients treated on the Medical Research Council United Kingdom Acute Lymphoblastic Leukaemia XI and ALL97 trials between 1991 and 2001, representing 2.2% of ALL relapses. Seventeen occurred as a first relapse, either in isolation or combined with relapse at another site, and three occurred as a second relapse. All patients with intraocular disease at first relapse were treated with both chemotherapy and radiotherapy, but the doses and protocols used varied. Eleven of these 17 patients are alive and in complete remission with a median follow up of 4 years 2 months from relapse. All 11 children that were treated with a full chemotherapy relapse protocol, together with local radiotherapy have survived. Patients treated with chemotherapy of shorter duration and intensity, despite radiotherapy and/or bone marrow transplantation, did poorly with only one survivor, currently in chronic relapse. Consequently, we suggest that children with eye relapse of ALL be treated with an intensive relapse chemotherapy protocol with local ocular radiotherapy, whether the relapse occurs in isolation or in combination with relapse at another site.     

Glycophorin A mutations and risk of secondary leukaemia in patients treated for childhood acute lymphoblastic leukaemia

Survivors of childhood acute lymphoblastic leukaemia (ALL) have a higher than expected risk of developing secondary acute myeloid leukaemia (AML). The glycophorin A (GPA) mutation assay measures the frequency of variant NO and NN erythrocytes in MN heterozygotes. A raised variant frequency (Vf) has been shown in patients treated with chemotherapy known to be at risk of secondary leukaemia. ALL patients were investigated for increased Vf using the GPA assay. Vf's at diagnosis were not significantly different from controls (NO Vf P  = 0.193; NN Vf P  = 0.790). During treatment Vf's increased significantly (NO Vf P  = 0.001; NN Vf P  = 0.001). NO Vf returned to control values ( P  = 0.169) within 5 years from diagnosis but NN Vf remained significantly raised ( P  = 0.014). Three study patients developed secondary AML. At diagnosis of AML all three had significantly increased Vf. The first had a significantly raised Vf at routine follow-up 19 years following diagnosis of ALL then developed AML 3.5 years later. The second had a significantly raised NN Vf at diagnosis of ALL indicating possibly prior exposure to a mutagen or defective DNA repair involving erythroid stem cells. We conclude that a raised Vf detected by the GPA assay can act as a marker for the development of secondary induced leukaemia and can be used to screen individuals at a known high risk of this complication.     

Cellular drug sensitivity in MLL-rearranged childhood acute leukaemia is correlated to partner genes and cell lineage

Rearrangements in the 11q23 region, the site of the mixed lineage leukaemia (MLL) gene, are found in both childhood acute myeloid (AML) and lymphoblastic (ALL) leukaemia. We studied the in vitro drug resistance by the fluorometric microculture cytotoxicity assay (FMCA) in 132 children with AML and 178 children with ALL (aged 0-17 years). In AML, children with t(9;11) (n = 10) were significantly more sensitive to cytarabine (P < 0.001) and doxorubicin (P = 0.005) than non-11q23 rearranged patients (n = 108). Children with other 11q23 rearrangements (n = 14) differed less from non-rearranged children. The 'AML-profile' common to all three groups included relative resistance to glucocorticoids and vincristine. In ALL, children with 11q23 rearrangement (n = 22) were significantly more sensitive to cytarabine (P = 0.026) than children without 11q23 rearrangement (n = 156), also after stratification for white blood cell count. In conclusion, the findings indicate that the cellular drug resistance is correlated to both the cell lineage and the type of 11q23 rearrangement. High cellular sensitivity to cytarabine and doxorubicin might explain the excellent treatment results in children with AML and t(9;11). The present study supports the strategy of contemporary protocols to include high-dose cytarabine in the treatment of 11q23-positive patients both in AML and ALL.     

Potentiation of in vitro ara-C cytotoxicity by ribonucleotide reductase inhibitors, cyclin-dependent kinase modulators and the DNA repair inhibitor aphidicolin in paediatric acute myeloid leukaemia

To modulate in vitro cytarabine (ara-C) resistance we combined ara-C with six potential resistance modifiers in 10 paediatric acute myeloid leukaemia (AML) patient samples (methyl thiazol tetrazolium assay). Drug interactions were determined by median drug effect analysis. Co-incubation of ara-C/aphidicolin showed strong synergism. The combinations of ara-C/cladribine and ara-C/gemcitabine were synergistic. Nearly additive and moderately synergistic interactions were observed between ara-C/flavopiridol and ara-C/UCN-01. The combination of ara-C/decitabine was antagonistic. In conclusion, favourable interactions were observed between ara-C and aphidicolin, cladribine, gemcitabine and also with flavopiridol and UCN-01, supporting the evaluation of these combinations in clinical trials with AML patients.     

Childhood acute myeloid leukaemia

Although acute myeloid leukaemia (AML) has long been recognized for its morphological and cytogenetic heterogeneity, recent high-resolution genomic profiling has demonstrated a complexity even greater than previously imagined. This complexity can be seen in the number and diversity of genetic alterations, epigenetic modifications, and characteristics of the leukaemic stem cells. The broad range of abnormalities across different AML subtypes suggests that improvements in clinical outcome will require the development of targeted therapies for each subtype of disease and the design of novel clinical trials to test these strategies. It is highly unlikely that further gains in long-term survival rates will be possible by mere intensification of conventional chemotherapy. In this review, we summarize recent studies that provide new insight into the genetics and biology of AML, discuss risk stratification and therapy for this disease, and profile some of the therapeutic agents currently under investigation.     

Treatment outcome of CRLF2 -rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups

The prognostic relevance of CRLF2 ‐rearrangements in childhood acute B‐cell precursor lymphoblastic leukaemia (ALL), was assessed by a comparative analysis of 114 non‐Down‐syndrome patients (99 P2RY8‐CRLF2+ , 15 IGH@‐CRLF2+ ), 76 from the AIEOP‐BFM ALL 2000 and 38 from the MRC ALL97 trials. The 6‐year cumulative relapse incidence of P2RY8‐CRLF2+ patients treated on the two trials was not statistically different: 0·37 ± 0·06 vs. 0·25 ± 0·08 (P = 0·194). In contrast, 0/9 IGH@‐CRLF2+ AIEOP‐BFM, but 5/6 ALL97 patients relapsed. Conclusively, P2RY8‐CRLF2+ patients had an intermediate protocol‐independent outcome while the different prognosis of IGH@‐CRLF2+ patients could be related to the different structures of the applied treatment protocols.     

The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: a report on the medical research council UKALLR1 study

We have examined the toxicity and overall outcome of the Medical Research Council UKALL R1 protocol for 256 patients with relapsed childhood acute lymphoblastic leukaemia (ALL). Second remission was achieved in over 95% of patients. Two patients died during induction and seven patients died of resistant disease. The overall actuarial event-free survival (EFS) at 5 years for all patients experiencing a first relapse was 46% (95% CI 40-52). Duration of first remission, site of relapse, age at diagnosis and sex emerged as factors of prognostic significance. Five-year EFS was only 7% for children relapsing in the bone marrow within 2 years of diagnosis, but was 77% for those relapsing without bone marrow involvement > 2.5 years from diagnosis. All analyses in this report are by treatment received. For those receiving chemotherapy alone, the 5-year EFS was 48%; for autologous bone marrow transplantation (BMT), the 5-year EFS was 47%; for unrelated donor BMT, it was 52%; and for related donor BMT, the 5-year EFS was 45%. The groups, however, were not comparable with respect to risk factor profile, and therefore direct comparison of EFS is misleading. Adjustment for time to transplant and prognostic factors was used to reduce the effects of biases between treatment groups, but did not suggest benefit for any particular treatment. There was failure of our planned randomization scheme in this trial with only 9% of those eligible being randomized, which highlights the difficulties in running randomized trials especially in patients who have relapsed from a previous trial. The optimal treatment for relapsed ALL therefore remains uncertain. Alternative approaches are clearly needed for those with early bone marrow relapse if outcome is to improve.     

Strategies for reducing the treatment‐related physical burden of childhood acute myeloid leukaemia – a review

Over the last four decades the survival of paediatric patients with acute myeloid leukaemia has gradually increased to 70% in high‐income countries. The therapy is very intensive and associated with many acute and long‐term side effects. The early death rate has been reduced to 1–4%. The acute toxicity is a limiting factor for improving survival in low‐income countries. Transplant is associated with more endocrinological late effects while cardiotoxicity is more common after relapse. Reducing the physical costs of therapy without jeopardizing survival may be accomplished by optimal supportive care, less cardiotoxic anthracyclines, less consolidation courses and strict indications for stem cell transplantation. Analysing scenarios with different frequency of transplantation in first complete remission show similar overall survival rates, indicating that almost all patients can be spared the procedure in first remission. Reducing relapse risk is an effective way of reducing toxicity and more targeted therapy and improved risk group stratifications are needed.     

Classical and molecular cytogenetic abnormalities and outcome of childhood acute myeloid leukaemia: report from a referral centre in Israel

The incidence of cytogenetic abnormalities in childhood de novo acute myeloid leukaemia (AML) and its prognostic significance was assessed in an Israeli paediatric referral centre. Cytogenetic analysis was successful in 86 of 97 children (< 20 years of age) diagnosed between 1988 and 2002 with de novo AML. Fluorescence in situ hybridization analysis detected new information in 11 of them, leading to reassignment in cytogenetic group classification. The incidence of the various cytogenetic subgroups was as follows: normal - 9%; t(11q23) - 22%; t(8;21) - 13%; t(15;17) - 8%; inv(16) - 3.4%; abn(3q) - 4.6%; 7/7q-(sole or main) - 5.8%; del(9q)(sole) and +21(sole) - 4.6% each; t(8;16) - 2.3%; t(6;9), t(1;22), +8(sole) - 1.1% each; and miscellaneous - 18%. The overall survival (OS) and event-free survival (EFS) (4 years) for 94 patients treated with the modified Berlin-Frankfürt-Münster (BFM) AML protocols (non-irradiated) were 59.9% (SE = 5%) and 55.7% (SE = 5%), respectively, and for the favourable t(8;21), t(15;17) and inv(16), OS was 60% (SE = 15%), 83% (SE = 15%) and 100% respectively. For the normal group it was 62% (SE = 17%), miscellaneous 64% (SE = 12%), t(11q23) 44.6% (SE = 11%) and of the -7/7q-, del(9q)(sole) or t(6;9), none had survived at 4 years. The incidence of cytogenetic subgroups in the Israeli childhood AML population and their outcome were similar to other recently reported paediatric series. Cytogenetic abnormalities still carry clinical relevance for treatment stratification in the context of modern chemotherapy.     

Long-term follow-up of relapsed childhood acute lymphoblastic leukaemia

We have reviewed the outcome after relapse in a cohort of 505 children with acute lymphoblastic leukaemia (ALL) seen at a single institution. The majority of relapses (74%) occurred within 3 years from diagnosis, and most involved the bone marrow alone or with overt extramedullary relapse. Early relapse was more common in children with T-ALL and those with unfavourable cytogenetics. Factors influencing second remission included length of first remission and type of relapse. Children who had not received previous cranial irradiation had a superior survival. The German relapse score involving length of first remission, site of relapse and immunophenotype was highly predictive of outcome: event-free survival with 95% confidence intervals at 6 years for patients who received modern treatment [intensive chemotherapy or bone marrow transplantation (BMT)] was 78% (51-92%) for standard risk, 41% (33-49%) for intermediate risk and 19% (10-31%) for highest risk. Retrospective comparison of BMT with chemotherapy showed no difference in the intermediate-risk group but a possible advantage in the highest risk group. Follow-up of 235 patients who relapsed after chemotherapy and received a third course of treatment showed an extremely high early attrition rate, but a small number of patients survived in third remission. We conclude that new approaches are needed to individualize therapy in intermediate-risk patients and to improve the outcome for those in the highest risk group. Only a small number of children can be treated effectively in third remission.     

Mutational analysis of the DNA mismatch repair gene hMLH1 in myeloid leukaemias

Mutations of the DNA mismatch repair (MMR) gene hMLH1 have recently been linked to the development of some hereditary and sporadic cancers which frequently display widespread microsatellite instability (MSI). Conflicting results regarding the extent of MSI in myeloid leukaemias prompted us to perform mutational analysis of all 19 exons of the hMLH1 gene by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) and sequence analysis in a total of 133 patients with acute and chronic myeloid leukaemia. Apart from one exonic and one intronic polymorphism, no mutations were detected in any of the samples indicating that the major MMR gene hMLH1 is not involved in the pathogenesis or progression of myeloid malignancies.