Variable mercaptopurine metabolism and treatment outcome in childhood lymphoblastic leukemia.

Intracellular thioguanine nucleotides (6-TGN) are the major cytotoxic metabolites of mercaptopurine (6-MP). Red blood cell (RBC) 6-TGN concentrations were measured in a group of 120 consecutive children with lymphoblastic leukemia (ALL) to assess interpatient variability and its clinical importance. Assays were performed after at least 2 months 6-MP maintanance chemotherapy and a minimum 7 days unattenuated protocol dose of 75 mg/m2. Observed 6-TGN concentrations ranged from 126 to 832 pmol/8 x 10(8) RBCs (median, 275). There was a correlation between 6-TGN and neutropenia 14 days postassay (rs = .51; P less than .0005), and an inverse correlation between 6-TGN and the length of time uninterrupted full protocol dose was tolerated without neutropenia (rs = -.3; P less than .01). After a median follow-up of 49 months, 19 children had relapsed, of whom 17 (89%) had 6-TGN concentrations below the group median (log-rank chi 2 = 11.9; P less than .001). Multivariate analysis using Cox's proportional hazards regression showed the 6-TGN effect on disease control to be independent of diagnostic WBC count, sex, age, immunological cell type, French-American-British (FAB) type, variation in other antineoplastic therapy, and duration of remission at the time of 6-TGN assay. Children with ALL taking the same dose of 6-MP show great variability in its measurable cytotoxic effect, and this variability is apparently important in predicting treatment outcome.

Acute lymphoblastic leukaemia in infancy: experience in MRC UKALL trials. Report from the Medical Research Council Working Party on Childhood Leukaemia.

Acute lymphoblastic leukaemia (ALL) is rare under 1 year and has a poor prognosis. Only 14 of 48 infants treated on two consecutive MRC UKALL trials remain alive in first remission. Forty infants have subsequently been treated on a protocol incorporating further intensification with an option for high-dose chemotherapy and autologous or allogeneic bone marrow transplantation. The results show no improvement over previous trials largely due to the number of remission deaths; four of nine being associated with toxicity of 5 days of etoposide and cytarabine. Only three of 11 children treated by high-dose chemotherapy and transplantation remain alive in remission. Multivariate analysis of the factors influencing prognosis in all 88 infants showed that only age was significant. Event-free survival was 40% at 5 years for children over 26 weeks but under 10% for younger children. These results show the susceptibility of infants to the toxicity of intensive chemotherapy and do not support the use of short term high-dose chemotherapy alone in the management of infant leukaemia. Despite the unique biological features of infant ALL it appears that these patients also may benefit from longer courses of treatment with a maintenance (continuing) phase.

Childhood leukemia: cerebrospinal fluid enolase isoenzymes in central nervous system infiltration.

Enolase isoenzymes were measured in the cerebrospinal fluid of seven consecutive children with lymphoblastic leukemia who developed meningeal (CNS) leukemia. Assays were performed at the time CNS disease was discovered and during the subsequent 4 weeks. Three of the seven were also examined 1-3 months before CNS relapse was confirmed. Fourteen children on similar systemic therapy without CNS infiltration served as controls. Prior to and at the onset of CNS disease alpha enolase was elevated in all patients studied. The gamma form was raised in only one beforehand and only three at the time of relapse. The alpha isoenzyme was related to the blast cell count and fell during therapy in all but one child, whereas the gamma was not and showed no significant change. The three patients with raised gamma enolase were the only children with other than common lymphoblastic leukemia. There was no clear indication whether either enzyme concentration had any importance in terms of disease outcome, although one child developed a further CNS relapse 10 months later. He was the only patient whose alpha enolase rose following intrathecal methotrexate. Neuronal disruption due to common lymphoblastic leukemia in the CNS appears to be minimal. Other types of leukemia may give rise to more neuronal damage. The alpha isoenzyme, from glial tissue and malignant cells, may be elevated even in the absence of detectable blasts in the cerebrospinal fluid and may be a sensitive marker of CNS infiltration in such circumstances.

Long-term follow-up of the United Kingdom Medical Research Council protocols for childhood acute lymphoblastic leukaemia, 1980-1997. Medical Research Council Childhood Leukaemia Working Party.

Results of three consecutive completed UK trials (1980-1997) for childhood lymphoblastic leukaemia are presented. National accrual has progressively increased so that over 90% of all the country's ALL cases were treated on the latest trial reported, UKALLXI. From 1980 to 1990, event-free and overall survival progressively improved, following adoption of an American therapy template and use of two post-remission intensification modules. Since 1990 despite demonstration of the benefit of a third intensification module overall event-free survival (EFS) has not improved further. Survival remains high due to a good retrieval rate especially for those relapsing off treatment after receipt of two intensification pulses. Possible reasons for the plateau in event-free survival (including type and dose of induction steroid, dropping of induction anthracycline, type and dose of asparaginase, gaps early in therapy following intensification, and overall lack of compliance in maintenance) are being explored in the latest protocol ALL '97. Cranial irradiation had been successfully replaced by a long course of intrathecal methotrexate injections for the majority of patients. Age (<1 year >10 years) sex (male) and white count >50 x 10(9)/l plus slow initial bone marrow clearance were consistently the most important independent prognostic indicators during this time period. Rome/NCI criteria accurately predict standard and high-risk groups for B cell lineage, but not consistently for T cell disease. This international collaborative venture might help us to define those truly at highest risk, and how we can optimise therapy for specific subgroups including T-ALL and those with unfavourable cytogenetics.

Benefit of intensified treatment for all children with acute lymphoblastic leukaemia: results from MRC UKALL XI and MRC ALL97 randomised trials. UK Medical Research Council's Working Party on Childhood Leukaemia.

Treatment of children with acute lymphoblastic leukaemia (ALL) aims to cure all patients with as little toxicity as possible and, if possible, to restrict further intensification of chemotherapy to patients with an increased risk of relapse. However in Medical Research Council (MRC) trial UKALL X two short myeloablative blocks of intensification therapy given at weeks 5 and 20 were of benefit to children in all risk groups. The successor trials, MRC UKALL XI and MRC ALL97, tested whether further intensification would continue to benefit all patients by randomising them to receive, or not, an extended third intensification block at week 35. After a median follow-up of 4 years (range 5 months to 8 years), 5 year projected event-free survival was superior at 68% for the 894 patients allocated a third intensification compared with 60% for the 887 patients who did not receive one (odds ratio 0.75, 95% CI 0.63-0.90, 2P = 0.002). This difference was almost entirely due to a reduced incidence of bone marrow relapses in the third intensification arm (140 of 891 in the third intensification arm vs. 171 of 883 in the no third intensification, 2P = 0.02). Subgroup analysis suggests benefit of the third intensification for all risk categories. Overall survival to date is no different in the two arms, indicating that a greater proportion of those not receiving a third intensification arm and subsequently relapsing can be salvaged. These results indicate that there is benefit of additional intensification for all risk subgroups of childhood ALL.

Oral 6-mercaptopurine in childhood leukemia: parent drug pharmacokinetics and active metabolite concentrations.

6-Mercaptopurine (6MP) pharmacokinetics and red blood cell 6-thioguanine nucleotide (TGN) concentrations were studied in 19 children receiving remission maintenance treatment for lymphoblastic leukemia. There was a high interpatient variation in all the pharmacokinetic parameters measured. The pharmacokinetic parameters measured in two children who subsequently had relapses were within the 95% confidence limits of the 17 other children. There was no difference in 6MP pharmacokinetic parameters with respect to neutropenia either after or before the study. The children who developed neutropenia 10 to 19 days after study had significantly higher TGN concentrations (U = 8; P less than 0.001) and had spent a longer time receiving reduced 6MP dosage in the 12 weeks before the study (U = 19.5; P less than 0.025). TGN concentrations are a better index of a child's ability to form active cytotoxic metabolites than 6MP dose or plasma concentrations.

Thiopurine pharmacogenetics in leukemia: correlation of erythrocyte thiopurine methyltransferase activity and 6-thioguanine nucleotide concentrations.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP) and azathioprine. Human erythrocyte (RBC) TPMT activity is controlled by a common genetic polymorphism. On a genetic basis approximately one in every 300 subjects lacks TPMT activity, and 11% of subjects have intermediate activities. 6-Thioguanine nucleotides (6-TGN) are major metabolites of 6-MP and azathioprine in humans. RBC 6-TGN concentrations are correlated directly with risk for the development of leukopenia in patients treated with thiopurine drugs. Our studies were performed to determine whether there was a relationship between genetically controlled levels of RBC TPMT activity and RBC concentrations of 6-TGN. We found a significant negative correlation between RBC TPMT activity and 6-TGN concentrations in blood samples from 40 children with acute lymphoblastic leukemia receiving long-term therapy with 6-MP (rs = -0.474; P less than 0.005). In addition, RBC TPMT activities were significantly higher in blood samples from these patients than in blood samples from adult control subjects (P less than 0.0001) or children with acute lymphoblastic leukemia who were in remission but were not receiving drug therapy (P less than 0.0001). Finally, three adult patients were studied who developed very high RBC 6-TGN concentrations and thiopurine-induced leukopenia. Two of the three patients had no detectable RBC TPMT activity--presumably on a genetic basis. These results indicate that low TPMT activity may be a risk factor for the occurrence of elevated 6-TGN concentrations and for the development of severe leukopenia in patients treated with thiopurine drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparinised clotting factor concentrates in patients with Christmas disease and liver disease.

Evidence has been sought of activation of the coagulation system in two groups of patients following the infusion of two heparinised clotting factor concentrates. No changes were detected in 13 patients with mild hepatic dysfunction. In six studies on patients with Christmas disease induced abnormalities occurred in only one. Activation of the coagulation mechanism did not occur in another individual who had received the same batch of material.

T-lymphoblastic leukaemia and aspartate aminotransferase.

AST levels from 11 untreated children with T-ALL were found to be significantly higher than those from 74 children with non-T disease. The enzyme was not related to haemoglobin or bilirubin levels nor to the presence of hepatosplenomegaly in any of the patients. It was correlated with the white cell count, but only in the T-cell group and not the remainder. It was also correlated with a parallel (but lesser) rise in ALT, but again only in the T-cell group. The blast cells themselves contained little or no transaminase activity, so it is probable that T-ALL produces more extramedullary tissue damage than non-T disease.

Frequency of coincident iron deficiency and beta-thalassaemia trait in British Asian children.

A study was carried out to determine the frequency of combined iron deficiency and beta-thalassaemia trait in a cohort of British Asian children to see whether the trait protects iron status. Of 470 consecutive children with red cell microcytosis, 77 had beta-thalassaemia trait and 26 (34%) of these also had evidence of iron deficiency. It was most common and profound in children under five years of age where the prevalence was 16 in 33 (48.5%). This suggests that iron deficiency is no less common in Asian children with beta-thalassaemia trait than in those without. It should not be presumed that the trait protects iron status or that the two are in any way mutually exclusive, at least in the early years.

Granular acute lymphoblastic leukaemia of childhood: a morphological phenomenon.

Three hundred and twenty consecutive children with lymphoblastic leukaemia (ALL), treated on the Medical Research Council UKALL VIII schedule, had their Romanowsky stained diagnostic marrows reviewed for the presence of azurophil granules in blast cell cytoplasm. Twenty patients (7%) had greater than 5% blasts showing this feature; 19 had the cell phenotype of "common ALL." Male children and those with French-American-British (FAB) L2 morphology predominantly showed this feature. There was also a strong correlation between granularity and non-diffuse acid phosphate positivity, but no obvious difference between the 20 patients in their response to treatment emerged during a minimum follow up of 15 months. The "granular" variant occurs in around 7% of children with ALL, but has no clear prognostic importance. Morphologists should be aware of its existence and incidence to avoid confusion with acute myeloid leukaemia.

Mouse red cell rosette formation and the colchicine sensitivity test: relative usefulness in the differential diagnosis of chronic lymphocytic leukaemia and B lymphocytic lymphoma.

Mouse erythrocyte (M) rosette formation and colchicine sensitivity were compared for their ability to differentiate chronic lymphocytic leukaemia (CLL) from B cell non-Hodgkin's lymphoma (NHL) with overspill. Twenty-two cases of CLL and eight of NHL were studied along with 31 normal adults. Results from the patients in both tests differed significantly from the controls but colchicine sensitivity failed to differentiate them further. M Rosettes, on the other hand, while increased in some patients with NHL were, without overlap, much more numerous in those with CLL, and clearly distinguished the two conditions. A significant autolymphocytotoxic effect of plasma from both study groups was also noted which was not found in the controls.

Lymphocyte subset counts in skin puncture and venous blood compared.

To determine whether skin puncture blood can be used reliably for CD4 lymphocyte counts, the numbers of the major subsets of lymphocytes were assessed in paired venous and skin puncture blood samples from 22 children and 10 adults. Paired values were highly correlated, with skin puncture values being about 7% higher than venous values for each cell type. Differences were of borderline statistical significance for total lymphocytes and for each subset except CD3+ CD8+ T lymphocytes. Nevertheless, the magnitude of the differences was small and unlikely to be of clinical importance, and it seems that skin puncture samples may be preferable for CD4 counts in children or adults with difficult venous access.

Morphological metamorphosis in relapsing lymphoblastic leukemia.

Blast cell morphology was assessed at diagnosis and subsequent bone marrow relapse in 33 unselected patients with lymphoblastic leukemia (ALL). Each marrow was classified 'blind' according to the French-American-British (FAB) criteria, and it was found that 19 of 24 (79%) patients initially typed as FAB L1 changed to FAB L2 during the course of their disease, but no patient made the reverse morphological change (p is less than 0.001). Five patients retained FAB L1 appearances; these included three of the four who had T-cell markers. One patient typed as FAB L3 did so consistently. This study indicates that FAB L2 ALL frequently emerges as a treatment-resistant offshoot of FAB L1 and provides further evidence that this marks a more aggressive form of the disease.

Periodic acid-Schiff reaction and prognosis in lymphoblastic leukaemia.

Diagnostic bone marrow smears from 132 patients with acute lymphoblastic leukaemia, (ALL) were stained simultaneously by the periodic acid-Schiff (PAS) reaction, and the blast cell positivity was assessed quantitatively. The patients fell naturally into two unequal groups: those with more than 20% PAS-positive blast cells (44 patients) and those with less (88 patients). There was no relation between the degree of positivity and age, sex, or presenting leucocyte count. Actuarial survival studies showed that the group with more than 20% PAS-positive blast cells survived longer, but that this difference assumed statistical significance only after the exclusion of patients over 14 years old and those with high white cell counts at the time of diagnosis. It appears that the PAS reaction can identify long survivors among patients with ALL, but not in the absence of features strongly associated with a poor prognosis.

Factor VIII inhibitor assay using skin puncture blood samples.

A method was developed for the measurement of factor VIII inhibitors by the Bethesda technique using small volumes of skin puncture blood. Human and porcine inhibitor concentrations on paired venous and skin puncture samples measured on 10 occasions were highly correlated, with no significant difference between them. Assays on four inhibitor negative haemophiliac patients were also negative by the skin puncture method. Sample dilution at the collection stage results in loss of sensitivity to inhibitor concentrations below 1 unit/ml, but the technique will be of value in monitoring higher concentrations when venous access is difficult and must be saved for parenteral treatment.

Application of fluorescence in situ hybridisation to chromosome analysis of aged bone marrow smears.

AIMS: To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years. METHODS: A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded. RESULTS: All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case. CONCLUSION: When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.

Chromosomes in childhood acute lymphoblastic leukaemia: karyotypic patterns in disease subtypes.

To define further the clinical importance of cytogenetic analysis in acute lymphoblastic leukaemia (ALL) a prospective study was performed on 139 unselected children. Analyses were considered adequate in 104, of whom 35 were normal and 69 had clonal abnormalities. Abnormalities were categorised according to banded chromosome analysis as well as chromosome count. Karyotypes were correlated with clinical and laboratory features at diagnosis and with survival. Of the successful analyses, thirty five (34%) children had no abnormalities; this group contained an excess of T cell disease. Twenty five (24%) had a "characteristic" hyperdiploid karyotype and as a group had lower presenting white counts, a tendency to CD10, and periodic acid schiff positivity of the blast cells and smaller spleens. None was an infant and only one was over 10 years old. Seven (7%) children with t(9; 22), t(8; 14), or t(4; 11) translocations were grouped together as "specific" translocations. Collectively they had a significantly worse prognosis than the remainder. Nine children developed central nervous system relapse, six of whom had either t(4; 11) or abnormalities of 9p or 19p. A descriptive classification taking into account chromosome bonding pattern is cytogenetically more appropriate and may be more clinically useful than grouping children simply by chromosome number. As knowledge and techniques improve, the classification of cytogenetic abnormalities in ALL will need to be kept under frequent review.

French American British (FAB) morphological classification of childhood lymphoblastic leukaemia and its clinical importance.

As part of the Medical Research Council Leukaemia Trial UKALL VIII, 738 unselected children with acute lymphoblastic leukaemia (ALL) had the morphology of their marrow blast cells reviewed by a panel of three haematologists. Ninety four (13%) showed appearances classifiable as type L2 by the French American and British (FAB) cooperative group's criteria, five (0.7%) were typed L3, and the remaining 639 (86%) as L1. Disregarding the patients classified as L3, those with the L2 variant showed an inferior disease free survival to that of the remainder (p less than 0.01), and more of them failed to remit after receiving "standard" remission induction treatment (p less than 0.01). They included an excess of older children (p less than 0.01) with less profound marrow failure at diagnosis, and fewer of them expressed the common ALL antigen (p = 0.05). There was no association between L2 morphology and the diagnostic white cell count, sex, or the presence of a mediastinal mass. These findings confirm earlier reports that FAB L2 ALL is associated with a poor prognosis and that it occurs more commonly in older children. The high remission failure rate is a recent observation and indicates that alternative early treatment may be appropriate for such patients.

Sex and acid phosphatase in childhood non-T lymphoblastic leukaemia.

A semiquantitative assessment of blast cell acid phosphatase activity, expressed as a score, was made in 41 unselected children with newly diagnosed and untreated non-T acute lymphoblastic leukaemia (ALL). Despite a wide range of enzyme activity in both sexes boys had significantly higher scores than girls, and, in view of the known association between males and T ALL on the one hand, and between acid phosphatase and T ALL on the other, these findings raise the possibility that boys may have a predisposition to a type of pre-T ALL which could contribute to the as yet unexplained difference in prognosis between the sexes.