Granulocytopoiesis in children with acute lymphoblastic leukaemia.

Numbers, proliferative potential, and differentiative capacity of bone marrow granulocyte-macrophage precursor cells were studied in 130 children with acute lymphoblastic leukaemia (ALL), including 77 children in an acute phase of the disease and 53 in remission. Bone marrow samples from 65 children without haematopoietic abnormalities were used as controls. The numbers of clonogenic precursors were found to be below normal in all phases of ALL, particularly during the acute period when the bone marrow was heavily infiltrated with leukaemic cells. It is shown that the decreases in the numbers and proliferative potential of the precursor cells during the acute phases was associated with the effects of leukaemic blast cells, but that in remission the observed reduction in the precursor cell pool was due to the cytostatic effect of therapy. The differentiative capacity of clonogenic granulocyte and macrophage precursors was not altered in children with ALL.     

Methotrexate-induced malabsorption in children with acute lymphoblastic leukaemia.

A one-hour D-xylose absorption test was performed on 18 children with acute lymphoblastic leukaemia. Xylose absorption was normal in children who had not received methotrexate, but there was a significant degree of malabsorption in those who had taken methotrexate within the previous seven days. There was a progressive and significant increase in malabsorption related to the cumulative dose of methotrexate. These findings provide further evidence that regular methotrexate treatment every seven days is more toxic than if it is more widely spaced. The spacing of treatment is currently under investigation.     

NUDT15 polymorphism and NT5C2 and PRPS1 mutations influence thiopurine sensitivity in acute lymphoblastic leukaemia cells

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.     

Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects.     

Prognostic value of adenosine deaminase activity in children with acute lymphoblastic leukaemia

Adenosine daminase (ADA) activity was measured in 70 children with ALL. On the basis of a 5 1/2 years observation, it was found that children with low ADA activity in serum before treatment have a greater chance for longer CCR duration than children with high ADA activity.     

The clinical impact of thiopurine methyltransferase polymorphisms on thiopurine treatment

Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood. Although current treatment results in long term survival in over 70% of cases there is evidence that as many as 50% could have been cured using a less complex regimen with a lower incidence of long term side effects. In previous studies it has been found that thiopurines given as part of continuing therapy are key agents in preventing relapse. However, optimal administration during continuing therapy is often not achieved. Variation in the level of thiopurine methyltransferase (TPMT) activity appears to be a major molecular determinant of the extent of thiopurine metabolism. TPMT activity shows a trimodal distribution pattern. A lack of activity is found in approximately one in 300 Caucasians; approximately 11% have intermediate activity and the remaining 89% high activity. Congenital loss of activity is associated with grossly elevated levels of active drug and profound myelosuppression on exposure to thiopurines. This loss of activity has been attributed to single nucleotide polymorphisms (SNPs) within the TPMT gene. The frequency of SNPs is related to ethnicity, with the most common in Caucasians being TPMT*3A which is characterized by a G to A transition at position 460 with a substitution of alanine for tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719 resulting in a change of tyrosine to cysteine at position 240 (Y240C). Polymorphisms have also been identified within the 5' flanking promoter region of the TPMT gene due to a variable number of tandem repeats (VNTR*3-*8). An overview of the polymorphisms identified to date, their implication on the metabolism of the thiopurine drugs and therapeutic importance will be discussed.     

HL-A genotype of patients with acute lymphoblastic leukaemia

Summary 10 patients with acute lymphoblastic leukaemia were tissue-typed for 21 HL-A specificities. Of these, genotypes of 9 pateints were determined by family analyses. Haplotype HL-A1,8 occurred in 5 out of 18 instances. On phenotype basis, a slight increase was observed in the incidence of antigens HL-A1 and HL-A8. No loss of HL-A specificities could be detected on lymphocytes through family analyses.     

Immunological detection of residual leukaemic disease in the bone marrow of children with acute lymphoblastic leukaemia

Peripheral blood lymphocytes incubated with tumour cells or extracts may undergo blastogenesis. This is the basis of a technique studied in children with acute lymphoblastic leukaemia (ALL) in childhood in an attempt to predict relapse. Samples of peripheral blood and bone marrow from 82 children with varying degrees of ALL were analysed. Cultures were prepared by incubating a lymphocyte suspension with an autologous bone-marrow suspension. Final ratios of lymphocytes to bone-marrow cells (L: BM) were 1: 1 and 2: 1. Control wells received bone-marrow or lymphocyte suspension only. Cultures were incubated for 72, 96, and 120 hours. All were pulse-labelled with ³H-TdR and radioactivity was measured by scintillation counting. Results were expressed as the stimulation index, calculated by dividing the mean counts per minute (cpm) of wells containing both lymphocytes and bone-marrow cells by the sum of the mean cpm for control wells. If the stimulation index exceeded 1 at 72, 96, or 120 hours at either L: BM ratio a positive response was recorded.Seventy-six children were in clinical remission at the time of testing (group A) and six were in clinical relapse (group B). In group A 24 patients showed stimulation and relapsed later at a mean time of 3·8 months (21 with marrow disease, two with testicular infiltration, and one with lung infiltration). Sixteen patients showed stimulation and had up to 4% blasts in their bone marrow but remained in remission. Nineteen other patients showed a positive response and several factors may have contributed to this: two underwent a “rebound” lymphocytosis after stopping treatment, nine had current or intercurrent infections, two had persistent unexplained bone-marrow lymphocytosis, but six had no causative symptoms and thus their responses were “true false-positives.” Seventeen patients from group A showed no response and remained in remission for a mean of 22·9 months after testing. None of the six children in group B responded, and at testing had 17-85% blasts in their bone marrow.During the study no patient relapsed who had not shown a positive response. The technique merits further study as a guide to the presence of leukaemic cells.     

No induction of thiopurine methyltransferase during thiopurine treatment in inflammatory bowel disease

The aim of this study was to follow, during standardized initiation of thiopurine treatment, thiopurine methyltransferase (TPMT) gene expression and enzyme activity and thiopurine metabolite concentrations, and to study the role of TPMT and ITPA 94C > A polymorphisms for the development of adverse drug reactions. Sixty patients with ulcerative colitis or Crohn's disease were included in this open and prospective multi-center study. Thiopurine na?ve patients were prescribed azathioprine (AZA), patients previously intolerant to AZA received 6-mercaptopurine (6-MP). The patients followed a predetermined dose escalation schedule, reaching target dose at Week 3; 2.5 and 1.25 mg/kg body weight for AZA and 6-MP, respectively. The patients were followed every week during Weeks 1-8 from baseline and then every 4 weeks until 20 weeks. TPMT activity and thiopurine metabolites were determined in erythrocytes, TPMT and ITPA genotypes, and TPMT gene expression were determined in whole blood. One homozygous TPMT-deficient patient was excluded. Five non compliant patients were withdrawn during the first weeks. Twenty-seven patients completed the study per protocol; 27 patients were withdrawn because of adverse events. Sixty-seven percent of the withdrawn patients tolerated thiopurines at a lower dose at Week 20. There was no difference in baseline TPMT enzyme activity between individuals completing the study and those withdrawn for adverse events (p = 0.45). A significant decrease in TPMT gene expression (TPMT/huCYC ratio, p = 0.02) was found, however TPMT enzyme activity did not change. TPMT heterozygous individuals had a lower probability of remaining in the study on the predetermined dose (p = 0.039). The ITPA 94C > A polymorphism was not predictive of adverse events (p = 0.35).     

Medical cost of curing childhood acute lymphoblastic leukaemia

Between 1970 and 1979 acute lymphoblastic leukaemia was diagnosed in 378 children at this hospital. The outcome for the 181 survivors was examined six or more years after diagnosis to assess morbidity in an unselected group of long term survivors. One hundred and thirty seven of the survivors were in first remission and probably cured (group I). Forty four (group II) had had one or more relapses, some of whom, who had isolated extramedullary relapses, also have a good chance of cure.In group I 136 patients had prophylactic cranial or craniospinal irradiation, while patients in group II, in addition to having that treatment, received local testicular (17) or craniospinal radiation (seven) for testicular or central nervous system relapse. Eight had additional prophylactic cranial radiotherapy after bone marrow relapse, and six had total body irradiation before bone marrow transplantation. The incidence of clinically important growth and endocrine morbidity was 20% in group I and 68% in group II. The morbidity in patients in group I was mainly attributable to early pubertal maturation. In group II 30 patients had growth failure, of whom 19 had gonadal failure from testicular or total body irradiation, 14 had growth hormone deficiency after doses of cranial irradiation of over 2400 cGy, and 10 had spinal growth impairment after craniospinal irradiation. Two also had early pubertal maturation. Five out of six patients who received total body irradiation had multiple endocrine deficiency. Neuropsychological sequelae of treatment were seen in 40 (42%) of 96 schoolchildren in group I and in 12 (38%) of 32 schoolchildren in group II. Postinfective sequelae of treatment were found in patients in both groups.These results show that the survivors who were in their first remission had a 42% residual morbidity related to treatment compared with an 82% morbidity in the survivors of one or more relapses who had multiple treatments.     

Remission induction with cytosine arabinoside and L-Asparaginase in acute lymphoblastic leukaemia

Nine patients with acute lymphoblastic leukaemia in relapse were treated with a course of cytosine arabinoside followed immediately by a course of L-asparaginase. Eight patients achieved complete remission of their disease. This combination of drugs is sufficiently effective to suggest that further trial is needed. It is possible that the combination has a synergistic effect.     

Poor prognosis of acute lymphoblastic leukaemia in Asian children living in the United Kingdom.

A study of the results of treatment of acute lymphoblastic leukaemia in Asian (Indian and Pakistani) children living in the United Kingdom showed that they had a poorer prognosis than native white children due mainly to deaths during remission. Similar proportions of Asian and white children suffered relapse. Lower socioeconomic status, poor nutrition, and difficulties in communication may have contributed to the worse outcome in the Asian children.