J.P.M. Bökkerink

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).

Modern treatment strategies, consisting of intensive chemotherapy and cranial irradiation, have remarkably improved the prognosis for children with acute lymphoblastic leukemia. However, patients with a potential for cure are at risk of severe acute and late adverse effects of treatment. Furthermore, in 25–30% of patients treatment still fails. The objectives of the DCLSG study ALL 8 were to decrease the toxicity and to increase the effectivity of BFM-oriented treatment. Decrease of toxicity was aimed at by confirmation of the results of the previous DCLSG study ALL-7, showing that the majority (94%) of children with ALL can succesfully be treated with BFM-oriented therapy without cranial irradiation, and by reduction of treatment for standard risk (SRG) patients. To increase the cure rate in medium risk (MRG) patients the efficacy of high doses of intravenous 6-mercaptopurine (HD-6MP) during protocol M and in SRG patients the efficacy of high doses of L-asparaginase (HD-L-ASP) during maintenance treatment was studied in randomized studies. Patient stratification and treatment were identical to protocol ALL-BFM90, with the following differences: no prophylactic cranial irradiation, SRG patients received only phase 1 of protocol I. Four hundred and sixty-seven patients entered the protocol: 170 SRG, 241 MRG and 56 HRG patients. The 5 years event-free survival rate for all patients was 73% (s.e. 2%); for SRG, MRG and HRG patients 85% (s.e. 3%), 73% (s.e. 3%) and 39% (s.e. 7%), respectively. In patients >1 year of age at diagnosis unfavorable prognostic factors were male sex, >25% blasts in the bone marrow at day 15 and initial white blood cell count (WBC) >50 × 109/l. The cumulative risk of CNS relapse rate was 5% (s.e. 1%) at 5 years. These results confirm that the omission of cranial irradiation in BFM-oriented treatment does not jeopardize the overall good treatment results, nor does early reduction of chemotherapy in SRG patients. No benefit was observed from treatment intensification with HD-L-ASP in SRG patients, nor from HD-6MP in MRG patients.

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10(9) cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

Treatment strategy and results in children treated on three Dutch Childhood Oncology Group acute myeloid leukemia trials.

This report describes the long-term follow-up data of three consecutive Dutch Childhood Oncology Group acute myeloid leukemia (AML) protocols. A total of 303 children were diagnosed with AML, of whom 209 were eligible for this report. The first study was the AML-82 protocol. Results were inferior (5-year probability of overall survival (pOS) 31%) to other available regimes. Study AML-87 was based on the BFM-87 protocol, with prophylactic cranial irradiation in high-risk patients only, and without maintenance therapy. This led to a higher cumulative incidence of relapse than that reported by the Berlin–Frankfurt–Münster (BFM), but survival was similar (5-year pOS 47%), suggesting successful retrieval at relapse. The subsequent study AML-92/94 consisted of a modified BFM-93 protocol, that is, without maintenance therapy and prophylactic cranial irradiation. However, all patients were to be transplanted (auto- or allogeneic), although compliance was poor. Antileukemic efficacy was offset by an increase in the cumulative incidence of nonrelapse mortality, especially in remission patients, and survival did not improve (5-year pOS 44%). Our results demonstrate that outcome in childhood AML is still unsatisfactory, and that further intensification of therapy carries the risk of enhanced toxicity. Our patients are currently included in the MRC AML studies, based on the results of their AML 10 trial.

DNA methylation patterns in the calcitonin gene region at first diagnosis and at relapse of acute lymphoblastic leukemia (ALL)

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5′ region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5′ region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (P < 0.01). each of six all patients who were investigated at initial diagnosis and at relapse showed alterations in dna methylation between the two stages. these six cases were also investigated by southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in dna methylation in only four of the six cases. the dna sequencing method thus appears to be better suited to assess alterations of dna methylation than southern blot analysis. there are marked regional differences in the frequency of methylation of individual cpg sites and in the frequency of alterations between the two stages. our results show that alterations in dna methylation continue to occur from the initial stage to the relapse stage of all, suggesting that aberrant dna methylation may play a role in tumor progression.

Metabolism of intravenously administered high-dose 6-mercaptopurine with and without allopurinol treatment in patients with non-Hodgkin lymphoma.

Purpose: We investigated the metabolism of high dose 6 mer-captopurine (HD-6MP) infusions and its influenee on the metabolism by allopurinol, an inhibitor of xanthinc oxidase, the enzyme that catabolizes 6MP into thioxanthine and thiourie acid. Patients and Methods: Nine patients (aged 2− 11 years) with non-Hodgkin lymphoma (NHL) were treated with HD-6MP (1300 mg/m2.24 h) within a therapeutic window after diagnosis. Four patients received oral allopurinol (200 mg/m2.day) to prevent urate nephropathy, and five did not. Plasma and RBC were isolated before and 4, 20, 24, 28, and 48 h after the start of the infusion. All measurements were performed with HPLC. Results: Considerable variations were found in the plasma levels of 6MP. thioxanthine. and thiourie acid and of RBC-MeTIN levels. 6MP-ribosidc was not detectable, and MeMP and MeMPR levels were <1.3 M in the plasma. In general, 6MP, thioxanthine, and McMP levels in plasma were higher, and thiourie acid plasma levels and RBC-MeTIN levels were lower in the patients treated with allopurinol compared to those who did not receive allopurinol. Conclusions: 6MP is extensively metabolized in patients with NHL treated with HD-6MP. Thiopurine mcthylation, at the levels of nuclcotide. nucleoside. and base, is an important metabolic pathway after HD-6MP. Co-administration of allopurinol can result in both a decreased catabolism and anabolism of 6MP compared to treatment with HD-6MP alone. This observation may have consequences for the therapeutic efficacy and toxic effects of 6MP in combination with allopurinol.

Validity of self-reported data on pregnancies for childhood cancer survivors: a comparison with data from a nationwide population-based registry

STUDY QUESTION To what degree do records registered in the Netherlands Perinatal Registry (PRN) agree with self-report in a study questionnaire on pregnancy outcomes in childhood cancer survivors (CCSs)? SUMMARY ANSWER This study suggests that self-reported pregnancy outcomes of CCSs agree well with registry data and that outcomes reported by CCSs agree better with registry data than do those of controls. WHAT IS KNOWN ALREADY Many studies have shown that childhood cancer treatment may affect fertility outcomes in female CCSs; however, these conclusions were often based on questionnaire data, and it remains unclear whether self-report agrees well with more objective sources of information. STUDY DESIGN, SIZE, DURATION In an nationwide cohort study on fertility (inclusion period January 2008 and April 2011, trial number: NTR2922), 1420 CCSs and 354 sibling controls were invited to complete a questionnaire regarding socio-demographic characteristics and reproductive history. In total, 879 CCSs (62%) and 287 controls (81%) returned the questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS The current validation study compared the agreement between pregnancy outcomes as registered in the PRN and self-reported outcomes in the study questionnaire. A total of 589 pregnancies were reported in CCSs, and 300 pregnancies in sibling controls, of which 524 could be linked to the PRN. MAIN RESULTS AND THE ROLE OF CHANCE A high intra-class correlation coefficient (ICC) was found for birthweight (BW) (0.94 and 0.87 for CCSs and controls, respectively). The self-reported BWs tended to be higher than reported in the PRN. For gestational age (GA), the ICC was high for CCSs (0.88), but moderate for controls (0.49). CCSs overestimated GA more often than controls. The Kappa values for method of conception and for method of delivery were moderate to good. Multilevel analyses on the mean difference with regard to BW and GA showed no differences associated with time since pregnancy or educational level. LIMITATIONS, REASONS FOR CAUTION Not all pregnancies reported could be linked to the registry data. In addition, the completeness of the PRN could not be assessed precisely, because there is no information on the number of missing records. Finally, for some outcomes there were high proportions of missing values in the PRN registry. WIDER IMPLICATIONS OF THE FINDINGS Our study suggests that questionnaires are a reliable method of data collection, and that for most variables, self-report agrees well with registry data. STUDY FUNDING/COMPETING INTEREST This work was supported by the Dutch Cancer Society (grant no. VU 2006-3622) and by Foundation Children Cancer Free. None of the authors report a conflict of interest. TRIAL REGISTRATION NUMBER NTR2922 http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2922.

Biochemical Evidence for Synergistic Combination Treatment with Methotrexate and 6-Mercaptopurine in Acute Lymphoblastic Leukemia

Methotrexate (MTX) and 6-mercaptopurine (6 MP) are common antimetabolites in the treatment of acute lymphoblastic leukemia (ALL) since their introduction in 1947 and 1952 [1, 2]. Originally, these agents were administered separately. However, after empirical evidence for a potentiating efficacy of the combination of both in mice leukemia [3], MTX and 6 MP have also been used in combination in childhood ALL. The enhanced efficacy of combination treatment in children was demonstrated in a study of the Acute Leukemia Group B in 1961 [4].

Thiopurine Treatment in Childhood Leukemia

The thiopurine antimetabolites 6-mercaptopurine (6MP) and 6-thioguanine (TG) are important chemotherapeutic drugs in the treatment of acute lymphobastic leukemia. Both drugs are initially converted by the purine enzyme hypoxanthine guanine phos-phoribosyl transferase (HGPRT) into 6-thio-inosine monophoshate (TIMP) and 6-thio-guanosine monophosphate (TGMP), respectively. Further conversion of TIMP to TGMP via 6-thio-xanthine monophosphate (TXMP) is catalyzed by two enzymes, IMP-dehydro-genase (IMPDH) and GMP-synthetase, respectively. IMPDH is the rate-limiting enzyme in these two enzymatic steps (1). Subsequently, TGMP is converted by consecutive steps to the 6-thio-guanosine triphosphates: 6-thio-GTP (TGTP) and 6-thio-deoxy-GTP (TdGTP), which are incorporated into RNA and DNA, respectively.

Intracellular Pharmacology and Biochemistry of Methotrexate and 6-Mercaptopurine in Childhood Acute Lymphoblastic Leukemia

After treatment with methotrexate (MTX), 6-mercaptopurine (6MP) demonstrated a synergistic effect and an enhanced incorporation into DNA and RNA in human lymphoblastic cell lines1.

6-Mercaptopurine Metabolism in Two Leukemic Cell Lines

6-Mercaptopurine (6MP), an analogue of the purine base hypoxanthine, is commonly used in the maintenance therapy of acute lymphoblastic leukemia (ALL) in children1.