Therapy prolongation improves outcome in multisystem Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH)-III tested risk-adjusted, intensified, longer treatment of multisystem LCH (MS-LCH), for which optimal therapy has been elusive. Stratified by risk organ involvement (high [RO+] or low [RO-] risk groups), > 400 patients were randomized. RO+ patients received 1 to 2 six-week courses of vinblastine+prednisone (Arm A) or vinblastine + prednisone + methotrexate (Arm B). Response triggered milder continuation therapy with the same combinations, plus 6-mercaptopurine, for 12 months total treatment. 6/12-week response rates (mean, 71%) and 5-year survival (84%) and reactivation rates (27%) were similar in both arms. Notably, historical comparisons revealed survival superior to that of identically stratified RO+ patients treated for 6 months in predecessor trials LCH-I (62%) or LCH-II (69%, P < .001), and lower 5-year reactivation rates than in LCH-I (55%) or LCH-II (44%, P < .001). RO- patients received vinblastine+prednisone throughout. Response by 6 weeks triggered randomization to 6 or 12 months total treatment. Significantly lower 5-year reactivation rates characterized the 12-month Arm D (37%) compared with 6-month Arm C (54%, P = .03) or to 6-month schedules in LCH-I (52%) and LCH-II (48%, P < .001). Thus, prolonging treatment decreased RO- patient reactivations in LCH-III, and although methotrexate added no benefit, RO+ patient survival and reactivation rates have substantially improved in the 3 sequential trials. (Trial No. NCT00276757 www.ClinicalTrials.gov).

Doxorubicin or daunorubicin given upfront in a therapeutic window are equally effective in children with newly diagnosed acute lymphoblastic leukemia. A randomized comparison in trial CoALL 07-03.

BACKGROUND: The anthracyclines daunorubicin (DNR) and doxorubicin (DOX) are among the most important drugs in the treatment of childhood acute lymphoblastic leukemia, however there are conflicting in vitro data about the comparative efficacy and equivalent doses of both anthracyclines. To address the question of in vivo efficacy of both anthracyclines, patients enrolled in the CoALL 07-03 trial were randomized to receive one single dose of either doxorubicin 30 mg/m(2) , daunorubicin 30 mg/m(2) , or daunorubicin 40 mg/m(2) upfront induction therapy. PROCEDURE: Children with newly diagnosed B-Precursor ALL or T-ALL were eligible for the randomized comparison. From the percentage of blasts and the white blood cell count (WBC) the absolute number of leukemic cells per µl peripheral blood (PB) was calculated and the initial value before DOX/DNR infusion equated as 100%. Main target criterion of this study was the leukemic cell decrease from Day 0 to Day 7. RESULTS: Seven hundred forty three patients were randomized: 247 to the DOX; 252 to the DNR 30 mg/m(2) ; and DNR to the 40 mg/m(2) arm. The in vivo response was similar in all three treatment arms with a comparable blast decline in the peripheral blood. The percentages of patients with a clear non-response (M3 marrow) and moreover, the level of minimal residual disease (MRD) on Day 15 or at the end of induction were similar. CONCLUSION: In vivo efficacy of a single dose daunorubicin 30 or 40 mg/m(2) is similar to that of doxorubicin given in a dose of 30 mg/m(2) .

Identification of genes associated with chemotherapy crossresistance and treatment response in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) can be cured with combination chemotherapy in over 75% of children, but the cause of treatment failure in the remaining patients is unknown. We determined the sensitivity of ALL cells to individual antileukemic agents in 441 patients and used a genome-wide approach to identify 45 genes differentially expressed in ALL exhibiting crossresistance to prednisolone, vincristine, asparaginase, and daunorubicin. We also identified a distinct phenotype of discordant resistance to asparaginase and vincristine and 139 genes whose expression was associated with this novel phenotype. The expression of these genes discriminated treatment outcome in two independent patient populations, identifying a subset of patients with a markedly inferior outcome (37% +/- 13% 5 year DFS).

Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of infants with mixed-lineage-leukemia (MLL)-rearranged acute lymphoblastic leukemia: results from the Interfant-99 Study.

To define a role for hematopoietic stem cell transplantation (HSCT) in infants with acute lymphoblastic leukemia and rearrangements of the mixed-lineage-leukemia gene (MLL(+)), we compared the outcome of MLL(+) patients from trial Interfant-99 who either received chemotherapy only or HSCT. Of 376 patients with a known MLL status in the trial, 297 (79%) were MLL(+). Among the 277 of 297 MLL(+) patients (93%) in first remission (CR), there appeared to be a significant difference in disease-free survival (adjusted by waiting time to HSCT) between the 37 (13%) who received HSCT and the 240 (87%) who received chemotherapy only (P = .03). However, the advantage was restricted to a subgroup with 2 additional unfavorable prognostic features: age less than 6 months and either poor response to steroids at day 8 or leukocytes more than or equal to 300 g/L. Ninety-seven of 297 MLL(+) patients (33%) had such high-risk criteria, with 87 achieving CR. In this group, HSCT was associated with a 64% reduction in the risk of failure resulting from relapse or death in CR (hazard ratio = 0.36, 95% confidence interval, 0.15-0.86). In the remaining patients, there was no advantage for HSCT over chemotherapy only. In summary, HSCT seems to be a valuable option for a subgroup of infant MLL(+) acute lymphoblastic leukemia carrying further poor prognostic factors. The trial was registered at www.clinicaltrials.gov as #NCT00015873 and at www.controlled-trials.com as #ISRCTN24251487.

Promising therapy results for lymphoid malignancies in children with chromosomal breakage syndromes (Ataxia teleangiectasia or Nijmegen-breakage syndrome): a retrospective survey.

Children with chromosomal instability syndromes have an increased risk of developing lymphoma and leukaemia. The treatment of these malignancies is hampered by therapy-associated toxicity and infectious complications. This retrospective analysis evaluated the therapy outcome of 38 children with Ataxia teleangiectasia or Nijmegen-breakage syndrome with acute lymphoblastic leukaemia (ALL, n = 9), Non-Hodgkin lymphoma (NHL, n = 28) and Hodgkin lymphoma (HL, n = 1). All patients with NHL or ALL were treated in accordance to Berlin-Frankfurt-Münster (BFM)- or Co-operative study group for childhood ALL (CoALL)-oriented chemotherapy schedules. 22 patients received significantly reduced-intensity chemotherapy. After a median follow-up of 3·7 years the 10-year overall survival was 58%. Dosage-reduction of chemotherapeutic drugs seemed to have no disadvantages and reduced toxic side effects. On the other hand, reduced-intensity chemotherapy did not prevent second malignancies, which occurred in ten patients with a 10-year incidence of 25%. After individual treatment approaches three of these patients with second malignancies were in complete clinical remission for more than 5 years. We conclude that BFM- or CoALL-oriented chemotherapy is effective and can be administered in children with AT or NBS. Moreover, we show that even second lymphoid malignancies can successfully be treated in these patients.

The CD70/CD27 pathway is critical for stimulation of an effective cytotoxic T cell response against B cell precursor acute lymphoblastic leukemia.

For effective immunotherapy, maintaining the frequency and cytotoxic potential of effector cells is critical. In this context costimulation via the CD70/CD27 pathway has been proven essential. CD70 has been reported to be expressed to varying degrees on malignant B cells. However, in B cell precursor acute lymphoblastic leukemia, the most common childhood malignancy, the role of CD70 in stimulation of antileukemic T cell responses has so far not been delineated. Herein we demonstrate that in B cell precursor acute lymphoblastic leukemia expression of CD70 is low but can be induced upon blast activation via CD40. Both CD70 and CD80/CD86 up-regulated on CD40-stimulated blasts contribute to primary stimulation of T cell proliferation and cytokine production in an additive manner. These two signals also cooperate in the prevention of T cell anergy. In contrast to blockade of CD70 during the effector phase, inhibition of CD70-mediated costimulation during generation of antileukemic T cells prevents effector cell proliferation and reduces their cytotoxic capacity. Modulation of the CD70/CD27 pathway may thus represent a novel therapeutic approach for augmenting magnitude and quality of the antileukemic response in B cell precursor acute lymphoblastic leukemia.

High expression of CD40 on B-cell precursor acute lymphoblastic leukemia blasts is an independent risk factor associated with improved survival and enhanced capacity to up-regulate the death receptor CD95.

CD40 and CD27, members of the tumor necrosis factor receptor (TNFR) family, are critical regulators of lymphocyte growth and differentiation. In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), we prospectively assessed the impact of CD40 and CD27 on outcome in 121 children treated according to the CoALL06-97 protocol. Expression of both CD40 and CD27 was found to be significantly higher in low- than in high-risk patients as defined by standard clinical risk parameters such as age and white blood cell count. In addition, in multivariable analysis, a very high percentage of CD40(+) blasts at diagnosis was identified as an independent favorable prognostic factor for relapse-free survival. Of note, high CD40 expression particularly protected against late relapse. In B cells, CD40 is known to enhance both antigen-presenting capacity and sensitivity to proapoptotic signals. Yet, although CD40 ligation does result in significant up-regulation of CD80/CD86 in our cohort, it is up-regulation of the death receptor CD95 that significantly correlates with the percentage of CD40(+) blasts. Thus very high expression of CD40 on BCP-ALL blasts is an independent prognostic marker indicative of superior relapse-free survival that may in part be due to CD40-dependent death receptor up-regulation.

Ongoing remission after intensive ALL-type chemotherapy in pediatric intestinal T-cell lymphoma.

A rare case of primary intestinal T-cell lymphoma (ITL) of an 8-year-old boy is reported. Medium- to large-sized tumor cells were betaF1+, CD3+, CD8+. TIA-1+, but CD4-, CD5-, CD30-, CD56-, CD20-, CD79a-, TdT-, consistent with an intraepithelial lymphocyte (IEL) origin. They showed monoclonal rearrangement of the T-cell receptor gamma-chain and no evidence of EBV infection. No clinical, histologic, laboratory, or genetic evidence of celiac disease was detected. In adults, ITL is often associated with enteropathy and has a very poor outcome. Our patient remains in first remission 30 months after finishing the acute lymphoblastic leukemia protocol COALL-07-03 high risk standard.

A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome-wide classification study.

BACKGROUND: Genetic subtypes of acute lymphoblastic leukaemia (ALL) are used to determine risk and treatment in children. 25% of precursor B-ALL cases are genetically unclassified and have intermediate prognosis. We aimed to use a genome-wide study to improve prognostic classification of ALL in children. METHODS: We constructed a classifier based on gene expression in 190 children with newly diagnosed ALL (German Cooperative ALL [COALL] discovery cohort) by use of double-loop cross-validation and validated this in an independent cohort of 107 newly diagnosed patients (Dutch Childhood Oncology Group [DCOG] independent validation cohort). Hierarchical cluster analysis with classifying gene-probe sets revealed a new ALL subtype, the underlying genetic abnormalities of which were characterised by comparative genomic hybridisation-arrays and molecular cytogenetics. FINDINGS: Our classifier predicted ALL subtype with a median accuracy of 90.0% (IQR 88.3-91.7) in the discovery cohort and correctly identified 94 of 107 patients (accuracy 87.9%) in the independent validation cohort. Without our classifier, 44 children in the COALL cohort and 33 children in the DCOG cohort would have been classified as B-other. However, hierarchical clustering showed that many of these genetically unclassified cases clustered with BCR-ABL1-positive cases: 30 (19%) of 154 children with precursor B-ALL in the COALL cohort and 14 (15%) of 92 children with precursor B-ALL in the DCOG cohort had this BCR-ABL1-like disease. In the COALL cohort, these patients had unfavourable outcome (5-year disease-free survival 59.5%, 95% CI 37.1-81.9) compared with patients with other precursor B-ALL (84.4%, 76.8-92.1%; p=0.012), a prognosis similar to that of patients with BCR-ABL1-positive ALL (51.9%, 23.1-80.6%). In the DCOG cohort, the prognosis of BCR-ABL1-like disease (57.1%, 31.2-83.1%) was worse than that of other precursor B-ALL (79.2%, 70.2-88.3%; p=0.026), and similar to that of BCR-ABL1-positive ALL (32.5%, 2.3-62.7%). 36 (82%) of the patients with BCR-ABL1-like disease had deletions in genes involved in B-cell development, including IKZF1, TCF3, EBF1, PAX5, and VPREB1; only nine (36%) of 25 patients with B-other ALL had deletions in these genes (p=0.0002). Compared with other precursor B-ALL cells, BCR-ABL1-like cells were 73 times more resistant to L-asparaginase (p=0.001) and 1.6 times more resistant to daunorubicin (p=0.017), but toxicity of prednisolone and vincristine did not differ. INTERPRETATION: New treatment strategies are needed to improve outcome for this newly identified high-risk subtype of ALL. FUNDING: Dutch Cancer Society, Sophia Foundation for Medical Research, Paediatric Oncology Foundation Rotterdam, Centre of Medical Systems Biology of the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research, American National Institute of Health, American National Cancer Institute, and American Lebanese Syrian Associated Charities.

Case-control study on the therapy of childhood cancer and the occurrence of second malignant neoplasms in Germany.

We report on a nested case-control study with 328 cases with second malignant neoplasm (SMN) following childhood cancer and 639 matched controls based on the German Childhood Cancer Registry. In the adjusted overall analysis, the odds ratio (OR) for SMN following any radiotherapy or chemotherapy is 2.1 [95% confidence interval (CI): 1.8-3.3] and 1.8 (95% CI: 0.98-3.1), respectively. The strongest effect is seen for alkylating agents (OR=2.0, 95% CI: 1.2-3.3). The risk of SMN after leukemia is pronounced for antimetabolites (OR=17.2, 95% CI: 1.7-177) and asparaginase (OR=4.3, 95% CI: 1.7-11.0). Following solid tumors, the greatest effect is seen for platinum derivatives (OR=4.1, 95% CI: 1.7-10.1). For anthracyclines, a decreased risk is observed (OR=0.3, 95% CI: 0.1-0.6). Secondary solid tumors are mainly associated with radiotherapy (OR=4.5, 95% CI: 2.5-8.0), especially secondary carcinomas. Secondary acute myeloid leukemia and myelodysplastic syndrome are mainly associated with alkylating agents (OR=8.5, 95% CI: 0.97-74.8), asparaginase (OR=6.8, 95% CI: 2.3-20.6), and platinum derivatives (OR=4.5, 95% CI: 1.5-13.6). The observed risks are in many instances lower than the ones published in previous studies relating to earlier treatment eras of the primary diseases. These differences may be attributed to less toxic but still effective treatment regimes but also to differences in the length of follow-up.

Pharmacokinetics of high-dose methotrexate in infants treated for acute lymphoblastic leukemia.

BACKGROUND: Interfant-99 was an international collaborative treatment protocol for infants with acute lymphoblastic leukemia (ALL). PROCEDURE: We collected data on 103 infants at the time of their first treatment with high-dose methotrexate (HD MTX), 5 g/m(2). Children <6 months of age received two-third of the calculated dose based on body surface area (BSA), children 6-12 months three-fourth of the calculated dose, and children >12 months full dose. RESULTS: The median steady-state MTX concentration at the end of the 24-hr infusion was 57.8 microM (range 9.5-313). The median systemic clearance was 6.22 L/hr/m(2) BSA, and tended to increase with age (P = 0.099). Boys had higher clearance than girls, 6.77 and 5.28 L/hr/m(2) (P = 0.030), and tended to have lower median MTX concentration at 24 hr. Eight infants had MTX levels below 20 microM, a level judged to be sufficient in B-lineage ALL in children >1 year of age. All infants tolerated the dose well enough to receive a second dose of HD MTX without dose reduction. We found no significant effect on disease-free survival for MTX steady-state concentration, MTX clearance, or time to MTX below 0.2 microM. CONCLUSIONS: Our data provide no support for a change in the dosing rules for MTX used in Interfant-99. However, in view of the poor treatment results for infants, one might consider increase in the dose for patients who reach plasma levels below median after the first MTX dose.

Results of CoALL 07-03 study childhood ALL based on combined risk assessment by in vivo and in vitro pharmacosensitivity.

We conducted a clinical trial and report the long-term outcome of 773 children with acute lymphoblastic leukemia upon risk-adapted therapy accrued in trial CoALL 07-03 (from the Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia). In a 2-step stratification, patients were allocated to receive either low- or high-risk treatment, based on initial white blood cell count, age, and immunophenotype. A second stratification was performed according to the results of in vitro pharmacosensitivity toward prednisolone, vincristine, and asparaginase (PVA score) and in vivo response after induction therapy (minimal residual disease [MRD]). Therapy was reduced for both risk groups in patients with a low PVA score or negative MRD result, and intensified in patients with a high PVA score. Overall outcome improved significantly compared with the predecessor CoALL 06-97 trial, with identical therapy backbone despite treatment reduction in 15.8% of patients (10-year probability of event-free survival, 83.5% vs 73.9%; overall survival, 90.7% vs 83.8%). Outcome for patients in the reduced treatment arms was superior to that of patients in the standard arms, associated with a profound reduction in frequency and severity of infectious complications. Importantly, we observed a lack of correlation between in vitro and in vivo drug response, as well as a lower predictive value of in vitro drug testing, reflecting an intrinsic limitation of this methodology that prevents its use for treatment stratification in future trials. In conclusion, it might be possible to reduce chemotherapy in children with acute lymphoblastic leukemia selected by stringent in vivo measurement of MRD without jeopardizing overall outcome.

Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment.

BACKGROUND: Childhood acute lymphoblastic leukemia (ALL) is curable with chemotherapy in approximately 80 percent of patients. However, the cause of treatment failure in the remaining 20 percent of patients is largely unknown. METHODS: We tested leukemia cells from 173 children for sensitivity in vitro to prednisolone, vincristine, asparaginase, and daunorubicin. The cells were then subjected to an assessment of gene expression with the use of 14,500 probe sets to identify differentially expressed genes in drug-sensitive and drug-resistant ALL. Gene-expression patterns that differed according to sensitivity or resistance to the four drugs were compared with treatment outcome in the original 173 patients and an independent cohort of 98 children treated with the same drugs at another institution. RESULTS: We identified sets of differentially expressed genes in B-lineage ALL that were sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), or daunorubicin (20 genes). A combined gene-expression score of resistance to the four drugs, as compared with sensitivity to the four, was significantly and independently related to treatment outcome in a multivariate analysis (hazard ratio for relapse, 3.0; P=0.027). Results were confirmed in an independent population of patients treated with the same medications (hazard ratio for relapse, 11.85; P=0.019). Of the 124 genes identified, 121 have not previously been associated with resistance to the four drugs we tested. CONCLUSIONS: Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

Survivin and its prognostic significance in pediatric acute B-cell precursor lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: Impaired apoptosis, mediated by members of the inhibitor of apoptosis proteins (IAP) family such as survivin, is thought to contribute to leukemic cell survival. In contrast to low expression of survivin in normal differentiated adult tissues, very high levels of survivin have been described in a number of different tumors. Overexpression of survivin was found to correlate with poor prognosis in a variety of cancers including hematologic malignancies. To date, however, there is no information available on the prognostic role of survivin in pediatric precursor B-cell acute lymphocytic leukemia (BCP-ALL), the most frequent malignancy in childhood. DESIGN AND METHODS: In a retrospective study including 66 pediatric patients we analyzed the impact of survivin protein levels on outcome in BCP-ALL. RESULTS: Survivin overexpression, with an up to ten-fold increase of the normal level, was detected in 65% of the leukemic samples in contrast to negligible expression in non-malignant hematopoietic cells. Despite considerable variety of expression levels in ALL cells, there was no association of survivin levels with established risk factors. However, patients suffering relapse of disease or death had significantly higher survivin expression than those with a favorable outcome. Overexpression of survivin is a significant prognostic marker for 3 year relapse free, event-free and overall survival, again independent of the established prognostic factors in ALL, such as age and leukocyte count at diagnosis as assessed in multivariate analysis. INTERPRETATION AND CONCLUSIONS: Overexpression of survivin in BCP-ALL identifies patients with a high risk of early relapse. Upon confirmation in a prospective analysis, survivin expression may, in the future, serve to further refine treatment stratification with intensification of therapy in those patients prone to relapse.

Long-term outcome in children with relapsed ALL by risk-stratified salvage therapy: results of trial acute lymphoblastic leukemia-relapse study of the Berlin-Frankfurt-Münster Group 87.

PURPOSE: Approximately 20% of children with acute lymphoblastic leukemia (ALL) suffer a relapse, and their prognosis is unfavorable. Between 1987 and 1990, the multicenter trial Acute Lymphoblastic Leukemia-Relapse Study of the Berlin-Frankfurt-Münster Group (ALL-REZ BFM) 87 was conducted to establish a uniform treatment for these children in Germany and Austria. PATIENTS AND METHODS: Of 207 registered patients, 183 patients were stratified into three groups according to the protocol: A, early bone marrow (BM) relapse (n = 56); B, late BM relapse (n = 101); C, isolated extramedullary relapse (n = 26). Treatment consisted of risk-adapted alternating short-course multiagent systemic and intrathecal chemotherapy, cranial irradiation, if indicated, and conventional maintenance therapy. Additionally, 24 patients with an exceptionally poor prognosis (early BM or any relapse of T-cell ALL) were treated with individual regimens. In 35 patients, stem-cell transplantation was performed. RESULTS: The probability of event-free survival (EFS) and overall survival of all registered patients at 15 years was 0.30 +/- 0.03 and 0.37 +/- 0.03, respectively, with significant differences between the strategic groups (A, 0.18 +/- 0.05 and 0.20 +/- 0.05; B, 0.44 +/- 0.05 and 0.52 +/- 0.05; C, 0.35 +/- 0.09 and 0.42 +/- 0.10). Despite risk-adapted treatment, an early time point of relapse and T-lineage immunophenotype were significant predictors of inferior EFS in uni- and multivariate analyses. CONCLUSION: With the ALL-REZ BFM 87 protocol, more than one-third of patients may be regarded as cured from recurrent ALL with second complete remissions lasting more than 10 years. Immunophenotype and time point of relapse are important prognostic factors that allow us to adapt more precisely treatment intensity to individual prognosis in future trials.

In vitro sensitivity and cross-resistance to deoxynucleoside analogs in childhood acute leukemia.

BACKGROUND AND OBJECTIVES: Cytarabine (ara-C) is a key drug in the treatment of acute leukemia. Resistance to ara-C might be circumvented by the use of other deoxynucleoside analogs. DESIGN AND METHODS: Using the MTT assay, we determined in vitro sensitivity and cross-resistance to deoxynucleoside analogs in 362 acute leukemia samples from untreated children and 32 normal bone marrow mononuclear cell samples. RESULTS: Normal bone marrow samples were significantly more resistant to ara-C, cladribine and fludarabine than were acute myeloid leukemia (AML) samples and significantly more resistant to ara-C and fludarabine than were acute lymphoblastic leukemia (ALL) samples. The only drug to which AML samples were more sensitive in vitro than ALL was cladribine. AML FAB M5 was significantly more sensitive in vitro to ara-C and cladribine than FAB M1/2 or FAB M4. T-ALL was significantly more resistant to cladribine than B-cell precursor ALL. A paired analysis of 60 AML and 99 ALL samples demonstrated significant cross-resistance between all four deoxynucleoside analogs. Cross-resistance was also observed between ara-C and etoposide (Rp=0.54, p<0.0001), and ara-C and daunorubicin (Rp=0.48, p<0.0001) in AML. In ALL blasts, cross-resistance was observed between ara-C and vincristine (Rp=0.50; p<0.0001), and between ara-C and daunorubicin and L-asparaginase (Rp=0.25; p=0.01; Rp=0.28; p=0.005). INTERPRETATION AND CONCLUSIONS: Cladribine appears to be a useful drug in AML, particularly in FAB M5. We observed cross-resistance between ara-C and other deoxynucleoside analogs, as well as between ara-C and drugs with different modes of action in childhood acute leukemia.

Validation and application of a high-performance liquid chromatographic-based assay for determination of the inosine 5'-monophosphate dehydrogenase activity in erythrocytes.

Thiopurine drug monitoring has become an important issue in treating children with acute lymphoblastic leukaemia (ALL). In this population, a genetic polymorphism causes wide differences in the activity of thiopurine S-methyletransferase (TPMT)--the rate-limiting enzyme of the thiopurine degradation metabolism--leading to the necessity of drug dose adjustments. It is not yet known if similar differences exist in the inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme of the thiopurine synthesis. To test this, we established and validated a high-performance liquid chromatographic (HPLC)-based assay to determine the IMPDH enzyme activity in erythrocytes. The remarkable features of this assay are its simple erythrocyte separation/haemolysis and assay conditions and a distinct segregation of xanthosine 5'-monophosphate (XMP) from the clear supernatant after precipitation. The probes were processed without a time-consuming extraction and heating procedure and the assay demonstrated a good intra- and interday stability as well as a recovery rate of approximately 100%. The IMPDH enzyme activity was measured in erythrocytes of 75 children with diagnosis of ALL before starting antileukaemic therapy and their activity compared to those of 35 healthy adult controls. The measured enzyme activity was wide ranging in both groups. The individual enzyme activity differences observed in children with ALL might led to differences in the thionucleotide levels in those undergoing the standard thiopurine dose regimen.

Expression levels of TEL, AML1, and the fusion products TEL-AML1 and AML1-TEL versus drug sensitivity and clinical outcome in t(12;21)-positive pediatric acute lymphoblastic leukemia.

PURPOSE: t(12;21)(p13; q22), present in approximately 25% of pediatric precursor B-ALL, is highly sensitivity to L-asparaginase and the prognosis depends on the intensity of the treatment protocol. This study analyzes the relationship between the mRNA expression of the genes and fusion products involved in t(12;21), in vitro sensitivity to prednisolone, vincristine, and L-asparaginase, and long-term clinical outcome in t(12;21)+ acute lymphoblastic leukemia (ALL) patients. EXPERIMENTAL DESIGN: Long-term clinical outcome in 45 t(12;21)+ ALL patients was related to mRNA expression of TEL, AML1, TEL-AML1, and AML1-TEL, determined by real-time quantitative PCR, and the in vitro sensitivity to prednisolone, vincristine, and L-asparaginase, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. RESULTS: A significant approximately 3.5-fold lower TEL expression in t(12;21)+ compared with t(12;21)- ALL samples (P = 0.006) and normal controls (P = 0.004) was found. Expression of AML1 did not differ between t(12;21)+ and t(12;21)- ALL. However, AML1 expression in the leukemic cells was 2-fold higher compared with normal controls (P = 0.02). The TEL-AML1 fusion product was expressed in all t(12;21)+ cases, whereas the reciprocal fusion product AML1-TEL was expressed in only 76%. High expression levels of TEL-AML1 [hazard ratio (HR), 1.3; 95% confidence interval (95% CI), 1.10-1.57; P = 0.003], AML1-TEL (HR, 4.9; 95% CI, 1.99-12.40; P = 0.001) and AML1 (HR, 1.1; 95% CI, 1.03-1.22; P = 0.006) were associated with a poor long-term clinical outcome within t(12;21)+ ALL. Cellular drug resistance towards prednisolone, vincristine, and L-asparaginase could not explain this predictive value. Multivariate analysis including age and WBC showed that only high AML1-TEL expression is an independent poor prognostic factor in t(12;21)+ childhood ALL. CONCLUSION: High AML1-TEL expression is an independent poor prognostic factor in t(12;21)+ childhood ALL.

Effect of the histone deacetylase inhibitor depsipeptide on B-cell differentiation in both TEL-AML1-positive and negative childhood acute lymphoblastic leukemia.

The fusion protein TEL-AML1 in t(12;21)+ acute lymphoblastic leukemia (ALL) recruits co-repressors and histone deacetylases (HDAC), which transrepress AML1 target genes. Normal bone marrow cells were more resistant to HDAC inhibitor FK228 induced cell killing than were cells from ALL patients with or without t(12;21). FK228 induced differentiation in ALL, irrespective of the presence of t(12;21).

Interference free and simplyfied liquid chromatography-based determination of thiopurine S-methyltransferase activity in erythrocytes.

The determination of the thiopurine S-methyltransferase activity (TPMT; EC 2.1.1.67) has become an important issue during thiopurine therapy due to its known genetic polymorphism resulting in a wide range of TPMT activity. Therefore, the standard thiopurine drug regimen is associated with increased hematopoetic toxicity in patients with low or absent TPMT activity, whereas patients with high activity may be insufficiently treated. However, presently available methods are labour intensive and time consuming and tend towards too high or too low enzyme activity due to their methodological approach. The use of instable substrate solutions (6-MP or 6-TG), organic solvents like dimethyl sulfoxide and too high substrate and co-substrate saturation concentrations contribute to this phenomenon. We therefore, established an optimized and fast isocratic HPLC linked TPMT assay based on the enzymatic methylation of mercaptopurine or thioguanine in RBC lysates with S-adenosyl-l-methionine as methyl donor. Unspecific non-enzymatic methylation was not detectable. The recovery of 6-methyl-mercaptopurine was 97-102%, the intra- and interday variation between 1.0 and 5.0%, respectively. The assay dispenses with a time consuming extraction procedure with organic solvents, a heating step, and a gradient elution and is therefore, favourable for clinical routine application. The TPMT activity was measured in 62 untreated children with acute lymphoblastic leucemia at the time of diagnosis (activity = 34.0+/-10.6 nmol/g Hb/h, range: 11.5-55.4 nmol/g Hb/h) and in 12 adult healthy volunteers (62.8+/-7.7 nmol/g Hb/h, range: 48-82 nmol/g Hb/h) reflecting the wide measurable TPMT activity found in erythrocytes.